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MECHANISMS LEADING TO TRANSLATION ARREST IN
AUTOGRAPHA CALIFORNICA MULTINUCLEOCAPSID
NUCLEOPOLYHEDROVIRUS-INFECTED LYMANTRIA

DISPAR CELLS

presented by

Christy Mecey

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MECHANISMS LEADING TO TRANSLATION ARREST
IN A UT OGRAPHA CALIF ORNICA MULTINUCLEOCAPSID
NUCLEOPOLYHEDROVIRUS-INFECTED
LYAMNTRIA DISPAR 652Y CELLS

By

Christy Mecey

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment for the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Genetics Program

2006

ABSTRACT

MECHANISMS LEADING TO TRANSLATION ARREST
IN AUTOGRAPHA CALIFORNICA MULTINUCLEOCAPSID
NUCLEOPOLYHEDROVIRUS-INFECTED
LYMANTRIA DISPAR 652Y CELLS
By

Christy Mecey

Infection of the Lymantria dispar 652Y (Ld652Y) cell line with the baculovirus
Autographa calzfornica Multinuoleocapsid nucleopolyhedrovirus (AcMNPV) results in
global translation arrest, initiation of apoptosis in the absence of a functional, virus-
encoded, apoptotic suppressor and a non-productive infection. While AcMNPV enters
Ld652Y cells and transcription of virus genes occurs, virus progeny are not produced.
Expression of a single gene, host range factor-1 (hrf-I), isolated from Lymantria dispar
Multinuoleocapsid Nucleopolyhedrovirus (LdMNPV), precludes translation arrest in
AcMNPV-infected Ld652Y cells further allowing permissive replication of the virus.
HRF-l also enables replication of Hyphantria cunea NPV, Bombyx mori NPV and
Spodoptera exigua NPV in Ld652Y cells indicating that hrf-I is an essential host range
factor for baculovirus infection in Ld652Y cells. Database and motif searches conducted
with the hrf-I sequence provide no indication to its function. The goal of this study was
to determine the mechanism of translation arrest in AcMNPV-infected Ld652Y cells to
further identify possible functions for HRF-l. We Show that eIF20. phosphorylation at
serine 51 occurs in AcMNPV-infected deSZY cells at 12 h.p.i. correlating to the onset

of translation arrest. Ld652Y cells infected with vAchrf-l Show a reduced level of

eIFZa phosphorylation for the full-length peIF20tserSl protein at 6, 12, 24 and 48 h.p.i. as
compared to mock-infected cells. PK2, the AcMNPV eIF20t kinase inhibitor, was

expressed in AcMNPV-infected Ld652Y cells but did not prevent eIF20t

ser5 l

phosphorylation. An apparent proteolytic cleavage of peIF20t was observed beginning
at 12 h.p.i.; this cleavage event was investigated further. In Sf21 cells, a permissive cell
line for AcMNPV replication, peIF20tscr51 was cleaved in both AcMNPV and vAchrf-l-
infected cells indicating that HRF-l was not directly responsible for the cleavage. Cell

5ch l

permeable protease inhibitors did not block cleavage of peIFZa . However, the

proteasome inhibitor epoxomicin did reduce phosphorylation of eIF2a at serine 51, block
cleavage of peIFZIIIschl and inhibit virus replication in AcMNPV and vAchrf-l-infected
8121 cells and vAchrf-l-infected Ld652Y cells. The proteasome inhibitor M0132
mimicked the effects of epoxomicin in vAchrf-l-infected Ld652Y cells. However,
treatment with MG132 in AcMNPV and vAchrf-l-infected SfZl cells resulted in reduced

virus titers and no block of peIFZItscr51

cleavage. This study suggests a role for the
proteasome in enabling baculovirus replication. A cross-reactive antibody to the human
eIF2 kinase, PKR, showed enhanced expression of a protein in vAchrf-l-infected cells at
12-24 h.p.i. Mass spectrometry analysis identified this protein as a 90 kDa heat shock
protein (HSP90). Inhibition of HSP90 with geldanamycin resulted in a block of occluded
virus production in baculovirus-infected $121 and Ld652Y cells at 48 h.p.i. Cleavage of
occurs in productively-infected cells in the presence of geldanamycin indicating that

cleavage is not dependent on HSP90 activity. The data suggest a possible role for HSP90

during the very late stages of baculovirus infection.

Cepyright by
Christy Mecey

2006

ACKNOWLEDGEMENTS

I would like to thank my principal investigator, Dr. Suzanne Thiem. Her efforts
have helped me to develop the critical thinking skills that are required to be an effective
scientist. Her unwavering requirement for absolute fact in the interpretation of scientific
data is demanding and valuable. Dr. Thiem has also taught me how to run a functional
laboratory with limited resources and her determination in the face of set-back is
inspirational.

Many thanks to my remarkable committee: Dr. David Amosti, Dr. Rebecca
Grumet, Dr. Steve Triezenberg and Dr. Suzanne Thiem. You were all chosen based not
only on your tremendous contributions to science but on, your equal commitment to
science education. Input from the committee was important, relevant and timely. I would
like to especially thank Steve and Rebecca for their time and effort in coordinating the
GAANN fellowship program. Your extended efforts enabled the funding of my graduate
education and increased science literacy amongst educators in the state of Michigan.

I would like to genuinely thank my husband Richard who is always kind even in
the wrath of my wildest mood swings. Without his help I would never have been able to
navigate through the daily challenges of being a mother and a graduate student.

I would like to thank Montgomery for all of the weekends she had to spend
playing in the lab instead of playing in the park. I am so fortunate that you are my

daughter.

I would like to thank my parents, who thought that getting a Ph.D. was the silliest
idea I’ve ever had, for their continued support and practicality.

Many thanks to the members of the Allison lab, past and present, Jamie, Cheryl,
Meredith, David, Katie and Julia for their friendship and technical support. Thank you
Cheryl Calhoun for teaching me that balance really is the most important aspect of life.

I would also like to thank the members of the Thiem lab, past and present, Ale,
J ian, Aubrey and Heather for their friendship, assistance and input.

I have genuine, heartfelt appreciation for Jeannine Lee, the most talented program
assistant at MSU. There was never a question she could not find an answer to or a
problem that she could not solve.

I would like to thank all of the friends that I have made throughout my graduate
experience for their supportive discussions, their insight, their backgrounds and their
vision for the future. A special thank you for Linda Danhof, master coordinator and
technician for the PRL, there are so many technical procedures that Linda either taught
me or found someone to teach me that it would be impossible to list them all as a part of
this document.

I would like to dedicate this effort to the family members that we have lost in the
past year that are missed and remembered: Lee McIntosh, Florence Allison and my 34
year old sister, Jamie Dixon. There is not a day that passes that I don’t wish I could trade

places with my sister and return her to her lost family.

-vi-

TABLE OF CONTENTS

List of Figures ............................................................................................. x
List of Tables ........................................................................................... xii
List of Abbreviations .............................................................................. xiii
Rationale for Study .................................................................................. 1
Chapter 1:
Literature Review .................................................................................... 4
1.1. Baculovirus pathogenesis ........................................................................ 5
1.2. Baculovirus replication ........................................................................... 7
1.3. Baculovirus host range ......................................................................... 11
1.3.1. Invertebrate innate immunity to virus infection ................................. 12
1.3.2. Developmental resistance to baculovirus .......................................... 14
1.3.3. Species-mediated resistance to baculovirus ...................................... 14
1 .3 3. 1. Host cell factor-l (HCF-l) .............................................. 14
l .3 3. 2. p143... 15
1. 3. 3. 3. Apoptotic shppreSsors and host range .................................. 16
1..3 3. 4. Host range factor-1 (HRF- -1) ............................................ 18
1.3.3.5. Other baculovirus proteins associated
with host range ............................................................. 20
1.4. Translation modifications in response to stress21
1.4.1. Translation initiation ............................................................... 22
1.4.2. Initiation stage translational modifications in
response to stress ................................................................... 23
1.4.2.1. Phosphorylation of eIF20t ............................................... 24
1.4.2.2. 4E-BP and eIF4E ......................................................... 25
1.4.2.3. Proteolytic cleavage of eIF4GI, eIF4GII
and other initiation factors. 26
1.4.3. Virus strategies for overcoming host-mediated
translation arrest .................................................................... 27
1.4.4. Preferential translation of viral transcripts ...................................... 29
1.5. Stress response pathways and virus infection ............................................ 30
1.5. l. The unfolded protein response .................................................... 30
1.5.1.1. The unfolded protein response and virus infection ...................... 34

-vii-

1.5.1 . 1.1. Host-encoded molecular chaperone requirements for virus

infection ............................................................... 35
1.6. Proteasome activation and virus infection ................................................... 36
1.6.1. The COP9 signalosome , protein degradation and kinase signaling ........ 38
Chapter 2:
In baculovirus infected Lepidoptera cells, proteasome inhibitors reduce virus
production and eIFZa phosphorylation at serine 51 ........................................ 40
2.1. Introduction ..................................................................................... 41
2.2. Materials and methods ......................................................................... 44
2.2.1. Viruses and cell lines ............................................................... 44
2.2.2. Cytoplasmic fraction extraction and polysome profiling ..................... 44
2.2.3. Cell stress induction assays ......................................................... 45
2.2.4. Protease and proteasome inhibitor assays ....................................... 46
2.2.5. Western blot analysis ............................................................... 46
2.2.6. Plaque assays ............... . ........................................................ 48
2.3. Results ....................................................................................... 48
2.3.1. Translation arrest in AcMNPV-infected
Ld652Y cells occurs at the initiation stage ............................................. 48
2.3.2. Translation arrest in AcMNPV-infected Ld652Y cells correlates with
eIF2a phosphorylation at serine 51 .............................................. 50
2.3.3. Proteolytic cleavage of peIF 20Lscr51 occurs in AcMNPV and vAchrf-l
infected Sf21 cells ................................................................. 55
2.3.4. PK2 is expressed in AcMNPV-infected Ld652Y cells ....................... 56
2.3.5. Cleavage of peIF2ocsets l in Sf21 and Ld652Y cell lines is not induced by
stress-inducing agents ............................................................. 57
2.3.6. Cleavage of peIF20tserSI is blocked by aphidicolin but not by protease
inhibitors ............................................................................ 59

2.3.7. Proteasome inhibition reduces phosphorylation of eIF20t at serine 51 and
virus production in lepidopteran cells productively-infected with

baculovirus ......................................................................... 62

2.4. Discussion ................................................................................. 68
Chapter 3:

HSP90 expression is enhanced in vAchrf-l-infected Ld652Y cells ...................... 75

3.1. Introduction ..................................................................................... 76

3.2. Materials and methods ......................................................................... 81

3.2.1. Viruses and cell lines ............................................................... 81

3.2.2. Irnmunoprecipitation and mass spectrometry analysis ........................ 81

- viii -

3.2.3. MALDI-TOF-MS .................................................................. 82

3.2.4. LC-ESF-MS ........................................................................ 82

3.2.5. Western blot analysis ............................................................... 83

3.3. Results ........................................................................................... 84
3.3.1. HSP90 is activated in Ld652Y cells that are productively-infected

with vAchrf-l ....................................................................... 84

3.3.2. Inhibition of HSP90 with geldanamycin inhibits polyhedrin protein
synthesis and occlusion body production in permissively-infected
cells at 48 h.p.i. but has little effect on budded virus production. . . . ........86
3.3.3. Proteolytic cleavage of peIF2aser51 precedes and is independent of

increased HSP90 expression in permissively-infected Ld652Y cells ...... 92
3.4. Discussion .................................................................................. 94
Chapter 4:
Summary and Future Directions ............................................................... 97
4.1. Research Summary ........................................................................... 98
4.2. Future Directions .............................................. , ............................... 100
Appendix A: Supplementary Data ............................................................ 107
References .......................................................................................... 1 13

-ix-

LIST OF FIGURES

Figure 1. Global translation arrests and virus replication is blocked in AcMNPV-infected

Ld652Y cells .......................................................................................... 2
Figure 2. Baculovirus infection cycle .............................................................. 6
Figure 3. Transcriptional regulation of baculovirus genes ...................................... 8

Figure 4. Early and late events that occur during AcMNPV infection
of Ld652Y cells ....................................................................................... 20

Figure 5. Initiation of protein synthesis ......................................................... 23

Figure 6. Phosphorylation of eIF20t and viral mechanisms to overcome
translation arrest that occur as a result of eIFZu phosphorylation ............................. 25

Figure 7. The unfolded protein response (UPR). . . . . . . . . . . . . . ................................ 34

Figure 8. Translation arrest occurs at translation initiation in
AcMNPV-infected Ld652Y cells ..................................................................... 50

Figure 9. Phosphorylation of full length eukaryotic initiation factor 2a (eIF20t) at serine
51 is reduced in vAchrf-l-infected Ld652Y cells as compared to AcMNPV-infected
Ld652Y cells ............................................................................................ 53

Figure 10. Phosphorylated eIF20tserSI is proteolytically cleaved in cell lines that are
productively infected with AcMNPV or vAchrf-l ............................................... 54

Figure 11. PK2 is present at late time points post-infection in Ld652Y cells infected with
AcMNPV or vAchrf-l ............................................................................... 57

Figure 12. Proteolytic cleavage of peIF20ts°'5 ' does not occur in Sf21 or Ld652Y cells
that are treated with stress-inducing agents ....................................................... 58

Figure 13. Phosphorylation and proteolytic cleavage of pelFZoU"chI is inhibited in
Lepidoptera cells that are productively-infected with baculovirus and treated with
serSl -

aphidicolin. Proteolytic cleavage of peIF20t 1s not blocked by protease
inhibitors .............................................................................................. 60

Figure 14. Proteasome inhibitors, MG132 and epoxomicin, inhibit phosphorylation and
proteolytic cleavage of peIF20tscr51 in vAchrf-l-infected Ld652Y cells ...................... 64

Figure 15. The very late baculovirus occlusion body protein, polyhedrin, is not produced
in productively-infected cells that are treated with epoxomicin ............................... 65

Figure 16. Proteasome inhibitors prevent budded virus production in vAchrf-l-infected
Ld652Y cells .......................................................................................... 67

Figure 17. Epoxomicin prevents AcMNPV or vAchrf-l budded virus production in 891
cells, M0132 reduces budded virus production in those same infected cells ................... 68

Figure 18. HSP90 levels increase in vAchrf-l-infected Ld652Y cells from 12-24
h.p.i ................................................................................................... 85

Figure 19. Geldanamycin delays or prevents occlusion body formation in lepidopteran
cells permissively-infected with baculovirus .................................................... 89

Figure 20. In cells treated with the HSP90 inhibitor, geldanamycin, polyhedrin protein
levels are reduced in Sf21 and Ld652Y cells productively-infected with
baculovirus ........................................................... . ................................. 90

Figure 21. Geldanamycin reduces budded virus production in vAchrf-l-infected Ld652Y

cells ................................................................................................... 92
Figure 22. Proteolytic cleavage of peIF20tscrsl occurs in Ld652Y and Sf21 cells
productively-infected with AcMNPV or vAchrf-l .............................................. 93
Figure 23. Proposed model of post-translational modification of eIF20t in response to
baculovirus infection ............................................................................... 101
Figure 24. The interaction of eIF2a with eIFZB ............................................. 104

Figure 25. Proposed model for HRF-l function in vAchrf-l-infected Ld652Y cells. . . .106

Figure 26. AcMNPV infection in Ld652Y cells does not impair the m7G cap-binding
ability of eIF4E .................................................................................... 128

Figure 27. 4E-BP is not up-regulated in response to AcMNPV infection in Ld652Y
cells .................................................................................................. 130

Figure 28. The lepidopteran kinase, 86K, is expressed in AcMNPV-infected Ld652Y
cells at a time that correlates to the onset of global translation arrest ...................... 131

Figure 29. Anti-PKR immunoprecipitation gels for mass spectrometry analysis... . . ....132

-xi-

LIST OF TABLES

Table l. Sequest summary of mass spectrometry analysis for
immunoprecipitation with anti-PKR antibodies in vAchrf-l
infected Ld652Y cells at 12 h.p.i .................................................................. 86

-xii-

4E-BP

AcMNPV

ATF
ATP
BeK
BiP
BmN
BmNPV
BV
Cf-203

CflVINPV

COP9 / CSN
Dm

DNA
Dnapol
dsRNA

EDEM

EDTA

KEY TO SYMBOLS OR ABBREVIATIONS

eIF4E binding protein
amino acid

Autographa califomica Multicapsid nuclear
Polyhedrosis Virus

alkaline nuclease

activating transcription factor

adenosine triphosphate

Bombyx mori eIF2a kinase

glucose regulated protein 78

Bombyx mori N cell line

Bombyx mori Multi Nucleocapsid Polyhedrovirus
budded virus '

Choristoneurafumiferana 203 cell line

Choristoneurafumiferana Multi Nucleocapsid
Polyhedrovirus

COP9 signalosome
Drosophila melanogaster
deoxyribonucleic acid
DNA polymerase
double-stranded RNA

ER degradation—enhancing a-mannosidase-like
protein

(ethylenedinitrilo) tetraacetic acid

- xiii -

eIF

ER

ERAD

ERSE

GCN

GDP

GTP

HCF

HCMV

HCV

HHV

HIV

HPI

HPPV

HRI

HSC

HSP

HSV

IAP

eukaryotic initiation factor

endoplasmic reticulum

endoplasmic reticulum-associated degradation
endOplasmic reticulum stress responsive element
general control non-derepressible

guanosine diphosphate

guanosine triphosphate

host cell factor

human cytomegalovirus

hepatitis C virus

human herpes virus

human immunodeficiency virus

hours post-infection

human papillomavirus

hours post-treatment

homologous repeat

host range factor

heme-regulated inhibitor of protein synthesis
heat shock cognate protein

heat shock protein

herpes simplex virus

inhibitor of apoptosis

~xiv-

IE

IRE
IRES
JEV

JN K
LEF
Ld652Y

LdMNPV

Met
MOI
mRNA

mTOR

ODV

OpMNPV

OV

P35

P39

PABP
PERK/PEK

PK2

immediate-early gene
inositol-requiring enzyme
internal ribosomal entry site
Japanese encephalitis virus
c-Jun kinase

late expression factor
Lymantria dispar 652Y cell line

Lymantria dispar Multi Nucleocapsid
Polyhedrovirus

methionine

multiplicity of infection
messenger RNA

mammalian target of raparnycin
nuclear factor-kappa B
occlusion derived virus

Orygia pseudotsugata Multi Nucleocapsid
Polyhedrovirus

occluded virus

baculovirus 35 kD apoptotic suppressor protein
baculovirus 39 kD capsid protein

poly-A binding protein

RNA-dependent-like endoplasmic reticulum kinase
AcMNPV inhibitor of eIF2a kinases

ribonucleic acid

-xv-

RNAi
RNA Pol. II

SeMNPV

St‘9
Sf21
Tn368
TNF
TRAF
tRNA
UPR
UTR

vAcAp35

VLF
vRNA Pol

XBP

RNA interference
RNA polymerase II

Spodoptera exigua Multi Nucleocapsid
Polyhedrovirus

Spodopterafiugiperda 9 cell line
Spodopterafrugiperda 21 cell line
Trichoplusia m' 368 cell line
tumor necrosis factor
TNF-receptor kinase

transfer RNA

unfolded protein response
untranslated region

AcMNPV strain with the apoptotic suppressor, p35,
deleted

very late expression factor
viral RNA polymerase

X-box binding protein

-xvi-

Rationale for Study

In 2001, annual international insecticide sales totaled over 9 billion dollars
(Donaldson et al., 2004). Chemical insecticides are often effective but can act broadly
and harm non-target species. Many chemical insecticides have deleterious effects on the
environment and have been removed from the commercial market or relicensed to limit
use (Donaldson et al., 2004). Biopesticides and biological control agents are living
organisms or products from living organisms used to suppress pest populations.
Biopesticides offer an alternative to chemical-based insecticides and generally affect a
narrower range of target species (Thiem, 1997). Baculoviruses primarily infect
Lepidopteran insects during the larval stage of development and are a biological control
agent used commercially to control soybean pests. Narrow host range is the primary
limiting factor to the further utilization and subsequent development of baculoviruses as
biopesticides (Maeda, 1995; Moscardi, 1989; Moscardi, 1999; Thiem, 1997).

Unlike bacterial and fungal host-pathogen systems, the molecular mechanisms
that govern invertebrate host-specificity to virus infection are not understood. In cell
culture, many baculoviruses are able to enter non-host invertebrate cells, virus replication
is initiated but then terminated during the late stages of infection (Du and Thiem, 1997a;
Lu and Miller, 1996). The Gypsy Moth, Lymantria dispar, is a non-permissive host to
Autographa caliform'ca Multinucleocapsid Nucleopolyhedrovirus (AcMNPV), the type
virus for the Baculoviridae family (McClintock, Dougherty, and Weiner, 1986). Virus
replication begins in Lymantria dispar 652Y (Ld652Y) cells that are infected with
AcMNPV but global translation arrest is initiated by 9 h.p.i. correlating to the late stages

of infection and no virus progeny are produced (Fig. 1A) (Guzo et al., 1992). Expression

of a Single gene, host range factor-I (HRF-l), isolated from Lymantria dispar
Multinucleocapsid nucleopolyhedrovirus (LdMNPV), blocks translation arrest and
enables the production of virus progeny in AcMNPV-infected Ld652Y cells and L.
dispar larvae (Fig. 13) (Chen et al., 1998; Du and Thiem, 1997a; Du and Thiem, 1997b;
Thiem et al., 1996). Homology and motif searches conducted with the hrf-I sequence

provide no indications to its function.

AcMNPV Infection in Ld652Y cells 8. HRF-1

Late Stages of Infection
Global , ‘3
Translation . '

/ Arrest
Ld652Y Cells N_o Progeny
Virus Produced

 

Virus enters cell,
viral transcripts are
made, translation of

 

 

early transcribed
genes OCCUI’S .
Ld652Y Cells | I
B ' @I % 0
. I I
AcMNPV ' ‘- - 0
+ 7- .. _ g or w o n . 0 .
H RF-‘I A Productive Virus
(LdMNPV) Lymantria dispar Infection

larvae

Figure 1. Global translation arrests and virus replication is blocked in AcMNPV-
infected Ld652Y cells (A). Expression of the LdMNPV gene, host range factor-1
(HRF-l), precludes translation arrest in AcMNPV-infected Ld652Y cells and
Lymantria dispar larvae further enabling the production of virus progeny (B).
(Image Presented in Color)

It has not been determined whether global translation arrest is the direct mechanism

leading to a block in the production of virus progeny in AcMNPV-infected Ld652Y cells

or simply a correlative event resulting from infection. Global translation arrest may be a
host-mediated innate immune response to control virus replication or a host-induced
response to stress. In mammalian systems, viruses use a variety of mechanisms to block
translation of host-encoded genes as a means of ensuring preferential translation of viral
transcripts (Jang et al., 1988; Joachims, Van Breugel, and Lloyd, 1999; Kerekatte et al.,
1999; Pelletier and Sonenberg, 1988). This is unlikely in baculovirus-infected cells as
host macromolecular synthesis appears to be controlled at the transcriptional level
(Nobiron, O'Reilly, and Olszewski, 2003; Ooi and Miller, 1988).

In this study, my goal was to identify the mechanism(s) that leads to translation
arrest in AcMNPV-infected Ld652Y cells. I hypothesized that identifying this
mechanism might provide some indication to the function of HRF-l. Furthermore, we
hoped to gain insight into invertebrate host responses that are initiated during non-
permissive infections to further understand the components that determine baculovirus

host range.

Chapter 1

Literature Review

Introduction
1.1 Baculovirus Pathogenesis

Baculoviruses are large, double-stranded DNA viruses that primarily infect
lepdiopteran insects during the larval grth stage. Autographa californica Multicapsid
Nuclear Polyhedrosis virus (AcMNPV) is the type virus for the family Baculoviridae.
Baculoviruses have a biphasic replication cycle which results in rod-shaped virions being
packaged into a budded and occluded form (Theilmann et al., 2004). Budded virus,
produced from a primary infection, is enveloped at the plasma membrane and infects
neighboring cells by adsorptive endocytosis (Blissard and Wenz, 1992; Volkman and
Goldsmith, 1988) resulting in a systemic infection. Synthesis of budded virus is reduced
by 24 hours post-infection (h.p.i.) and there is a switch to occluded virus production
(V olkman and Knudson, 1986). The occluded form of the virus (0V) is environmentally
stable and consists of multiple, enveloped nucleocapsids that are packaged into a
polyhedrin protein-rich matrix. Virions condensed in a polyhedrin matrix are wrapped in
a virus-derived, carbohydrate-rich, calyx which forms a unilamellar structure with spike-
like projections, forming the occlusion body. Caterpillars ingest the occluded form of the
virus, the polyhedra dissolve rapidly within the alkaline pH of the midgut (Summers,
1971; Summers and Amott, 1969) and the virions pass through the peritrophic membrane
of the insect. The virus enters cells via fusion of virion envelope with the microvillar
membrane (Granados, Lawler, and Burand, 1981). The virus then travels along the
microvillus aided by microtubules (Charlton and Volkman, 1993; Granados, Lawler, and
Burand, 1981) and enters the nucleus where it uncoats at the nuclear pore or just inside

the nuclear membrane (Bilimoria, 1991) (For review of the baculovirus infection cycle

see Fig. 2) (Blissard and Rohrmann, 1990). Advanced infection of the larvae results in

complete cellular destruction and a condition known as “melted” (Federici, 1997).

     
   

Nuclear Membrane
Nuclear Pore

Virogenic Stroma

O Occlusion Body

  

 

Primary . .
’ Infectiom . Budded Vlrus i lngested by caterpillar
21'3“" vaI'I‘ Secondary lnfection-
é ' gm ° “50W" End°°Yt°sis é Polyhedra solubilized
= .‘ ______ , in the midgut-released
when insect tissues
deteriorate

Figure 2. Schematic diagram of the baculovirus infection cycle adapted from
Blissard and Rohrmann 1990 (Image Presented in Color)

1.2 Baculovirus Replication

Within the nucleus of the midgut epithelial cells the infection process begins with
immediate and early gene transcription of the virus followed by DNA replication, late
gene expression, the production and release of budded virus, very late gene expression
and the synthesis of occluded virus (See Fig. 3). Baculoviruses encode a DNA
polymerase gene that is transcribed early in the infection process and is essential to
replication of the virus (V anarsdall, Okano, and Rohrmann, 2005). Additional viral-
encoded genes required for virus replication include: immediate-early gene-1 (ieI), late
expression factor flef) I and 3, alkaline nuclease (an), p143 and p35 (Ito et al., 2004;
LaCount and Friesen, 1997; McDougal and Guarino, 2000; Mikhailov and Rohrmann,

2002; Stewart et al., 2005; Todd, Passarelli, and Miller, 1995).

lnfectlon

Beglns

I Early Late Very Late

3 fl : fl :fl
Transcription § ie1, ieO, RNA Pol", § 5 i
Mediated by: HR repeats, host :

E Transcription factors, Virus-encoded

5 other host factors 5 alpha-atnantin resistant

I RNA polymerase
Virus Genes ie1, ieO, let-1, lef-2, , .
Transcribed: § lef-3, lei-7, hcf-1, § Structural Proteins. § P°'Yh°d""

p143, p35, ie2, DNA —

E pol. and pe-38 ‘ ‘
DNA Replication :
(Genes required leI, Ief-1, Ief-3, p143,§
for replication) 5 p35, DNA 5 """"""""""""" ’

polymerase I

Most RNA Pol. ll
Transcription

Downregulated

Budded Virus

Production

Occluded Virus I I I— I

Production ; : : 5

Figure 3. Schematic diagram of transcriptional regulation that occurs during the
baculovirus replication cycle adapted from Rohrman 1992

Transcription of early expressed baculovirus genes is regulated by host RNA
polymerase II and IE1. IE1 has a splicing variant known as IE0. Individually either IE1
or IEO can support virus replication but both are required to achieve a wildtype infection
(Stewart et al., 2005). Homologous repeat (HR) sequences that serve as transcriptional
enhancer regions can be found in eight locations of the AcMNPV genome (Rasmussen et
al., 1996) and HR regions have been identified in all sequenced baculoviruses. IE1

regulates early virus transcription in cis by binding a 28-mer palindromic site in the HR

regions and host transcription factors also bind within the HR region (Landais et al.,
2005). LEF—1 is a DNA primase (Mikhailov and Rohrmann, 2002) and p143 a DNA
helicase (McDougal and Guarino, 2000) that associates with LEF-3 (Evans et al., 1999),
a single-stranded DNA binding protein, for nuclear transport and localization (Chen and
Carstens, 2005). It has been proposed that the HR sites also serve as origins of DNA
replication, indeed HR sites are essential for virus DNA replication (Leisy et al., 1995).
Immunoprecipitation followed by crosslinking has shown that IE-l , P143 and LEF-3 bind
viral chromatin at closely linked sites, suggesting that replication complexes form at the
homologous repeat regions (Ito et al., 2004). Baculoviruses also encode an early
expressed alkaline nuclease (AN) which is essential for virus replication. AN has 5’-to-3’
endonuclease activity on single-stranded DNA and 5’-to-3’ exonuclease activity when
associated with LEF-3. The data suggests that AN plays a role in DNA recombination
and the resolution of replication intermediates (Mikhailov, Okano, and Rohrmann, 2004).
The apoptotic suppressor, p35 (Clem, Fechheimer, and Miller, 1991) directly binds
caspases, blocking substrate proteolysis. P35 functions at early and late timepoints post-
infection (LaCount and Friesen, 1997). Baculovirus IE2, LEF-7, HCF-1 and P38
stimulate viral replication but are not essential to a productive infection (Miller, 1997).
The late stage events of baculovirus infection are defined as those that occur after
DNA replication of the virus. The production of budded virus temporally correlates to a
time immediately following DNA replication and the production of occluded virus is the
result of the late stages of infection in cells infected with baculovirus (Friesen and Miller,
1986; Rice and Miller, 1986). Characterized late genes are transcribed at six to twenty-

four hours post-infection (h.p.i.), the corresponding gene products are primarily structural

in nature and include the VP39 capsid protein (Thiem and Miller, 1989) and P69 core
protein (Wilson et al., 1987). Very late genes are transcribed in a burst of expression from
eighteen to seventy-two h.p.i. and include the polyhedrin occlusion body protein
(Rohrmann, 1986) and p10, which effects nuclear disintegration upon exit of the
occluded virus (Rohrmann, 1992).

Regulation of the late stages of infection is primarily transcriptional. The segue of
early-to-late gene transcription is coupled to a decrease in host mRNA levels at six to
eight h.p.i. (Nobiron, O'Reilly, and Olszewski, 2003; Ooi and Miller, 1988) and the
induction of a virus-encoded (Guarino et al., 1998), alpha-amantin resistant RNA
polymerase (vRNApol) (Beniya et al., 1996; Fuchs, 1983; Grula, 1981a; Grula, 1981b;
Huh and Weaver, 1990; Yang, Stetler, and Weaver, 1991). By 18 h.p.i. transcription is
exclusive to vRNApol (Fuchs, 1983; Huh and Weaver, 1990) (For a review of
baculovirus temporal gene regulation, see Fig. 2). The viral RNA polymerase specifically
binds the virus template at a consensus sequence of TAAG found in the promoters of late
and very late genes (Morris and Miller, 1994; Ooi, Rankin, and Miller, 1989; Rankin,
Ooi, and Miller, 1988; Thiem and Miller, 1989; Thiem and Miller, 1990). However, host
and virus transcription factors, including IE1, IEO and host GATA factors, bind the virus
DNA template in concert with the vRNApol (Chen, Tsai, and Chen, 2001; Hefferon,
2004; Krappa et al., 1992; Landais et al., 2005) and the HR enhancer elements play a
critical role in gene regulation even at late times during the infection process (Hefferon,
2004). Twenty viral early or constitutively expressed genes are required for late and very
late baculovirus gene expression, these include: ie-I, ie-2, dnapol, p143, p47, p35, 39K

and the late expression factors (LEFS) 1-12 (Hefferon, 2004; Li, Harwood, and

10

Rohrmann, 1999; Lu and Miller, 1995b). The baculovirus-encoded LEF-8 and LEF-9 are
homologous to subunits of RNA polymerase II. LEF-4 has guanyltransferase activity and
can be purified as a component of the vRNApol complex (Guarino, Jin, and Dong, 1998).
The very late expression factor 1 (V LF -1) is required for transcription from the
polyhedrin promoter (McLachlin and Miller, 1994; Mistretta and Guarino, 2005).
1.3 Baculovirus Host Range

Baculoviruses have been commercially developed as eukaryotic protein
expression vectors and as biopesticides. Hundreds of different baculoviruses have been
isolated from hundreds of species of infected lepidopteran larvae and it is predicted that
thousands more exist (Federici, 1997; Theilrnann et al., 2004). A small percentage of
characterized baculoviruses have the potential to be useful in controlling insect pests
(Moscardi, 1999; Yearian, 1982). The use of baculoviruses as insect control agents has
been successful for controlling a lepidopteran pest of soybeans (Moscardi, 1989). The
limited commercial success is due, in part, to the narrow host range of baculoviruses.
AcMNPV has the broadest host range and can permissively infect six Lepidopteran
species and semi-permissively infect another twenty-six (Federici, 1997). Permissive
hosts are denoted as those being highly susceptible to a particular virus infection while
semi-permissive are those that are less susceptible (Bishop, 1995). Environmentally, the
limited host range of baculoviruses is beneficial because effects on non-target species are
minimal to non-existent. Economically, the limited host range of baculoviruses is
detrimental because agricultural producers require a single broad-range insecticide to
maintain cost effectiveness. Substantial research has been devoted to expanding host

range and/or enhancing insecticidal properties through the development of recombinant

11

baculoviruses that express host range factors, insect regulatory proteins and/or general
insecticidal toxins (Black, 1997; Maeda, 1995).

In mammalian cells, virus host range is frequently determined by the interaction
of a viral protein with a host-encoded receptor. This does not appear to be true for
baculoviruses. In a number of non-permissive and semi-pennissive baculovirus infections
the virus enters the cell nucleus, viral DNA replication begins but infection is blocked
during the late stages of infection (Miller, 1997; Simon et al., 2004; Thiem, 1997;
Yanase, Yasunaga, and Kawarabata, 1998). As an example, AcMNPV infects, has some
late gene expression but does not productively replicate in cell lines from Bombyx mori,
Choristoneura firmiferana, Mamestra brassicae and Lymantria dispar. AcMNPV infects
Drosophila melanogaster cell lines but late gene expression does not occur (Morris and
Miller, 1992; Morris and Miller, 1993). AcMNPV can enter and evoke an immune
response in human cells (Abe et al., 2005) but viral DNA is not transcribed because viral
promoters are not recognized by host machinery (Thiem, 1997). Host immune response
also limits virus infection in mammals. In contrast, little is known about immune
response to virus infection in invertebrates.

1.3.1. Invertebrate Innate Immunity to Virus Infection

Due to a lack of a molecularly well-characterized virus pathogen and invertebrate
host model, the pathways leading to induction of invertebrate innate immune response in
response to virus infection have not been clearly elucidated. Recent evidence shows that
Toll pathways may contribute to antiviral response in Drosophila melanogaster. Adult
Drosophila dif mutants lacking a Toll pathway-activated NF-KB transcription factor were

found to have a decreased tolerance to Drosophila X virus (Zambon et al., 2005).

12

Microarray data from the midgut of Aedes aegypti vectoring the Sindbis virus indicate a
role for Toll and c-Jun amino terminal cascades in the infection process (Sanders et al.,
2005). The RNAi pathway in Anopheles gambiae can be silenced with dsRNA of the
ArgonauteZ gene. These Ag02 knock-down mosquitos are also more vulnerable to
infection with O’nyong-nyong virus, an RNA alphavirus, as compared to untreated
animals, which leads to the conclusion that RNAi is an integral antiviral response in A.
gambiae (Keene et al., 2004).

Heliothis virescens larvae are a fully permissive host to AcMNPV while
Helicoverpa zea larvae, a closely-related species, are semi-permissive, showing a 1000-
fold difference in susceptibility (Allen, 1969; Vail, 1978; Vail, 1982). However, larvae of
both species that were orally fed AcMNPV ODV show no difference in their
susceptibility during primary infection of the midgut cells or secondary infection of the
tracheal cells. However, by 48 h.p.i in Helicoverpa zea, hemocytes encapsulate
melanized infection foci in the trachea and clear the virus. Helicoverpa zea hemocytes
are resistant to AcMNPV infection while hemocytes of Heliothis virescens are not.
Encapsulation of infection foci does not occur in AcMNPV-infected Heliothis virescens.
Originally, virus encapsulation and clearing were believed to be an innate immune
response to baculovirus infection in Lepidoptera, however, further study indicates that
swelling and tracheal damage occur in response to AcMNPV infection in Helicoverpa
zea, making melanization and encapsulation a probable response to wounding that
occurs from virus-damaged tissues (Trudeau, Washburn, and Volkman, 2001; Washburn,

1996)

13

1.3.2. Developmental Resistance to Baculovirus

There appears to be some level of developmental resistance to baculovirus
infection. As larvae age they become more resistant to oral-ingested baculovirus
infection. This phenomena appears to be the result of midgut cell sloughing during the
molting process. Larvae in the temporal late stages of instar development, immediately
preceeding or during a molt, are less likely to establish a productive AcMNPV infection
than those at the early instar stage. This is in contrast to H. virescens or T richoplusia
m' larvae that are hemocoel-injected with one plaque forming unit of AcMNPV BV,
which results in 59-89% mortality. It would appear that infected midgut cells are shed
during the molting process before a systemic baculovirus infection can be established,
thus “clearing” the virus from the insect. This decreased susceptibility is mimicked in H.
virescens larvae that are fed on cotton where a plant-encoded foliar peroxidase initiates
midgut sloughing in the insect (Trudeau, Washburn, and Volkman, 2001; Washburn,
Kirkpatrick, and Volkman, 1995; Washburn, 1996).
1.3.3. Species-Mediated Resistance to Baculovirus

Based on the current data, many different mechanisms appear to mediate or
contribute to the species-specific host range of baculoviruses. In cell culture, host range
mechanisms that have been investigated in detail commonly show impaired baculovirus
replication and a block during the late stages of infection (Miller, 1997; Thiem, 1997).
1.3.3.1 Host Cell Factor-1 (HCF-l)

In Sf21 and TN368 cells, both of which are permissive to AcMNPV infection,
transient expression assays were conducted that combined a late viral promoter, a

reporter gene and expression of eighteen AcMNPV LEFs. In Sf21 cells, expression of the

14

eighteen characterized LEFs was sufficient to initiate transcription of the reporter gene.
However, in Tn368 cells transcription from a late and very late viral promoter required
the expression of an additional baculovirus gene, host cell factor-1 (hcf-I) (Lu and
Miller, 1995a), which is not homologous with mammalian hcf-I. Infection with
AcMNPV hcf-I deletion mutants had no effect on virulence, virus DNA replication or
temporal gene expression in SfZl cells. However, in T richoplusia m' 368 (Tn368) ovarian
cells infected with hcf-I null mutants, DNA replication and late gene expression of the
virus did not occur and an arrest of host and viral protein synthesis occurred by 18 h.p.i.
(Lu and Miller, 1996). Infection with hcf-I null mutants in Hi-5 cells, a cell line derived
from Trichoplusia ni eggs, and in T. ni larvae resulted in a reduced virulence of the virus.
A 50-fold decrease in infectivity was observed for the budded form of the mutant virus.
However, there was no reduction in oral infectivity for larvae that were fed polyhedra
lacking the hcf-l gene. Thus, HCF-1 has species and tissue-specific effects (Lu and
Miller, 1996). HCF-1 has been characterized as an early protein that localizes to the
nucleus in infected Tn368 cells and has been shown to interact with itself in
coprecipitation and yeast two-hybrid studies (Hefferon, 2004).
1.3.3.2. p143

Bombyx mori N (BmN) cells are permissive to Bombyx mori Multi Nucleocapsid
Polyhedrovirus (BmNPV) but Tn368, and the Spodoptera frugiperda Sf21 and CLS-79
cell lines are non-permissive to BmNPV infection. Conversely, BmN cells are non-
permissive to AcMNPV infection (Summers, 1978). Co-infection of BmNPV and
AcMNPV yielded a variant that was capable of replicating in all three cell types that were

non-permissive to BmNPV infection (Kondo and Maeda, 1991). Using construction of

15

recombinant BmNPV variants carrying AcMNPV genome fragments, the gene
responsible for the expanded host range was identified as p143-a putative DNA helicase
(Maeda, Kamita, and Kondo, 1993). The region within p143 associated with expanding
the host range of AcMNPV was narrowed'to 3 amino acids that allowed replication of
AcMNPV in B. mori larvae (Croizier et al., 1994). Eventually a single amino acid (AA)
was identified as being sufficient to expand the host range of AcMNPV (Karnita and
Maeda, 1997). At a high multiplicity of infection (MOI), AcMNPV variants carrying this
single AA substitution were capable of infecting Sf9 and BmN cell lines (Karnita and
Maeda, 1996). Since the original host range studies involving p143 were conducted, p143
has been confirmed as an in-vitro DNA helicase (McDougal and Guarino, 2000) with
ATP-binding (McDougal and Guarino, 2001) activity that can be found associated with
DNA in crosslinking studies (Ito et al., 2004). Suggestions as to how a DNA helicase
might alter host range have not been offered. However, it is worth noting that the DNA
polymerase from Lymantria dispar MNPV (LdMNPV) can functionally replace that of
AcMNPV in transient DNA replication assays in Sf21 cells; however, p143 from
LdMNPV can not replace AcMNPV p143 in the same assay (Ahrens and Rohrmann,
1996). The host specificity of the helicase indicates a potential role for its interaction with
host-encoded proteins within the DNA replication complex of the virus.
1.3.3.3. Apoptotic Suppressors and Host Range

There are multiple hypotheses to explain why apoptosis occurs in virus-infected
cells. The predominant hypothesis for invertebrates is that apoptosis results in a
premature cell lysis further preventing virus replication and subsequent infection of

neighboring host cells. Paradoxically, apoptosis may also function as a means for virus

16

dissemination and has thus, co-evolved with permissive virus infections (Friesen and
Miller, 1996). It is important to consider that invertebrates are not equipped with an
adaptive immune system and thus, must rely on innate immune responses to guard
against pathogen infection. While mammals also rely on innate immunity for protection
from pathogens, they are also equipped with immunological memory resulting in
antibody-based recognition of epitopes from invading pathogens.

The apoptotic suppressor, p3 5, is non-essential for AcMNPV replication in Tn368
cells (Clem, Fechheimer, and Miller, 1991; Clem and Miller, 1993; Clem and Miller,
1994). Virus replication occurs in Trichoplusia ni larvae infected with AcMNPV p35
deleted (vAcAp35) virus but there is a reduction in the production of occluded virus, with
less melting at the end of infection as compared to a wildtype infection (Clem and Miller,
1993; Clem and Miller, 1994). Spodoptera fiugiperda 21 (Sf21) cells that are infected
with vAcAp35 show severely impaired virus replication and a non-efficient shut-off of
host protein synthesis (Clem and Miller, 1993; Hershberger, Dickson, and Friesen, 1992).
In Sf21 cells infected with vAcAp35, p35 can be replaced with an inhibitor of apoptosis
(lap) gene from other baculoviruses and wildtype infection is restored (Bimbaum, Clem,
and Miller, 1994). Spodoptera fiugiperda larvae that are infected with vAcAp35 show a
25-fold decrease in rate of infection as compared to wildtype AcMNPV (Clem and
Miller, 1993; Clem and Miller, 1994). Spodoptera Iittoralis-Z (SI-2) (Chejanovsky and
Gershburg, 1995) and Charistoneura fitmrferana-203 (Cf-203) cells (Palli et al., 1996)
infected with AcMNPV undergo apoptosis, resulting in a decrease in budded virus
production and no occluded virus production. Both cell types are permissive to other

baculovirus infections. P35 may be insufficient to block apoptotic response in these cell

17

lines, as transcriptional analysis revealed that p35 expression is delayed in AcMNPV-
infected Cf-203 cells (Palli et al., 1996). Thus, baculovirus apoptotic suppressors are also
species and tissue-specific regulators of virus host range.

1.3.3.4. Host Range Factor-l (Inf-I)

AcMNPV enters but does not productively infect the Lymantria dispar 652Y
(Ld652Y) cell line (McClintock, Dougherty, and Weiner, 1986). Global translation arrest
begins in AcMNPV-infected Ld652Y cells by 8 h.p.i. correlating with the onset of DNA
replication and the late stages of virus infection. All temporal classes of AcMNPV
mRNA are synthesized and transported to the cytoplasm in infected Ld652Y cells and
these mRNAs can be translated in vitro (Guzo et al., 1992). Expression of single gene,
host range factor-1 (hrf-I ), isolated from Lymantria dispar Multinucleocapsid
Nucleopolyhedrovirus (LdMNPV) prevents translation arrest in AcMNPV-infected
Ld652Y cells and enables permissive replication of the virus in cell culture and infected
larvae (Chen et al., 1998; Du and Thiem, 1997b; Thiem et al., 1996). HRF-l also expands
the host range of the nonperrnissive Hyphantria cunea Multinucleocapsid
Nucleopolyhedrovirus (HcMNPV) and the semi-permissive BmNPV and Spodoptera
exigua Multinucleocapsid Nucleopolyhedrovirus (SeMNPV) (Ishikawa et al., 2004).
These data suggest that HRF-l is an essential host range factor for baculovirus infection
of Ld652Y cells. Motif and homology searches provide no indications as to the functional
role of HRF-l. A truncated conceptual protein of 78 amino acids with homology to
HRF-l exists in the genome of Orygia pseudotsugata Multinucleocapsid
Nucleopolyhedrovirus (OpMNPV) but does not complement for LdMNPV HRF-l

function in AcMNPV-infected Ld652Y cells (Ikeda, Reimbold, and Thiem, 2005). HRF-

18

1 contains a domain that is predicted to be highly acidic, mutations in this domain result
in varied changes in HRF-l enabled AcMNPV replication in Ld652Y cells suggesting a
role for the acidic domain in HRF-l function (Ikeda, Reimbold, and Thiem, 2005).

In the absence of p35 and HRF-l , translation arrest and apoptosis are still initiated
during the early stages of infection in AcMNPV-infected Ld652Y cells, apoptosis is
enhanced and global translation arrest occurs at a time that correlates to the onset of DNA
replication and the late stages of infection (Thiem and Chejanovsky, 2004). Blocking
DNA replication and the late phases of virus replication with aphidicolin precludes global
translation arrest in AcMNPV-infected Ld652Y cells, thus associating the arrest of
protein synthesis with the late stages of virus infection (Thiem and Chejanovsky, 2004).
Events correlating to the late stages of infection include the onset of DNA replication of
the virus, the expression of late viral proteins and the Shut-off of host RNA Polymerase
II-mediated transcription (Fig. 4) (Friesen and Miller, 1986; Thiem and Miller, 1989).
Global translation arrest and apoptosis appear to be linked in the response because
infecting Ld652Y cells with an AcMNPV mutant carrying a non-fimctional version of the
apoptotic suppressor p35 does not result in global translation arrest (Thiem and
Chejanovsky, 2004). The global translation arrest infection phenotype is restored when
the same AcMNPV mutant expresses Opiap, Cpiap or p49, apoptotic suppressors isolated
from other baculoviruses that have different modes of action as compared to p35 (Thiem
and Chejanovsky, 2004). Treatment of vAcAp3S-infected Ld652Y cells with peptide
caspase inhibitors does not result in global translation arrest and supports high levels of
budded virus production, suggesting that apoptosis must be initiated for global translation

arrest to occur (Thiem and Chejanovsky, 2004).

19

Earl Events Durin Infection Late Events Durin Infection

 

 

 

Blocked by p35 Blocked by p35
Apoptosis Enhanced Stages
Initiated? Of A tosis
/ \ DNA / 9°”
suffix: Replication
Of the Virus
In Ld652Y _
Cells \ Translation / \ Global Translation
Arrest . Arrest
Initiated Sensor leading to

late stage events:
DNA Recombination?
Host Transcription Shut-off?
Late Gene Expression of Virus?

Blocked by PK2? Blocked by HRF-1

 

 

 

Figure 4. Events that occur during AcMNPV-infection of Ld652Y cells (Image
Presented in Color)
1.3.3.5. Other Baculovirus Proteins Associated with Virus Host Range

The late expression factor, LEF-7, has homology to the herpes-virus UL29 family
of single-stranded binding proteins (Lu and Miller, 1995b). LEF-7 has been identified as
being an enhancer of viral DNA replication in Sf-21 cells (Chen and Thiem, 1997; Lu
and Miller, 1995a) and a contributor to homologous recombination in plasmid transfected
insect cells when in the presence of a hill complement of viral proteins (Crouch and
Passarelli, 2002). Viral replication was reduced to ten percent of wildtype AcMNPV
levels in Sf21 and SElc cells infected with a lef-7 deletion mutant. The same mutation
had no effect on virus replication in Tn368 cells indicating a species-specific role for host

range effects of LEF-7 (Chen and Thiem, 1997).

20

When larvae of Spodoptera frugr'perda and Trichoplusia m' are fed occluded
AcMNPV which lack the immediate early gene, ie2, virus infectivity is reduced 1000 to
100 fold, respectively. Changes in infectivity are attributed to a lack of virions packed in
the ODV of the mutant virus. However, based on this change oral infectivity, IE2 has
been denoted as a possible determinant of virus host range. Larvae infected via
intrahemocoelic injection with the same mutant had infection rates similar to wildtype
AcMNPV infections (Prikhod'ko et al., 1999). Like LEF-7, the immediate early gene, ie2,
of BmNPV and AcMNPV also enhances virus DNA replication of BmNPV in BmN cells
(Gomi et al., 1997) and AcMNPV in Sf21 cells (Lu and Miller, 1995a), respectively. IE2
has been shown to transactivate IE1 in AcMNPV-infected Sf21 cells (Yoo and Guarino,
1994a; Y00 and Guarino, 1994b), to block cell cycle progression in cell lines from
Spodoptera fiugiperda and Trichoplusia ni (Prikhod'ko and Miller, 1998) and IE2 from
BmNPV has E3 ubiquitin ligase activity (Irnai et al., 2003). The diverse firnctional
activity of IE2 in different virus-host relationships make it diffith to assess its possible
role in determining virus host range.

1.4. Translational Modifications in Response to Stress

Global translation is reduced in most, if not all, types of cellular stress (Holcik
and Sonenberg, 2005). Control of translation provides the cell with the ability to
immediately respond to a broad range of stress-induced signals. There is increasing
evidence, based on disproportionate amounts of mRN A as compared to coordinate
protein levels, which indicate an essential role for translational regulation and protein
stability in mediating the cellular response to stress (Gygi et al., 1999; Holcik and

Sonenberg, 2005; Rajasekhar and Holland, 2004; Rajasekhar et al., 2003). Translation

21

can be separated into three distinct phases: initiation, elongation and termination
(Mathews, 2000). The majority of examples that currently exist show translational
control occurring at the initiation stage as a cessation of translation (Holcik and
Sonenberg, 2005).
1.4.1. Translation Initiation

The initiation of translation is a complex process coordinated by numerous
protein subunits that join at the 5’ and 3’ ends of an mRNA transcript. In the cytoplasm,
ternary complexes are formed which consist of a GTP molecule, a methionine initiator
tRN A and the eukaryotic initiation factor 2 complex. The ternary structure joins with the
43S pre-initiation complex which consists of the 40S ribosomal subunit and eIF3, eIF 1,
eIFlA and eIF5. Binding of the pre-initiation complex to the5’ cap structure is believed
to require bridging interactions between eIF 3 and the eIF 4F protein complex which is
bound through eIF4E, the cap binding protein, at the m7GpppN cap structure. Other
subunits within the eIF4F complex include eIF4A, the dead-box RNA helicase, and
eIF4G which acts as a scaffold protein between eIF4E, eIF4A and eIF 3. The subunit
eIF4G also interacts with the poly-A binding protein (PABP) and this interaction between
proteins bound at the 5’ and 3’ end of the mRNA transcript, respectively, results in a
circularization of the template (See Fig. 5). After recruitment to the mRNA, it is believed
that the 43S initiation complex scans the template for an AUG translation initiation codon
(Pestova and Kolupaeva, 2002). The methionine initiator tRNA binds to the AUG start
site and the 43S ribosomal subunit is joined by the 60S ribosomal subunit to form the 808
ribosome. Ribosome formation is followed by the release of initiation factors for cellular

recycling (See Fig. 4) (Gebauer and Hentze, 2004).

22

<ATP
ADP+Pi

ATP

I< ADP+P.

408lnlflatlon Complex Met ‘ fl

elF5B\[%GTP
2xGDP+Pi

80$ Initiation Complex

       
   

   

    

 
  
 

 
    
 

.........

 
  

 

 

Large (60S) Ribosomal Subunit Joining

Figure 5. Schematic diagram of the initiation of protein synthesis in eukaryotes
adapted from Gebauer and Hentze 2004 (Image Presented in Color)

1.4.2. Initiation Stage Translational Modifications in Response to Stress
Translation arrest occurring at the initiation stage generally results from post-
translational modifications of eukaryotic initiation factors (eIFs). Common post-

translational modifications of eLFs include phosphorylation and/or proteolytic cleavage.

23

Modifications lead to blocked interactions with other components of the translation
initiation machinery and result in translation attenuation.
1.4.2.1. Phosphorylation of eIF2a

During translation initiation, as the methionine initiation tRNA joins the AUG
start codon, eIF2-bound-GTP undergoes hydrolysis yielding a GDP molecule. In order to
once again form an active ternary complex, eIF2-bound-GDP must be recharged to GTP
by the nucleotide exchange factor eIF2B. Phosphorylation of the alpha subunit of eIF2 at
serine 51 results in the sequestering of eIFZB which is available in limited quantities (See
Fig. 5 and 6). There are four characterized mammalian kinases of eIF2orse'51: the amino
acid regulated GCN2, the heme-regulated HRI, PERK-an eIF20t kinase that is activated
in response to stress in the endoplasmic reticulum (ER) and the virus-activated, double-
stranded RNA-dependent protein kinase (PKR). Homologues of GCN2 and PERK have
been characterized for invertebrates. Invertebrate PERK is functionally similar to its
mammalian homologue and is activated in response to ER stress but is also
developmentally regulated (Sood et al., 2000). A novel eIF2a kinase, BeK, was
characterized from the Lepidoptera, Bombyx mori. BeK exerts kinase activity in
transformed Drosophila Schneider cells as a result of heat shock and osmotic stress. BeK

is not activated in response to bacterial infection or induced ER or oxidative stress

(Prasad et al., 2003) (For overview see Fig. 6).

24

Baculovirus PK2
(Proposed dominant-negative

Amino Acid inhibitor of elF2a kinases)
Starvation ER Stress
”ypm‘” Masks dsRNA
Heat snook/ (HSV-1. Vaccinia.
® osmotic Stress Reovirus, Influenza)
GCN2
PERK Heme-Regulated
I BeK / Osmotic Shock Binds p KR &
(ELK / HRI ® Heat Shock blocks Its

Azg/ ® / activation
'4 r PKR (Adenovirus,
@ Virus Infection 7 \ HSV-1)

     

. 2 I. Induces PKR Pseudo-
? . substrate
4 degradatIon
(Poliovirus) For PKR
/ \ l (Vaccinia)
Met 2' Recruits a
“2: + Global Translation Selective Translation phosphatase
@é Arrest of (HSV-1)

Stress-Related Genes

 

Figure 6. Schematic diagram of eIF2a phosphorylation and viral strategies for
overcoming kinase-induced translation arrest adapted from Holcik and Sonenberg
2005 (Image Presented in Color)
1.4.2.2. 4E-BP and eIF4E

The cap-binding protein, eIF4E, is shaped like a bowl. Its concave surface directly
interacts with the m7GpppN structure at the 5’ end of the mRNA and its convex surface
interacts with the scaffold protein eIF4G. In proliferating cells, the 4E-binding protein
(4E-BP) is phosphorylated and unable to bind eIF4E. At times of stress and apoptotic
induction, 4E-BP becomes a target for phosphatases or is targeted to the proteasome.
Hypophosphorylated 4E-BP competes for binding of eIF4E with eIF4G and eventually

the cap binding protein is sequestered, attenuating translation. Translation resumes when

25

mTOR—activated kinases phosphorylate 4E-BP, causing the release of eIF4E (Gebauer
and Hentze, 2004; Holcik and Sonenberg, 2005).
1.4.2.3. Proteolytic Cleavage of eIF4GI, eIF4GII and Other Initiation Factors

Caspase-mediated proteolytic cleavage of several eukaryotic initiation factors can
occur in response to the induction of apoptotic programs in a variety of mammalian cells.
Different apoptotic pathways can be initiated as a result of ER stress, DNA damage or
specific ligand-receptor binding. However, all defined apoptotic pathways cascade to the
eventual production of low-level, effector caspases such as caspase 3 or 7. Caspase-3
mediated cleavage has been described for eIF20t, eIF4E, PABP and eIF4GI/eIF4GII
(Holcik and Sonenberg, 2005; Morley, Coldwell, and Clemens, 2005). This apoptotic-
induced cleavage of translation initiation factors generally leads to a global attenuation of
cap-dependent translation.

Caspase-mediated cleavage of eIF2a may be functionally significant as the
truncated form appears to no longer be dependent on GDP exchange by eIFZB (Marissen
et al., 2000). The truncated form of eIF20t, when over-expressed, was able to overcome
translational repression mediated by PKR (Satoh et al., 1999). Cleavage of eukaryotic
initiation factors has been proposed as a means for the cell to overcome translational
repression by favoring internal ribosomal entry site (IRES) or alternate scanning-based
translation of stress responsive genes such as GCN4 in yeast and ATF4 in human cells.
Caspase-driven cleavage of eIF4GI and eIF4GII has been reported for virus and stress-
induced cell lines (Belsham, McInemey, and Ross-Smith, 2000; Clemens, Bushell, and
Morley, 1998; Gradi et al., 1998; Svitkin et al., 1999). In BJAB cells treated with

cycloheximide, eIF4GI is cleaved and is incorporated into a long-term, stable eIF4F

26

complex prior to eventual degradation. This complex may support cap-dependent
translation (Bushell et al., 2000).
1.4.3. Virus Strategies for Overcoming Host-Mediated Translation Arrest

Viruses are dependent on host-encoded translation machinery to synthesize viral
proteins, thus viruses have evolved a number of mechanisms to overcome host-induced
translation arrest. Most characterized viral mechanisms for overcoming translation arrest
at the initiation stage affect PKR-induced eIF2a phosphorylation. In mammalian cells,
PKR exists as a dimer in the cytoplasm. Recognition and subsequent interaction with
dsRNA species induces a conformational change in PKR which results in its
autophosphorylation and activation as an eIF2a kinase (Kaufinan, 2000). Upon infection
and cell entry, dsRNA can result from both the transcription of RNA viruses and the dual
template transcription from DNA viruses (Schneider and Mohr, 2003). Viruses have
developed a number of clever strategies to overcome translational inhibition initiated by
PKR. Adenovirus and Epstein-Barr virus produce the abundant and highly structured
RNA species VARNA, (Schneider, 2000) and EBER RNAs (Clemens, 1993),
respectively that compete with dsRNA for binding of PKR. Herpes Simplex l (HSV-1)-
Usll (Khoo, Perez, and Mohr, 2002; Mulvey et al., 1999), Vaccinia virus—E3L (Beattie et
al., 1995), Reovirus-o3 (Lloyd and Shatkin, 1992) and Influenza-N81 (Salvatore et al.,
2002) are all proteins that bind and mask dsRNA, thus preventing its recognition by PKR.
VARNAl, Usll (Cassady and Gross, 2002; Peters et al., 2002) and the HIV TAR RNA
(Gunnery et al., 1990; Maitra et al., 1994) all directly bind PKR to prevent
autophosphorylation and activation. Vaccinia virus K3L acts as a pseudo-substrate for

PKR (Carroll et al., 1993) and there is some evidence that indicates that poliovirus is

27

capable of directing cellular proteases to degrade PKR (Gale, Tan, and Katze, 2000).
Additionally, the 734.5 protein of HSV-1 recruits a phosphatase to peIFZuS‘Ir51 which
prevents the sequestering of eIF2B allowing for GDP hydrolysis and continued
translation (He, Gross, and Roizrnan, 1997).

A PKR homologue has not been identified in insects. Double-stranded RNA
interference has been shown to be very effective in completely silencing virus and host-
encoded gene expression in invertebrate cell lines (Means, Mum, and Clem, 2003),
without initiating translation arrest, so, a classical PKR protein may not exist in
invertebrates. However, PKR is integral to antiviral response in mammalian cell lines
and RNA interference can be used to silence gene expression in those same cells (Cioca,
Aoki, and Kiyosawa, 2003; Spankuch and Strebhardt, 2005). Both baculovirus infection
and dsRNA used in an attempt to silence hemolin gene expression in Chinese Oak Silk
Moth resulted in an induction of hemolin expression, indicating the existence of a
lepidopteran molecular response pathway to dsRNA (Hirai et al., 2004). It is possible that
other eIF2u kinases, previously characterized for invertebrates, serve as surrogates to
PKR firnction or that another uncharacterized dsRNA response mechanism exists for
Lepidoptera. Interestingly, baculoviruses encode PK2, a protein with kinase subdomain
homology to PKR and all other eIF20t kinases. It is predicted that PK2 acts as a
dominant-negative inhibitor of eIF20t kinases (Li and Miller, 1995), suggesting that
eIF2or phosphorylation may be induced in response to baculovirus infection and that the
virus has evolved a mechanism(s) to overcome its effects. Indeed, eIF2or is protected
from phosphorylation at late times post-infection in SF9 cells infected with AcMNPV

(Dever et al., 1998) (Fig. 6).

28

1.4.4. Preferential Translation of Viral Transcripts

In addition to circumventing the effects of host-induced translation arrest
programs, viruses have developed a number of mechanisms to ensure preferential
translation of viral transcripts over host-encoded mRNA, thus pirating away the host’s
translational apparatus. Picomaviruses, such as poliovirus, encode a 2A protease which
cleaves the N-tenninus of eIF4G, thus abolishing the ability of eIF4G to interact with
PABP (which is also cleaved by 2A) and eIF4E, which eliminates cap—dependent
translation of host RNAS (Joachims, Van Breugel, and Lloyd, 1999; Kerekatte et al.,
1999). Picomaviruses enable translation of virus RNA with internal ribosomal entry sites
(IRES) and bootlegging of virus-modified, host translation factors (Jang et al., 1988;
Pelletier and Sonenberg, 1988). Modification and secondary structure of the 5’ UTR of
many virus transcripts is essential to ensuring preferential translation. IRES have been
identified for all picomaviruses, HIV, HCV and HHV 8 (Hellen and Sarnow, 2001). It is
interesting to note that IRES sites have been identified for stress-responsive mammalian
genes, thus suggesting a mechanism for cell recovery once translation programs have
been attenuated (Hellen and Sarnow, 2001). Adenovirus late expressed genes carry a
tripartite leader sequence in the 5’ UTR that is essential for translation (Schneider, 2000)
and the 5’ UTR of influenza virus attracts a cellular protein, GRSF-l , which stimulates
translation in-vitro (Park, Wilusz, and Katze, 1999). Other novel virus-mediated,
translation initiation factor modifications exist but all result in a shutdown of host

translation (Aragon et al., 2000; Feigenblum and Schneider, 1993; Piron et al., 1999).

29

1.5. Stress Response Pathways and Virus Infection

Hosts have evolved a number of molecular strategies to respond to the threat of
virus infection and other environmental and pathogenic stress inducers. Stress-activated
pathways share many components with characterized innate immunity pathways.
Likewise, viruses have evolved many mechanisms to overcome these strategies and in
some cases, viruses use host-induced defenses to benefit their own replication. Notably,
apoptosis and global translation arrest can occur in response to the UPR via endoplasmic
reticulum stress which can be induced in response to virus infection.

1.5.1. The Unfolded Protein Response (U PR)

Baculovirus host range systems have been characterized in cell culture.
Information gained from cell culture systems is limited because a response initiated in a
single type of cell may differ from other cell types from the same organism. Cells from
certain tissues may accommodate viral entry and in a living insect system, virus may be
cleared or an immune response launched prior to entry of the virus into the cell.
However, in characterized, cell culture-based systems, baculovirus host range appears to
be controlled during the late stages of infection. Viruses enter cells that are non-
permissive to virus replication, early genes are transcribed, DNA replication begins but
neither budded nor occluded virus is produced. This is true not only for AcMNPV in
Ld652Y cells but in other baculovirus host range systems as well (Thiem, 1997). Host
cell transcription is not down-regulated until the late stages of infection. In AcMNPV-
infected Ld652Y cells, global translation arrest and enhanced apoptosis do not occur until
the late stages of infection. Thus, it is logical to presume that an increasing number of

virus-encoded transcripts on the endoplasmic reticulum (ER) and cytosol of the host

30

places a measurable, increased load on the host’s translational machinery and protein
folding apparatus. An abundance of unfolded proteins in the ER can result in ER stress,
leading to the initiation of the unfolded protein response (UPR). The UPR has been
characterized in mammalian cell lines and yeast (Rutkowski and Kaufman, 2004).
Homologous components of the UPR have been identified and characterized for
invertebrates. The UPR response pathway has characterized in C. elegans and is
important in developmental regulation of the nematode (Shen et al., 2001). PERK, the ER
stress activated eIF2or kinase has been identified and characterized in Drosophila
melanogaster but not Lepiodptera (Olsen et al., 1998; Sood et al., 2000). Additionally,
the transcriptional activator ATFC has been identified as being activated in response to
UPR stress in Bombyx mori (Goo et al., 2004). Activation of the UPR results in a
decrease of global translation, an increase in the synthesis of chaperone proteins and
protein degradation coupled to endoplasmic reticulum-associated degradation (ERAD)
(Meusser et al., 2005; Rutkowski and Kaufman, 2004).

The endoplasmic reticulum is the site of synthesis, folding and post-translational
modification of cell-surface and secretory proteins and proteins that comprise the
secretory pathway. Folding of proteins destined for intracellular organelles and the cell
surface occurs in the ER and is accomplished by cellular chaperone proteins. The most
abundant cellular chaperone in the ER is the glucose-regulated, ER chaperone protein
BiP, which is also known as the heat shock 70 protein cognate 3 precursor (HSC70).
Three proximal sensors of accumulated unfolded proteins, PERK, IREl and ATF 6, exist
at the lumen of the ER (Bertolotti et al., 2000; Harding, Zhang, and Ron, 1999; Liu,

Schroder, and Kaufman, 2000). Long-term activation of PERK also results in cell cycle

31

arrest (Brewer and Diehl, 2000; Harding et al., 2000b; Harding et al., 2003; Okada et al.,
2002). Subsequent to PERK activation, ATF4 is preferentially translated despite eIF2u
phosphorylation and serves to activate genes important to stress recovery (Harding et al.,
2003). ATF 6 is cleaved from the ER in response to stress and is transported to the Golgi
body for processing, it is then transported to the nucleus where it acts as a transcriptional
activator for genes which alleviate ER stress, including chaperone proteins and XBPl
(Ye et al., 2000). At a step following PERK and ATF6 induction, the IREI pathway is
initiated resulting in differential splicing of XBPl which serves as a transcriptional
activator for genes associated with protein degradation (For overview see Fig. 7) (Calfon
et al., 2002; Lee et al., 2002; Rutkowski and Kaufinan, 2004; Shen et al., 2001; Yoshida
et al., 2003; Yoshida et al., 2001).

Extended activation of the unfolded protein response results in the induction of
two reversible, interactive, proapoptotic pathways. An intrinsic apoptotic pathway,
similar to that stimulated in response to DNA damage, can be initiated in response to the
UPR, which results in the mitochondrial release of cytochrome c, leading to caspase 9
activation and the eventual activation of effector caspases. The UPR also activates
caspase 9 in Apaf-1'7' cells where caspase 9 cannot be activated with the release of
cytochrome c (Rao et al., 2002). In these cells, UPR-induced apoptosis is initiated via a
disruption of IRE] with TRAF2 at the ER membrane, which results in the release of
caspase 12 by TRAF2. Caspase 12 subsequently activates caspase 9 and the apoptotic
cascade commences (Nakagawa et al., 2000; Yoneda et al., 2001). An extrinsic pathway,
as modeled by the association of cell surface receptors in response to ligation of tumor

necrosis factor, further stimulates a cascade of effector caspases via caspase 8 activation

32

and results in apoptosis. Both intrinsic and extrinsic type pathways seem to be required
for apoptosis to occur in response to the UPR. It is worthy to note that while apoptotic
pathways have been characterized in C. elegans, which has the core components of the
intrinsic pathway, the nematode lacks the homologous components of the extrinsic
pathway. Additionally, the intrinsic pathway in nematodes does not require cytochrome c
for activation of Ced-3 (caspase 9) (Richardson and Kumar, 2002; Zheng et al., 1999).
Damaged proteins that are retained in the ER during the UPR are eventually targeted for
degradation through the endoplasmic reticulum associated degradation (ERAD) pathway.
Misfolded and denatured proteins are translocated from the ER to the cyt0plasm through
special pores in the ER that are linked to ER-bound ubiquitin ligases which, in vitro, act
specifically on denatured proteins. Ubiquitin-conjugated proteins are then targeted to the

proteasome for degradation (For Review see Meusser et al., 2005).

33

Time

 

 

 

 

 

 

 

 

 

 

 

 

UPR Activation
; Translation Attenuation‘
:*
Cleaved a Chaperones 3
ATF6 3
:1.
® \ - Metabolism & 3
\ redox g
9
ii . ;
n A De dati
x391 RNA J are on 3-
x391 splicing 5

 

 

 

Figure 7. The unfolded protein response (UPR) in eukaryotes adapted from
Rutkowski and Kaufman 2004 (Image Presented in Color)
1.5.1.1. The UPR and Virus Infection

Recent research shows that permissive virus replication in human cell lines results
in an accumulation of viral proteins in the ER lumen (Cheng, Feng, and He, 2005; Su,
Liao, and Lin, 2002). The emerging data shows that the UPR is activated in response to
many different types of infections, and that viruses have either evolved to suppress the
pathway or to command components of the UPR to aid in virus replication. Herpes
Simplex Virus (HSV) activates PERK and regulates its dephosphorylation with the viral-

encoded protein 7134-5 (Cheng, Feng, and He, 2005) and PERK contributes to cellular

34

resistance to Vesicular Stomatitis Virus (VSV) as evidenced by enhanced virus
replication in PERK' cell lines (Baltzis et al., 2004). Hepatitis C Virus (HCV) activates
the IREl-XBPl pathway of the UPR which directs both refolding and degradation of
unfolded proteins in the ER lumen. While expression of XBPl is increased in cells
carrying HCV subgenomic replicons, its transactivating activity is blocked, resulting in
no transcriptional induction of the ER degradation-enhancing a-mannosidase-like protein
(EDEM) which is required for the degradation of misfolded proteins (Tardif et al., 2004).
Japanese Encephalitis Virus (JEV) induces the UPR in human fibroblast and neuronal
cells, resulting in eventual apoptotic cell death. Strangely, apoptotic-resistant K562 cells
show no UPR induction following JEV infection, suggesting a regulatory feedback 100p
for the virus-induced UPR (Su, Liao, and Lin, 2002). Perhaps the most intriguing
example of the UPR and virus infection is Human Cytomegalovirus (HCMV) which
selectively induces and suppresses individual mechanisms of the UPR. HCMV induces
the UPR and PERK but phosphorylation of eIF20t is limited and translation does not
attenuate. In contrast, ATF4, which normally lies downstream of PERK-mediated eIF2a
phosphorylation, is transcriptionally activated, allowing for transcriptional activation of
stress recovery genes. In HCMV infected cells, ATF6 activation is suppressed but
synthesis of chaperone proteins is increased. Additionally, splicing of XBP-l occurs,
indicating a prolonged UPR stress response, but transcriptional activation of EDEM is
suppressed (Isler, Skalet, and Alwine, 2005).
1.5.1.1.1. Host-Encoded Molecular Chaperone Requirements for Virus Infection

As previously mentioned, molecular chaperone proteins are transcriptionally up-

regulated as a part of the UPR to relieve the ER stress induced from an abundance of

35

unfolded proteins retained within the ER lumen. Additional translational regulation of
molecular chaperones may also exist. Several groups have documented the importance of
host-encoded chaperone proteins in the virus infection process. Heat shock protein 90
(HSP90) is required to enable the reverse transcriptase activity of Hepatitis B Virus (Hu
and Seeger, 1996). HSP90 also facilitates virus replication of Vaccinia Virus in human
cell lines (Hung, Chung, and Chang, 2002) and Flock House Virus in Drosophila cells
(Karnpmueller and Miller, 2005). HSC70, also known as BiP, is shown to be
transcriptionally activated in AcMNPV-infected SfZl cells at a time when most host-
encoded transcription is being repressed (Nobiron, O'Reilly, and Olszewski, 2003). It
follows that if the UPR stress response is integral to many different types of virus
infection, expression of the ER luminal chaperone, BiP, may also play a role in
baculovirus infection.
1.6. Proteasome Activation and Virus Infection

An increasing number of reports indicate that proteasome-mediated degradation is
a key mechanism utilized by viruses to enable permissive replication cycles. Likewise,
proteasome-mediated degradation is key to orchestrating other cellular functions such as
DNA repair, cell-cycle progression, signal transduction and immune response. In
eukaryotes, multi-subunit proteasomes reside in the nucleus and cytosol. Proteins are
targeted to the proteasome for degradation via ubiquitination by cellular localized E3
ubiqitin ligases. Ubiquitination leads to translocation of the targeted protein to the
proteasome where deubiquitination occurs prior to entry into the 268 active proteasome
(Jentsch and Schlenker, 1995). Proteasome-mediated degradation generates peptide

fragments of 7-8 amino acids in length (Lowe et al., 1995). The COP9 signalosome

36

provides another level of complexity to proteasome-mediated degradation. COP9 is
integral to many signaling pathways including pathogen response (Azevedo et al., 2002;
Liu et al., 2002), in a wide variety of organisms. Recent evidence suggests that the COP9
signalosome may serve as platform for kinases, kinase substrates and the proteasome thus
regulating the degradation of phosphorylated proteins (Harari-Steinberg and Charnovitz,
2004)

Viruses can alter the cell cycle to enable virus replication. The E6 protein of
human papillomavirus (HPV) binds the p53 tumor suppressor protein, targeting it for
proteasome degradation (Stewart et al., 2004) and pp71 of HCMV acts in a similar
fashion on the human retinoblastoma protein (Kalejta, Bechtel, and Shenk, 2003). In both
cases, the result is stimulation of DNA synthesis. Viruses also direct innate immunity
pathways through proteasome activation. HCV and Pararnyxovirus X direct proteasome-
dependent degradation of the IFN-signaling protein, STATl, thus disabling host innate
control of the virus (Lin et al., 2005; Nishio et al., 2005). HCMV directs ubiquitination
and degradation of [RB via the proteasome thus allowing activation of the Nf-icB pathway
and virus replication (Evers, Wang, and Huang, 2004). The RTA immediate-early (IE)
transcription factor acts as a E3 ubiquitin ligase that targets IFN7 for proteasomal
degradation, which further blocks induction of the interferon response in human cells
(Yu, Wang, and Hayward, 2005) . Of other interest is the requirement for proteasome
activation in coordinating Vmw110 modification and the switch from latent to lytic HSV
infection (Everett, Orr, and Preston, 1998). Additionally, proteasome inhibition results in

the accumulation of Murine Coronavirus in the endosome and denser lysosome of DBT

37

cells, indicating a role for the proteasome in mediating viral entry into the cytosol (Yu
and Lai, 2005).
1.6.1. The COP9 Signalosome, Protein Degradation and Kinase Signaling

The COP9 signalosome (CSN) is structurally similar to the regulatory lid of the
26S proteasome and the eukaryotic initiation factor 3 (eIF3) (Kim et al., 2001). The CSN
has been identified and functionally analyzed in fungi (Busch et al., 2003), fission yeast
(Mundt, Liu, and Carr, 2002; Mundt et al., 1999), mammals (Seeger et al., 1998; Wei and
Deng, 1998), plants and Drosophila. This complex has been shown to play a key role in
regulating the development of the latter two organisms (Freilich et al., 1999; Peng,
Serino, and Deng, 2001a; Peng, Serino, and Deng, 2001b). COP9 has also been identified
as playing a role in regulating cellular response to viral and fungal pathogens (Azevedo et
al., 2002; Liu et al., 2002), responding to environmental stress and directing growth
hormone signaling cascades in plants (Schwechheimer et al., 2001). The signalosome
functions through the two distinct but not mutually exclusive pathways of kinase
signaling and ubiquitin-dependent, proteasome-mediated degradation (Harari-Steinberg
and Chamovitz, 2004). The CSN acts as a target for kinases and also coordinates kinase
activity. It also has deneddylation and deubiquitination activities. The complex
coordinates ubiquitin conjugation of key components of regulatory signaling cascades.
CSN has kinase activity against IKB, p105 and c-JUN (Seeger et al., 1998) and the CSN-
associated proteins p53, p27kip and ICSBP are phosphorylated by CSN-associated kinases
(Bech-Otschir, Seeger, and Dubiel, 2002). C-JUN is normally activated in response to
environmental stress, radiation and growth factors resulting in an increase in AP-l

mediated transcription (Seeger et al., 1998). CSN-dependent JNK phosphorylation of c-

38

JUN stabilizes the transcription factor and protects it from proteasome-dependent
degradation. In contrast, the CSN maintains constant, proteasome-mediated degradation
of the p53 tumor suppressor protein, maintaining a low level of the protein until a variety
of cell stress can result in stabilization of p53 and activation of the cell cycle (Sharpless
and DePinho, 2002). Both c-JUN and p53 can be sumolated, which impacts proteasome-
mediated degradation (Schwechheimer and Deng, 2001). One possible model poses that
the CSN serves as a master docking station that coordinates the stabilization or
proteasome-mediated degradation of phosphorylated proteins through their interacting

kinases (Harari-Steinberg and Chamovitz, 2004).

39

Chapter 2
In baculovirus-infected Lepidoptera cells, proteasome
inhibitors reduce virus production and eIF2a
phosphorylation at serine 51

4O

2.1. Introduction

Baculoviruses infect lepidopteran larvae. Permissive baculovirus replication
results in the production of occluded and budded virus. Caterpillars ingest occluded virus
that is embedded in polyhedral occlusion bodies. Occludedviruses are released and infect
midgut cells. Infected midgut cells produce budded virus, which then spreads to other
tissues. Autographa californica Multinucleocapsid Nucleopolyhedrovirus (AcMNPV),
the type virus for the family Baculoviridae (Theilmann et al., 2004), enters but does not
productively infect the Lymantria dispar 652Y (Ld652Y) cell line (McClintock,
Dougherty, and Weiner, 1986). Translation arrests in AcMNPV-infected Ld652Y cells
beginning at approximately eight hours post-infection (h.p.i.) and correlates with the
onset of virus DNA replication (Goodwin, Tompkins, and McCawley, 197 8; Guzo et al.,
1992). Translation is rescued by the expression of a single gene, host range factor-I (hrf-
l) (Du and Thiem, 1997a; Thiem et al., 1996), which was isolated from Lymantria dispar
Multinucleocapsid Nucleopolyhedrovirus (LdMNPV), a baculovirus that productively
infects Ld652Y cells. Homology and motif searches conducted with the hrf-I sequence
do not provide any indications of its function.

Translational arrest is induced in response to a variety of stress signals and is
commonly controlled at initiation. A key mechanism mediating translation arrest is
phosphorylation of eIF2a at serine 51. Phosphorylated eIF20tscrs I binds and sequesters the
guanosine nucleotide exchange factor eIFZB thus preventing the formation of the active
ternary complex required for translation initiation (Hershey and Mathews, 1996). To
counter viral infection, mammalian cells utilize the Double-stranded RNA-dependent

Protein Kinase (PKR) which forms a dimer upon binding double-stranded RNA and is

41

activated by autophosphorylation. Activated PKR then phosphorylates eIF2or at serine 51
(Williams, 1999). Virus infection can also lead to eIF2a phosphorylation mediated by the
endoplasmic reticulum (ER) resident kinase (PERK or PEK) through the unfolded protein
response (UPR). ER stress occurs presumably as the result of large scale host synthesis of
virus proteins (Isler, Skalet, and Alwine, 2005). A PKR homolog has not been identified
in invertebrates. However, other eIF2a kinase homologs have been identified and
characterized from invertebrates including the amino acid regulated GCN2 (Olsen et al.,
1998; Santoyo et al., 1997), BR stress-regulated PERK/PEK (Sood et al., 2000) and
Bombyx mori elF20L kinase (BeK), a kinase specific to lepidopteran species that is
activated in response to heat shock and osmotic stress in transformed Drosophila
Schneider cells (Prasad et al., 2003).

Other host cell mechanisms that regulate protein synthesis in response to virus
infection include stress granule formation, cleavage of mRNAs and tRNAs, and specific
proteolytic cleavage of translation initiation factors (Clemens, 2005; Marissen et al.,
2000; Ohlmann et al., 2002; Satoh et al., 1999; Tee and Proud, 2002). Currently, host cell
mechanism(s) responsible for proteolysis of translation initiation factors are poorly
understood. However, the translation initiation factors eIF2a, 4E-BP, eIFGI and eIFGII
have been shown to be specifically cleaved by different caspases in response to the
induction of apoptosis (Clemens et al., 2000; Marissen et al., 2000; Satoh et al., 1999;
Tee and Proud, 2002). The host translation factor, eIF4Gl, is proteolytically cleaved in a
different pattern in picomavirus-infected cells by viral proteases (Larnphear et al., 1993)

and dissected into multiple fragments in-vitro by an HIV protease (Ohlmann et al., 2002).

42

Targeted ubiquitination leading to proteasome-mediated degradation is essential
to many cellular regulatory processes including DNA repair, cell cycle progression and
immune response (Duan et al., 2004; Greene et al., 2003; Hoege et al., 2002; Sarmento et
al., 2005). It has also been shown that proteasome-mediated degradation of target
proteins may be critical to successful replication of some viruses in mammalian systems.
For example, RTA, the immediate-early transcription factor of Kaposi’s Sarcoma-
Associated Herpesvirus (KSHV or HHV 8), has E3 ubiquitin ligase activity that targets
proteasome-mediated degradation of IRF-7, thus blocking the induction of a host innate
immune response (Yu, Wang, and Hayward, 2005). Proteasome activation is also
believed to be required for the transition of HSV-1 from a latent to a lytic infection
through Vmwl 10 modification (Everett, Orr, and Preston, 1998). Additionally, the
proteasome inhibitor, MG132, has been shown to block replication of human
cytomegalovirus (HCMV) at the immediate-early stage of infection (Prosch et al., 2003).
In this study we identify a virus-induced, proteasome-dependent, phosphorylation and
cleavage of eIFZot that accompanies permissive AcMNPV replication in two invertebrate
cell lines. Phosphorylation and cleavage occur during the late stages of the infection
cycle. We further show that proteasome inhibition with epoxomicin or MG132 blocked
vAchrf-l replication in Ld652Y cells. Epoxomicin was effective at blocking vAchrf-l
and AcMNPV replication in Sf21 cells but M6132 only reduced budded virus titers in

the same productively-infected Sf21 cells.

43

2.2. Materials and Methods
2.2.1. Viruses and Cell Lines.

Sf-21 cells (Vaughn et al., 1977) and Ld652Y cells (Goodwin, Tompkins, and
McCawley, 1978) were maintained in TClOO medium (JRH BioScience, Lenexa, KS)
supplemented with 10% fetal bovine serum and 0.26% tryptose broth. AcMNPV variant
L1 (Lee and Miller, 1978), vAcAPK2 (Li and Miller, 1995) and vAchrf-l (previously
designated vAchPD, Du and Thiem, 1997) were propagated in Sf21 cells with titers
being determined by plaque assay.

2.2.2. Cytoplasmic fraction extraction and polysome profiling.

Polysome profiles were prepared using a protocol adapted from Brecht and
Parsons (Brecht and Parsons, 1998). Five 100mm tissue culture plates were seeded with
5 x 106 Ld652Y cells per plate and infected with either AcMNPV L1 or vAchrf-l at an
MOI of 10 PFU per cell. After 1 h incubation, the inoculurn was removed and complete
medium was added to cells. The cells were then incubated at 27°C until the indicated
time. At 9 h.p.i., AcMNPV and vAcHrf-l infected Ld652Y cells were treated with 100
pg/ml cycloheximide for 5 min and the cells were harvested. The harvested cells were
washed with 10 ml of ice-cold phosphate buffered saline (PBS), then suspended in 750 pl
of ice-cold polysome buffer A (20mM Tris HCl [pH 7.4], 10 mM NaCl, 3 mM MgC12,
100 pg/ml cycloheximide, 1 pM DTT and 15 pg RNasin) and equilibrated on ice for 3
min. Cells were lysed by the addition of 250 pl of lysis buffer (polysome buffer A
containing 0.2% NP40 and 0.2M sucrose) followed by incubation on ice for 5 min with
occasional mixing. The lysates were centrifuged at 1000 x g at 4°C for 10 min to remove

nuclei and the supematants were transferred to a new tube containing 40 pl 4M NaCl,

44

 

Cl

 

an

 

100 p1 heparin (10 mg/ml) and 20 pg RNasin. 400 pl of the cytoplasmic extract was then
layered on a linear 15-50% sucrose gradient and centrifuged at 230,000 x g at 4°C for 1.5
h in a SW40 rotor. Following centrifugation, the gradients were harvested from the top
using an ISCO density gradient fractionator while continuously measuring the optical
density at 254nm.

2.2.3. Cell Stress Induction Assays.

Sf21 cells were seeded in 35 mm cell culture plates at a density of 1 x 106 or
Ld652Y cells at a density of 5 x 105 and each plate was overlaid with 3 ml of TC100
medium. Stress inducing compounds were added directly to cell culture media to reach
the following final concentrations: 2 pM epoxomicin (Calbiochem #324800), 50 pM
hydrogen peroxide, 90 pM methyl methanesulfonate (MMS) (Sigma #M 4016), 1 pg/ml
rapamycin (Sigma #R 0395), 1 pM thapsigargin (Sigma #T 9033) and 12 pM
tunicarnycin (Sigma #T 7765). After treatment, cultures were returned to 27°C
incubation. Cells receiving heat shock were cultured at 39°C. Media and cells were
collected at 6 and 24 hours post-treatment, centrifuged at 2,600 x g for 8 minutes, media
was removed and cells were lysed in 100 pl of NP40 buffer containing a protease and
phosphatase inhibitor cocktail of lmM benzamidine, 10 pg/ml aprotonin, 1 mM PMSF, 5
pg/ml leupeptin and 100 pM okadaic acid. Cells were lysed on ice for 30 minutes then
debris was collected via refrigerated centrifirgation at 17,900 x g for 8 minutes.
Supernatant was collected and stored at -80°C for future quantification and western blot

analysis.

45

2.2.4. Protease and Proteasome Inhibitor Assays.

Cell culture plates (60 mm) were seeded with either Sf21 cells at a density of 2 x
106 or Ld652Y cells at a density of 1 x 106. For aphidicolin treatment, cells are pre-
conditioned for one hour prior to infection with aphidicolin added directly to the cell
culture media to a final concentration of Spg/ml. After cell attachment, media was
removed and cells were infected at an MOI of 10 with AcMNPV or vAchrf-l
respectively. After a one hour infection, virus was removed and cells were refed with 4
ml of TC 1 00 media per plate. Inhibitors were added directly to cell culture media to reach
the following final concentrations: 1 pM aprotonin (Calbiochem #616370), 5 pg per ml
of aphidicolin (Sigma #A 0781), 1 pM E64d/EST (Calbiochem #330005), 10 mM

EDTA-Naz (Gibco, #15575-038), 10 pM pepstatin A (Calbiochem #516481) and cells for

the proteasome inhibitor assay were treated with 2 pM epoxomicin or 10 pM MG132.
Infected cell cultures that were assayed at 48 hours post-infection were re-treated with
fresh inhibitors at 24 hours post-infection to counteract possible inhibitor degradation.
Media and cells were collected at 24 and 48 hours post-infection, centrifuged at 2,800 x g
for 8 minutes, media was removed and cells were lysed as described above.
2.2.5. Western Blot Analysis. I

Protein samples were quantified using the DC Reagent System (Bio-Rad #500-
0116), a Lowry-based detergent compatible assay. Protein levels were measured in 96
well ELISA plates at 750 nm using a pQuant microplate reader (Witech AG,
Switzerland) and BioTek-KCJM,or Analysis software package (Fisher Scientific #BT-
5270501). Each sample containing 20 pg of total protein and 10 pl 2X SDS sample

buffer was boiled at 98°C for 10 nrinutes, briefly centrifuged to collect sample and then

46

 

 

loaded into a 15% his-glycine polyacrylamide gel. Samples were separated for 2 hours at
110 volts using SDS-PAGE (Laemmli, 1970) and then transferred to Immobilon—P,
PVDF membrane (Millipore Corp., Bedford, MA) overnight at 4°C in Towbin Buffer (25
mM Tris, 192 mM glycine and 20% methanol) at 40 volts. Membranes were blocked for
5 hours in 5% lowfat milk in 1X TBS-Tween (0.2 M NaCl, 66 mM Tris, 0.1% Tween).
Irnmunodetection was performed by incubating blots overnight with 2% lowfat milk in
1X TBS-Tween and one of the following primary antibodies: anti-eIF2or phosphor-SerSl
serum (Biosource, #44-728Z- lot 0103) diluted to 1:3000. The blot was then washed with
2% milk in 1X TBS-Tween and incubated with horseradish peroxidase conjugated anti-
rabbit antibody (Pierce, #31460) diluted to 1:20,000 in 2% lowfat milk in 1X TBS-Tween
for 2 hours. Bands were visualized using an ECL detection kit (Amersham-Pharrnacia
Biotech, #RPN 2209) and exposure on Classic Blue Sensitive autoradiography film
(Midwest Scientific). Following detection for peIF2a’"51, blots for proteasome inhibitor
assays were stripped by agitation at 37°C in Restore Western Blot Stripping Buffer
(Pierce, #21059), re-blocked for 3 hours and then incubated overnight in anti-polyhedra
serum (M. Ikeda) at a dilution of 1:20,000. Blots were sequentially probed with
secondary antibodies and the interaction was detected as described above. Blots for initial
eIF2a phosphorylation studies were probed with peIF2a3°ISI antibodies (Biosource, #44-
728Z- lot 0103), then stripped and reprobed with polyclonal antibodies to eIF2a (Santa
Cruz Biotechnology, #FL-315) at a dilution of 1:1000 and monoclonal or-tubulin
antibodies (Santa Cruz Biotechnology, #TU-02) diluted to 1:1000. Blots probed with

antibodies to or-tubulin were probed with horseradish peroxidase-linked anti-mouse

47

secondary antibodies (Pierce, 31430) at 1:20000. Incubation, stripping and ECL detection
were conducted as described above.
2.2.6. Plaque Assays.

Cell culture plates (60 mm) were seeded with either Sf21 cells at a density of 2 x
106 or Ld652Y cells at a density of 1 x 106. After cell attachment, media was removed
and cells were mock-infected or infected at an MOI of 10 with AcMNPV or vAchrf-l
respectively. After one hour, virus was removed and cells were washed twice with
TC100. Cell cultures were refed with 4 ml of TC100 media per plate, epoxomicin was
added directly to the media for indicated samples as previously described. Cells and
media were collected at indicated times and a standard plaque assay was used to
determine virus titers (O'Reilly, Miller, and Luckow, 1994). Plates were scored for 0ch
plaques at 4 and 6 days post-infection.

2.3. Results
2.3.1. Translation arrest in AcMNPV-infected Ld652Y cells occurs at the initiation
stage.

In order to determine the stage at which translation arrests in AcMNPV-infected
Ld652Y cells, polysome profiles of wild-type AcMNPV-infected cells were compared to
those of vAchrf-l-infected cells. vAchrf-l is a recombinant AcMNPV that expresses the
LdMNPV hrf-I gene (Du and Thiem, 1997b). At six hours post-infection polysome
profiles of AcMNPV and vAchrf-l-infected cells are identical (Fig. 8A). The six hour
time point was selected because translation arrest is not observed in AcMNPV-infected
Ld652Y cells at this time (Guzo et al., 1992; McClintock, Dougherty, and Weiner, 1986).

Figure 8A shows polysome profiles in infected cells prior to translation arrest. Figure

48

8B shows polysome profiles from AcMNPV and vAchrf-l-infected Ld652Y cells at 9
hours post-infection, during translation arrest. In these profiles a large increase in the size
of the monosome peak (arrow Fig. 8B) is evident in AcMNPV-infected cells as compared
to the same peak from the vAchrf-l-infected cells (Fig. 8B). The increase in the
monosome peak in AcMNPV-infected Ld652Y cells (Fig. 8B) is indicative of an arrest in
translation initiation. However, polysomes are still evident in AcMNPV-infected cells
(bracket, Fig. 8B). The most likely explanation for this observation is that translation
does not arrest unifonnly in the population of cells sampled. It is also possible that
polysomes persist as a result of proteins that are translated despite translation arrest such
as those synthesized during cap-independent translation (Pelletier and Sonenberg, 1988;
Pestova et al., 2001). (Polysome profiling experiments were completed by Wade A.

Williams, M.S.U. Dept. of Microbiology and Molecular Genetics.)

49

 

 

 

 

 

 

 

 

5 3I Wt AcMNPV E 1‘ Wt AcMNPV

.3. : Infected ff I. Infected
4 N ‘-

8 1 “mm 81M
. i

g vAchrf-1 g vAch rf-1

s; Infected § Infected

N o

8 0
Top _ Bottom Top . Bottom

A. 6 h.p.I. B. 9 h.p.I.

Figure 8. Translation arrest occurs at translation initiation in AcMNPV-infected
Ld652Y cells. Polysome profiles prepared from AcMNPV and vAcHrf-l-infected (Fig. 8
caption, cont.) Ld652Y cells at 6 h.p.i. (A) and 9h.p.i. (B) post-infection. Arrow in panel
B indicates a monosome peak in AcMNPV-infected cells at 9 h.p.i., the bracket indicates
polysomes. Infected cells were lysed and nuclei removed. Cytoplasmic components were
separated on 15-50% sucrose gradients and analyzed with an ICSO density gradient
fractionator. (Polysome profiling experiments were completed by Wade A. Williams,
M.S.U. Dept. of Microbiology and Molecular Genetics.)

2.3.2. Translation arrest in AcMNPV-infected Ld652Y cells temporally correlates to

phosphorylation of eIF2a at serine 51

Translation arrest is induced in response to various stress signals and is commonly
controlled at the initiation stage (Schneider and Mohr, 2003). Two well characterized

mechanisms are dephosphorylation of eIF4E binding proteins in response to nutritional
stress (Attardo, Hansen, and Raikhel, 2005; Attardo et al., 2003; Clemens, 2005; Hansen

et al., 2004; Miron et al., 2001) and phosphorylation of eIF20t at serine 51 in response to

50

amino acid depletion, heme-depletion, endoplasmic reticulum (ER) stress, or virus
infection (Chen, 2000; Chen and London, 1995; Harding et al., 2000a; Kaufman, 2000;
Mathews, 2000; Williams, 1999). Phosphorylation at serine 51 causes eIF20t to bind to
and competitively inhibit the nucleotide exchange factor eIF 2B, resulting in translation
arrest. Because it is the predominant mechanism mediating translation arrest at initiation,
we analyzed the phosphorylation state of eIF20t in mock and virus-infected cells using
western blots. For these experiments we infected cells with AcMNPV or with one of two
modified AcMNPV, vAchrf-l and vAcApk2. vAcAka is an AcMNPV deletion mutant
which lacks pk2, a gene encoding a truncated eIF20t kinase homolog (Li and Miller,
1995). PK2 functions as a dominant-negative inhibitor of the eIF20t kinases, GCN2 and
PKR, and presumably other members of this kinase family (Dever, 1998). Western blots
were generated using whole cell lysates and probed with polyclonal antibodies raised
against human eIF20t or against an eIF2a derived peptide phosphorylated at serine 51
that specifically recognizes phosphorylated eIFZot (peIF20I5ch 1) (Grundmann, Mosch, and
Braus, 2001; Kimball, 1999; Kumar et al., 2001; Li and Koromilas, 2001; Pataer et al.,
2002; Patel et al., 2000; Soboloff and Berger, 2002). These antibodies were expected to
recognize the L. dispar proteins, as the alpha subunit of eIF20t is highly conserved. In
BLAST comparisons between full length human (Genbank accession no. AAP36281)
and lepidopteran, S. fiugiperda (Genbank accession no. AA015491) eIF20t, proteins
there are 69% identical and 79% positive residues, with 100% identity in the region
between residues 35-64, surrounding the phosphorylation site at serine 51. In our
experiments, these antibodies recognized proteins of the expected size with no cross-

reactivity. At 6 h.p.i., we observe reduced levels of peIF2as‘I’5I in Ld652Y cells infected

51

with AcMNPV, vAchrf-l or vAcApk2 as compared to mock-infected cells (Fig. 9, lanes

sets 1

2, 3 and 4 as compared to lane 1) which suggests that peIF20t is dephosphorylated

during the early stages of baulovirus infecton. However, an increase in eIF20LS°I5I
phosphorylation is observed in AcMNPV and vAcAka-infected cells relative to mock-
infected cells at 12 hours post-infection (Fig. 9, compare lanes 6 and 8 with lane 5). The

similar phosphorylation levels of eIF2otsc'SI in AcMNPV and vAcApk2-infected cells

indicate that PK2 does not inhibit eIF2a phosphorylation in AcMNPV infected Ld652Y

sets 1

cells at 12 h.p.i. (Fig. 9, lanes 6 and 8). However, levels of peIF20I are apparently
reduced in Achrf-l-infected Ld652Y cells at 12 post-infection (Fig. 9, lane 7). At 24
h.p.i., phosphorylation levels of eIF2or in AcMNPV-infected Ld652Y cells are similar to

mock infected cells (Fig. 9, lanes 6 and 10). This reduction in the eIF2a phosphorylation
state in AcMNPV-infected Ld652Y cells at 24 h.p.i. may be due to an overall decrease in

eIF2a levels resulting from translation arrest.

sets I

The antibodies against peIF20I recognized a second band of ~26 kDa only in
vAchrf-l-infected Ld652Y cells (Fig. 9, lane 7 and 11). The ~26 kDa band was not
observed at 6 h.p.i. (Fig. 9, lane 3). The ~26 kDa band apparently represents a

“'5 I. When gels for western blots were run for shorter

proteolytic fragment of peIF20t
times and probed for eIF2a, a band of ~11 kDa was detected (Fig. 10, lane 3). The sum
of the ~26 kDa band detected on blots probed with antibodies to peIFZOLS‘Ir51 (Fig. 9, lanes
7 and 11, Fig. 10, lane 6) plus the 11 kDa band detected with antibodies to eIF2a total
~37 kDa, the band recognized by both antibodies, which is the predicted molecular mass
for S. fi'ugiperda eIF2a. These data support the idea that peIF20ts°ISI is proteolytically

cleaved in Ld652Y cells infected with vAchrf-l. In vAchrf-l-infected Ld652Y cells,

52

phosphorylation of eIF2a may serve as a signal for cleavage of the protein. Since eIF201
is phosphorylated but not cleaved in AcMNPV-infected Ld652Y cells, it appears that

cleavage is directly or indirectly enabled by HRF-l.

6hpi 12hpi 24hpi

E E. g E E E E s ‘94
...... --— ~ F“? firs-"ST eIFZor-P

,_,.;..-’;,‘:'f" '.;i ...-... ...“...

mm a Finn-1 ot-tubulin
10 11M" 12

 

Figure 9. Phosphorylation of full length, eukaryotic initiation factor 211 (eIF2a) at
serine 51 is reduced in vAchrf-l-infected Ld652Y cells as compared to AcMNPV-
infected, temporally correlating to the onset of translation arrest in AcMNPV-
infected Ld652Y cells. (Fig. 9 caption, cont.) Levels of total eIF2a were assayed by
using antibodies that specifically recognize the phosphorylated and total endogenous
forms of the protein. Total cell lysates were prepared from Ld652Y cells that were mock-
infected or infected with AcMNPV, vAchrf-l or vAcApk2. Infected cells were harvested
at 6, 12 h and 24 h post-infection. Proteins from whole cell lysates were separated on
15% SDS-PAGE gels and analyzed with immunoblotting. The same blot for each time
point was stripped and reprobed sequentially with antibodies to phosphorylated
eIF2aS°'5I, eIF 201, and or-tubulin. Samples from the 6 h.p.i. timepoint were collected from
a separate infection timecourse than the 12 and 24 h.p.i. samples.

53

Ld652Y Ld652Y Sf21

AB: eIFZa AB: pelF2aser51 AB: pelF2aser51

% i': E 1': g r:

2 '5 2 5 5

- o < - o <‘r’ — ‘3 °

5 < > 2 < > 2 < E
.- —- ...—.---.. -___~37kDa
- --~26kDa
"" ~11kDa

Figure 10. Phosphorylated eIF211’c'I5l is proteolytically cleaved in cell lines that are
productively infected with AcMNPV or vAchrf-l. Proteolytic cleavage of eIF2u was
assayed at 24 h.p.i. in mock, AcMNPV and vAchrf-l-infected Ld652Y and Sf21 cells
with antibodies that specifically recognize the phosphorylated or total endogenous forms
of the protein. Proteins from whole cell lysates were separated on 15% SDS-PAGE gels
and analyzed with immunoblotting. The same blot for Ld652Y cells was probed with
antibodies to peIF2oIschI (AB: peIFZawSI) (lanes 4-6) then stripped and reprobed with
antibodies to eIF2a (AB: eIF20r) (lanes 1-3). Cell lines and antibodies used are shown
above the panels and the molecular sizes of immune reactive bands are indicated to the
right of the panels.

In AcMNPV-infected Ld652Y cells, incubation of cell lysates with m7GTP-bound
sepharose followed by western blot analysis showed that the cap-binding ability of eIF4E

was not impaired (Fig. 26, Supplementary Data). In addition, western blots generated

54

with antibodies raised against Drosophila melanogaster 4E-BP (kindly provided by
Mathieu Miron) did not show an increase in 4E-BP protein levels in AcMNPV-infected
Ld652Y cells (Fig. 27, Supplementary Data). The results suggest that modification or
sequestering of eIF4E is not the primary mechanism leading to global translation arrest in
AcMNPV-infected Ld652Y cells.

To show that eIF2or phosphorylation was the mechanism leading to global
translation arrest in AcMNPV-infected Ld652Y cells, experiments were conducted to
silence endogenous eIF201 expression in invertebrate cell lines using RNAi technology.
Silenced Ld652Y cells were infected with a recombinant AcMNPV that constitutively
expressed the Spodopterafiugiperda eIF2a gene with an alanine substitution at serine 51
(vAcHAeIFZaaIISI). Isotopic labeling of protein synthesis in Ld652Y cells silenced for
eIF201 expression and infected with the vAcHAeIF 201aIaSI recombinant were inconclusive
(data not shown).

2.3.3. Proteolytic cleavage of peIFZamSI occurs in AcMNPV and vAchrf-l-infected

8121 cells.

serSl

Cleavage of peIF20I in vAchrf-l-infected cells suggested a possible

mechanism for HRF -1 to overcome translation arrest in AcMNPV-infected Ld652Y cells.

serS 1

One hypothesis was that cleavage of peIF201 could prevent its binding of the

guanosine nucleotide exchange factor eIFZB thus allowing protein synthesis to continue.

To determine whether cleavage of peIF20tser51

was mediated by HRF-l we compared
peIF2as°III cleavage in cell lysates of vAchrf-l- and AcMNPV-infected Sf21 cells, using

western blots probed with antibodies to peIF201selrs I. In contrast to Ld652Y cells, Sf21

55

“'5 I occurred in both

cells are permissive for AcMNPV replication. Cleavage of peIF2a
AcMNPV and vAchrf-l-infected Sf21 cells by 24 h.p.i. (Fig. 10, lanes 8 and 9) and not in
mock-infected cells (Fig. 10, lane 7) suggesting that cleavage is not directly-mediated by

sets 1

HRF-l. In the cell lines studied, cleavage of peIF20t occurs only in those that are

“'5‘ may be directly or

productively-infected with baculovirus. Cleavage of pelF2a
indirectly mediated by HRF-l in Ld652Y cells as a host protein may be functionally
homologous to HRF -1 during AcMNPV infection in Sf21 cells.

2.3.4. PK2 is expressed in AcMNPV-infected Ld652Y cells.

PK2 was expected to inhibit eIF2a kinases, yet similar levels of phosphorylation
were observed in cells infected with AcMNPV and vAcApk2 (Fig. 9, lanes 6 and 10 as
compared to lanes 8 and 12). To determine if PK2 was expressed in AcMNPV-infected
Ld652Y cells, we performed western blot analysis using antibodies to PK2 (R. Clem
from L.K. Miller collection). Although PK2 levels decreased over time, it was still
present in Ld652Y cells infected with AcMNPV and Achrf-l at 6, 12 and 24 hours post-
infection (Fig. 11, lanes 3, 4, 7, 8, 11 and 12). Thus, it is likely that concentrations of PK2
are too low to block eIF2u kinase activity at later time points. Alternatively, it is possible
that the eIFZot kinase activated by AcMNPV infection in Ld652Y cells is not inhibited by
PK2. There is a non-specific band of ~31 kD that is evident in all samples at 6 h.p.i. (Fig.

11, lanes 1, 2, 3 and 4) and has previously been observed with this antibody (Li and

Miller 1995).

56

       

ohm lznm 24hm
EEE EEE SEE
gaésgsés‘éEE-s
...E__§ < i E31: g 6-55.59?
“I: rang.-. ’I-ir-r' 344369”
123456789101112

Figure 11. PK2 is present at late time points post-infection in Ld652Y cells infected
with AcMNPV and Achrf-l. Infected cells were harvested at 6, 12 and 24 hours post-
infection, subjected to SDS-PAGE on 15% polyacrylamide gels and immunoblotted with
antibodies to PK2.

2.3.5. Cleavage of peIFZaI'I"51 in 8121 and Ld652Y cell lines is not induced by stress-

inducing agents.

To test if peIF201scr51 cleavage resulted from a general stress response, we treated

ser5 1

Sf21 and Ld652Y cells with stress-inducing agents and assayed for peIF20t cleavage
by western blot analysis. To screen for the effects of genotoxic stress, uninfected cells
were treated with methyl methanesulfonate. Nutritional stress was induced with
rapamycin, oxidative stress with hydrogen peroxide, proteasome inhibition with
epoxomicin and heat shock by incubation at 39° C. ER—stress and the unfolded protein
response (UPR) were induced using thapsigargin and tunicamycin. Assays were

“'5 I was not

conducted at 6 and 24 hours post-treatment (h.p.t.). Cleavage of peIF2a
observed in either Ld652Y or Sf21 cells, in response to any of these treatments (Fig. 12).

The reduction in eIFZot phosphorylation, in both Ld652Y and Sf21 cells treated with

57

hydrogen peroxide at 24 h.p.t. (Fig.12A, lane 8 and 12B, lane 8), may be the result of cell

damage initiated by reactive oxygen species.

A. Ld652Y Cells

Mock Epox. HS H202 MMS Rap. Thap. Tun. vAchrf-1

252:2:E=E=EEE3E33
omomomo§o§io§o§o§§ I
W~W~peIFZG

-

1234567891011121314151617

4'8"} .

B. Sf21 Cells

Mock Epox. HS H202 MMS Rap. Thap. Tun. AcMNPV

age§s=ssess=EcE£=
V V V
0N3N¢ONONON©§¢D§IO§§

-
‘I234567891011121314151617

Figure 12. Proteolytic cleavage of peIFZa’”51 does not occur in 8121 or Ld652Y cells
that are treated with stress-inducing agents. Ld652Y and Sf21 cells were treated with
stress inducing agents as indicated above each panel. Cells were harvested at 6 and 24
hours post-treatment. Proteins from whole cell lysates were fractionated on 15% SDSSl-
SCI

PAGE gels and analyzed on western blots probed with antibodies against peIF20t .
Cell lines and treatments are shown above the panels.

58

2.3.6. Cleavage of peIFZa’M’l is blocked by aphidicolin but not by protease
inhibitors.

Late events in the baculovirus replication cycle enhance apoptosis in AcMNPV-
infected Sf21 cells in the absence of the virally encoded apoptotic suppressor, p35
(LaCount and Friesen, 1997), and stimulate translation arrest in AcMNPV-infected
Ld652Y cells (Thiem and Chejanovsky, 2004). To test if peIF20Ise'5l cleavage correlated
with the onset of late viral gene expression, we treated vAchrf-l-infected Ld652Y cells

serS 1

with the DNA polymerase inhibitor aphidicolin and scored for peIF20t cleavage at 24

h.p.i. on western blots. Cleavage was not observed and eIF20t phosphorylation was
reduced in aphidicolin-treated, vAchrf-l-infected Ld652Y cells and AcMNPV-infected
Sf21 cells (Fig. 13, lanes 1 and 6) indicating that phosphorylation and cleavage of

peIF201I’c'5 I required a late event in the AcMNPV replication cycle.

59

 

Ld652Y/vAchrf-1 Sf21/AcMNPV

 

n

C
=5 5:5 ,5;
.38 938 E
Esngm23n<m
£55 Oneal-o.
0.0. 000.0. Dd)
<<UJUJO-<<u.iuio.

M ~37 kDa

.....- “- ~26 kDa
1 2 3 4 5 6 7 8 9 10

Figure 13. Phosphorylation and proteolytic cleavage of peIFZaWI’l is inhibited in

Lepidoptera cells that are productively-infected with baculovirus and treated with

aphidicolin. Proteolytic cleavage of peIF2am5' is not blocked by protease inhibitors.

(Figure 13 caption cont.) Ld652Y or Sf21 cells were mock-infected or infected with
either vAchrf-l or AcMNPV respectively. Cells were harvested at 24 h.p.i.. Proteins from
whole cell lysates were fractionated on 15% SDS-PAGE gels and analyzed on western
blots probed with antibodies against peIF2a’°'5I. Cell lines and treatments are shown
above the panels and the molecular sizes of immune reactive bands are indicated to the
right of the panels.

To identify a candidate protease responsible for cleavage of peIF201scr51 in

AcMNPV- and Achrf-l-infected Sf21 and vAchrf-l-infected Ld652Y cells, we attempted
to block cleavage with membrane-permeable protease inhibitors. Inhibitors were added
directly to the cell culture media. We used aprotonin to block serine protease activity,
E64d (EST) to inhibit cysteine proteases, (ethylenedinitrilo) tetraacetic acid (EDTA) for
metalloproteases and pepstatin to inhibit aspartic proteases. Inhibitor concentrations were
based on recommendations from previous studies (Buttle et al., 1992; Gray and Tsai,

1994; Janas, Marks, and LaRusso, 1994; Tamai et al., 1987; Thiem and Chejanovsky,

60

2004). We note, however, the pepstatin derivative we used has not been analyzed for
membrane permeability. None of these inhibitors prevented cleavage of peIF2ase'5I (Fig.
13, lanes 2-5 and 7-10). However, phosphorylation levels of full-length peIF201$ch I
appear to be reduced in vAchrf-l-infected Ld652Y cells that were also treated with a
protease inhibitor (Fig. 13, lanes 2-5). This reduction in peIF201schI levels is most
evident in vAchrf-l-infected Ld652Y cells that were treated with EDTA (Fig. 13, lane 4).
Due to the broad range effects that EDTA imposes on cellular processes, it is difficult to
determine if this reduction in peIF201I’"r51 levels is significant in EDTA-treated Ld652Y
cells during vAchrf-l infection. However, there is still a light ~26 kD band present in
EDTA-treated Ld652Y cells infected with vAchrf-l indicating that proteolytic cleavage

of peIF201s¢r51 still occurs and is not inhibited by EDTA.

We also tested possible roles for caspases and cathepsin caspase in peIF201"cr51

cleavage. Cell permeable, caspase and cathepsin inhibitors were added to the media of
AcMNPV-infected Sf21 cells or vAchrf-l-infected Ld652Y cells, as previously described
(Thiem and Chejanovsky, 2004). In addition, because the AcMNPV apoptotic suppressor
p35 may not be expressed at sufficient levels to completely block apoptosis (P. Friesen,
personal communication) we examined Sf21 cells infected with a recombinant AcMNPV
with enhanced expression of p35 (kindly provided by P. F riesen). Cells were harvested at
24 h.p.i. and lysates analyzed by western blot. Inhibiting either caspase or cathepsin

sets 1

activity did not prevent peIF20I cleavage (data not shown).

61

2.3.7. Proteasome inhibitors reduce phosphorylation of eIF2a at serine 51 and virus

production in lepidopteran cells productively-infected with baculovirus.

er5 1

Protease inhibition did not block cleavage of peIF20ts in invertebrate cell lines

productively-infected with baculovirus, so, we examined the proteasome as a possible

sets 1

mediator of proteolytic activity against peIF201 . The role of proteasome activity and

its relation to eIF201 phosphorylation and cleavage were investigated using the
proteasome-specific inhibitors epoxomicin and MG132. Epoxomicin blocks trypsin-like
(T-L) and chymotrypsin-like (CT-L) activities of the proteasome without inhibiting other
proteases. Additionally, epoxomicin blocks the peptidylglutamyl peptide hydrolyzing
(PGPH) caspase-like activity of the proteasome. Epoxomicin covalently binds the LMP-
7, MECLl and Z catalytic subunits of the proteasome and that direct interaction is a
probable mechanism for its inhibitor activity (Meng et al., 1999; Sin et al., 1999) .
MG132 is a peptide aldehyde and a less potent inhibitor of the proteasome. MG132
reduces the degradation of ubiquitin-conjugated proteins by the 26S complex without
inhibiting the isopeptidase or ATPase activities of the proteasome (Steinhilb, Turner, and
Gaut, 2001). Inhibitors were added directly to infected cell cultures and their effects on
eIF2a phosphorylation and cleavage assayed at 24 and 48 h.p.i. as previously described.
At 24 and 48 h.p.i., it appears that levels of full-length peIF20tS°I5I are reduced in both
Sf21 and Ld652Y cells that are infected with baculovirus and treated with a proteasome
inhibitor (Fig. 14A, lanes 2, 3, 8, 9, 11, 12, 17 and 18; 14B, lanes 2, 3, 8, 9, 11, 12, 17
and 18) as compared to mock-infected samples that are treated with a proteasome
inhibitor at the corresponding time points (Fig. 14A, lanes 5, 6, 14 and 15; 14B, lanes 5,

6, 14 and 15). Cleavage of peIFZOII‘I'51 was not observed in vAchrf-infected Ld652Y cells

62

or AcMNPV and vAchrf-l-infected Sf21 cells that were treated with epoxomycin (Fig.
14A, lanes 8 and 17; 14B, lanes 2, 8, 11 and 17). There is a faint band of ~30kDa
appearing in infected cells treated with proteasome inhibitors (Fig. 14A, lanes 6, 14, 15

serSl via an

and 17). This faint band may reflect proteolytic degradation of peIF20t
initiated proteolytic pathway that is not proteasome dependent. In virus-infected Ld652Y
and Sf21 cells, MG132 appears to reduce eIF201 phosphorylation levels (Fig. 14A, lanes
9 and 18; 14B, lanes 3, 9, 12 and 18) but cleavage of peIF201schI is prevented only in
vAchrf-l-infected Ld652Y cells (Fig. 14A, lanes 9 and 18). M6132 treatment did not
prevent peIF20II’I'51 cleavage in vAchrf-l and AcMNPV-infected Sf21 cells (Fig. 14B,

lanes 3, 9, 12 and 18).

63

 

A. Ld652Y Cells

 

.ZAJLILL. m
AcMNPV Mock VAChl‘f-I AcMNPV Mock VAChl’f-I
I- O) I— n .— ‘— ‘- s- ‘—
z m 2 Z UJ 2 z m §’ '2 “81’ E” 5 1.1% g I7: 1181' g
- g. « - “-—- c—I- .- 4 -’-.--' “r" ' ‘ pelF2aser51
- I D T

123 4 56 7 89101112131415161718

 

B. Sf21 Cells
24 |'_'I.Q,i. ML.
AcMNPV Mock vAchff-‘l AcMNPV Mock vAchrf-1
'- v- 1- v- ‘-
zmssmssossésséssés
...—...- . — - ...-H *" -- 'I-D --- .b-—- o—\ pelFZaser51
.- ..— - fl‘ - - g
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 14. Proteasome inhibitors, MG132 and epoxomicin, inhibit phosphorylation
and proteolytic cleavage of peIFZasersl in vAchrf-l-infected Ld652Y cells. Ld652Y or
Sf21 cells were treated with 2 pM epoxomicin or 10 pM MG132 from 1 hour preceding
infection to time of collection. Cells were mock-infected or infected with either
AcMNPV or vAchrf-l and harvested at 24 h.p.i. and 48 h.p.i. Proteins from whole cell
lysates were fractionated on 15% SDS-PAGE gels and analyzed on western blots probed
with antibodies against peIFZas‘IISI. Cell lines and treatments are shown above the

panels.

Because polyhedra were not observed in epoxomycin-treated cells, we examined
whether polyhedrin protein was synthesized. Immunoblots shown in Figure 14 were
stripped and reprobed with antibodies to the baculovirus polyhedrin protein. Polyhedra or
occlusion bodies are produced in the very late stages of permissive baculovirus infection

from the polyhedrin protein. Polyhedrin protein was not observed in either epoxomicin or

64

 

12“.. .

MG132-treated vAchrf-l-infected Ld652Y cells at 24 and 48 h.p.i. (Fig. 15A, lanes 8, 9,
17 and 18). Epoxomicin blocked production of the polyhedrin protein in AcMNPV and
vAchrf-l-infected Sf21 cells (Fig. 15B, lanes 2, 8, 11, and 17) but MG132 did not (Fig.
15B, lanes 3, 9, 12 and 18). Polyhedrin degradation products are evident on western blots

in non-treated, productively—infected Ld652Y and Sf21 cells by 48 h.p.i.

 

 

Ld652Y Cells
15.11.13.— 43.11.91...
AcMNPV Mock vAchrI-I AcMNPV Mock vAchrf-I
"I a "I I3 * 9" x' ”I x' 94 x' 91
Elise-Es SEE-51E: ESE?
" -. Whedfin

12 3 4 5 6 7 8 9101112131415161718

Sf21 Cells
m1. mm.

AcMNPV Mock vAchrf-I AcMNPV Mock vAchrf-I
.— '-
zmsréssaé

E...
- ' -*°' 1- - - ’- polyhedrin
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 15. The very late baculovirus occlusion body protein, polyhedrin, is not
produced in permissively-infected cells that are treated with epoxomicin. Ld652Y or
Sf21 cells were pre-treated with 2 pM epoxomicin or 10 pM MG132 from 1 hour
preceding infection to time of collection. Cells were mock-infected or infected with either
AcMNPV or vAchrf-l and harvested at 24 h.p.i. and 48 h.p.i.. Proteins from whole cell
lysates were fractionated on 15% SDS-PAGE gels and analyzed on western blots probed
with antibodies against peIF20ts‘I'5', stripped and sequentially probed with polyclonal
antibodies against polyhedrin. Cell lines and treatments are shown above the panels and
immune reactive bands are indicated to the right of the panels.

65

To determine if proteasome inhibition blocked virus replication, budded virus
(BV) production in the presence or absence of epoxomicin or MG132 was measured over
time by plaque assay. Experiments for all samples were plated in duplicate, titers noted in
Figure 16 and 17 represent the average titer for both samples. Epoxomicin treatment
eliminated or reduced BV production in vAchrf-l-infected Ld652Y cells (Fig. 16) and in
Sf21 cells infected with either vAchrf-l or AcMNPV (Fig. 17). Treatment of vAchrf-l-
infected Ld652Y cells with MG132, resulted in a block of budded virus production at 24
h.p.i. (Fig. 16). AcMNPV and vAchrf-l-infected SF21 cells treated with MG132 showed
reduced level of budded virus production of at least one order of magnitude as compared
to cells that were not treated with MG132 (Fig. 17). The different response observed in
8121 cells treated with epoxomicin as compared to those treated with MG132 may be a
direct measure of the potency of the inhibitor or may specifically relate to the mechanism
of the inhibitor. Epoxomicin blocks proteasome activity by directly binding catalytic
subunits of the proteasome while MG132 only reduces the degradation of ubiquitin-
conjugated proteins, so if MG132 treatment results in a partial block to virus production,
it would suggest that complete degradation of one or more proteins is essential to
establishing a productive baculovirus infection. Both proteasome inhibitors, epoxomicin
and MG132, are effective in blocking the production of virus progeny in vAchrf-l-
infected Ld652Y cells. However, there may be a small increase in budded virus
production in vAchrf-l-infected Ld652Y cells treated with MG132 by 48 h.p.i. The data
suggests a potential difference in proteasome response in permissively-infected Ld652Y

cells as compared to Sf21 (Fig. 16 as compared to Fig. 17). These experiments suggest

66

that the proteasome plays a role in promoting a productive baculovirus infection in cell

culture.

 

El vAchrf-1

Virus titers in Ld652Y cells

flvAchrf-1 wI Epox.

EEI vAchrf-1 wI MG132

IAcMNPV

E AcMNPV wI Epox.

pfulml

IAcMNPV wl
MG132

 

 

 

 

 

Figure 16. Proteasome inhibitors prevent budded virus production in vAchrf-l-
infected Ld652Y cells. vAchrf-l titers are reduced within at least one order of magnitude
in cells treated with either epoxomicin or MG132. Ld652Y cells were pre-treated with 2
pM epoxomicin or 10 pM MG132 from 1 hour preceding infection to time of collection.
Cells were mock-infected or infected with either AcMNPV or vAchrf-l and virus was
harvested at 24 h.p.i. and 48 h.p.i. Virus was titered on Sf21 cells using a standard
plaque assay (O'Reilly, Miller, and Luckow, 1994). Plates were scored for och plaques
at 4 and 6 days post-infection.

67

 

D vAchrf-1

Virus titers in Sf-21 cells

E vAchrf-1 wI Epox.

  

 

 

 

 

 

1-°°E+°3 - [BvAchrf-1wIMG132
100E+07 . .. ..
Te .3 ' - . ._ IAcllllNPV
E r ’9' a they,” . I ' . aAcMNPVwIEpox.
‘ 1ooe+os "JET II III I
..3 - , T - IAcMNPV wI
MG132
1 ooa+os -
100E+04 '1

Figure 17. Epoxomicin prevents AcMNPV or vAchrf—l budded virus production in
5121 cells, MG132 reduces budded virus production in those same infected cells.
AcMNPV and vAchrf-l titers are reduced within at least one order of magnitude in
infected Sf21 cells treated with epoxomicin. Sf21 cells were pre-treated with 2 pM
epoxomicin or 10 pM MGl32 from 1 hour preceding infection to time of collection.
Cells were mock-infected or infected with either AcMNPV or vAchrf-l and virus was
harvested at 24 h.p.i. and 48 h.p.i.. Virus was titered on Sf21 cells using a standard
plaque assay (O'Reilly, Miller, and Luckow, 1994). Plates were scored for occ+ plaques
at 4 and 6 days post-infection.

2.4. Discussion

Global translation arrest occurs in Ld652Y cells infected with the baculovirus
AcMNPV in a non-productive infection. The addition of a single gene, hrf-I, isolated
from LdMNPV, rescues translation arrest and promotes a productive virus infection. In
this study, while searching for mechanisms that lead to translation arrest in AcMNPV-
infected Ld652Y cells, we discovered that the eukaryotic initiation factor 201 is

phosphorylated at serine 51 (pe1F2a5°'5‘) in AcMNPV-infected Ld652Y cells (Fig. 9) but

68

 

that peIF2as°IS I is proteolytically cleaved during the late stages of infection in vAchrf-l-
infected Ld652Y cells (Fig. 9 and 10) . In this study, we investigated a possible role for

ser5 1

HRF-l in directing cleavage of peIF201 and sought to identify the responsible

protease. Subjecting cells to various stresses did not result in cleavage of peIF2015'"51 (Fig.
12). We found that cleavage occurred at the late stages of infection in productively-
infected cells, regardless of the presence of HRF-l (Fig. 10 and 13). In vAchrf-l-infected
Ld652Y cells, phosphorylation of eIFZot at serine 51 and cleavage of peIF201scr51 was
prevented by the proteasome inhibitors epoxomicin or M6132 (Fig. 14). Production of
virus progeny appears to be prevented in vAchrf-l-infected Ld652Y cells treated with
epoxomicin or M6132 and in AcMNPV and vAchrf-l-infected Sf21 cells treated with
epoxomicin (Fig. 16 and 17).

sets 1

Cleavage of peIF20I suggested a potential mechanism for HRF -1 to overcome

translation arrest in Ld652Y cells. One hypothesis was that cleaved peIF2as°I5I was
conformationally-altered and no longer able to bind and sequester the nucleotide

exchange factor eIF2B, thus preventing translation arrest. The fact that cleavage of

peIF201schI occurs in Sf21 cells infected with AcMNPV as well as in those infected with
vAchrf-l suggested that HRF-l is not directly responsible for inducing pelF2am5'
cleavage (Fig. 10). However, this may not be true if HRF-l is functionally replacing a
host-encoded protein available in Sf21 cells that does not exist in Ld652Y cells. This
question could be addressed by expressing a truncated eIF201 gene from a recombinant
AcMNPV virus, then infecting Ld652Y cells to determine if budded virus production was

enabled by the recombinant. Similar experiments could be conducted by transfecting

Ld652Y cells with an expression plasmid encoding a truncated form of eIF201. Earlier

69

reports which examined the phosphorylation state of eIF 201 in response to productive
AcMNPV infection in Sf9 cells, did not identify cleavage of the initiation factor (Dever
et al., 1998). However, in those studies, isoelectric focusing gels were used to determine
the phosphorylation state of eIF20t which would not have easily identified a cleavage
product.

Treatment with a range of chemical protease inhibitors had no effect on the
cleavage of peIF201sets I in $121 or Ld652Y cells permissively-infected with AcMNPV or
vAchrf-l, respectively (Fig. 13). However, the proteasome-specific inhibitor,
epoxomicin, prevented phosphorylation of eIF2or and cleavage of peIF2orserSI at 24 and
48 hours post-infection in vAchrf-l and AcMNPV-infected Sf21 cells and vAchrf-l-
infected Ld652Y cells (Fig. 14A, lanes 8 and 17; 14B, lanes 2, 8, 11 and 17). In contrast
to M6132-treated Sf21 cells, vAchrf-l-infected, M6132-treated, Ld652Y cells show
little to no increase in budded virus titers at 48 h.p.i. over initial inputs (Fig. 10) which
may indicate a role for HRF-l in mediating the proteasome response in Ld652Y cells.
The differential effect of M6132 on productive virus infection in Ld652Y cells as
compared to Sf21 cells may be significant. M6132 reduces the degradation of ubiquitin-
tagged proteins and M6132 prevents vAchrf-l replication in Ld652Y cells, suggesting
that degradation of a ubiquitinated protein, possibly elF2a, is essential to promoting a
productive infection in Ld652Y cells. A reduction in ubiquitin mediated degradation may
not be sufficient to block AcMNPV replication in Sf21 cells as AcMNPV may have a
mechanism to ensure degradation of certain proteins in that cell type. Epoxomicin blocks
proteasomal activity through an irreversible, direct interaction with proteasomal subunits,

ser5 1

our data shows that epoxomicin blocks all cleavage of peIF201 and virus replication in

70

vAchrf-l-infected Ld652Y cells and AcMNPV or vAchrf-l-infected Sf21 cells. In this
study, the impairment to budded virus production in productively-infected cells treated
with epoxomicin suggests that the virus has evolved to use the proteasome as a part of its
replication cycle (Fig. 16 and Fig. 17). To our knowledge, this is the first account of a
proteasome—dependent cleavage of a eukaryotic translation initiation factor. Virus-
mediated proteasome utilization is not without precedent. Human cytomegalovirus
mediates proteasomal destruction of hypophosphorylated host retinoblastoma proteins in
a ubiquitin-independent manner (Kalejta and Shenk, 2003) which results in stimulation of
cell cycle progression thus allowing virus replication (Kalejta, Bechtel, and Shenk, 2003).
Herpes simplex virus I is unable to transition from a latent to lytic state in the presence of
proteosome inhibitors and Hepatitis C virus evades the host antiviral interferon response
by directing proteasomal degradation of phosphorylated STAT] (Lin et al., 2005).

It is possible that activation of the proteasome is part of an orchestrated host
response to the late stages of virus infection. Proteasome enabled cleavage of pelF201’"5I
in Ld652Y and Sf21 cells infected with vAchrf-l and AcMNPV, respectively, is blocked
in the presence of aphidicolin (Fig. 13, lanes 1 and 6). In those same productively-
infected cells that are treated with aphidicolin or proteasome inhibitors, eIF201 is not
phosphorylated (Fig. 13, lanes 1 and 6, Fig. 14A, lanes 8, 9, 17 and 18; 14B, lanes 2, 3, 8,
9, 11, 12, 17 and 18 ). This indicates that eIF201 phosphorylation is the result of an event
or events that occur during the late stages of infection and may be a signal for the
initiation of cleavage. Other studies have shown late stages in the baculovirus infection
cycle, including viral DNA replication or late viral gene expression (LaCount and

Friesen, 1997), to promote apoptosis and translation arrest (Thiem and Chejanovsky,

7l

2004). It is interesting to note the phosphorylation state of eIF20t in productively-infected
cells treated with proteasome inhibitors compared to those same treated cells that are not
infected. Reduced eIF201 phosphorylation levels occur only in baculovirus infected cells
at 6 h.p.i. (Fig. 9, lanes 2, 3 and 4) and in those cells that are baculovirus-infected and
treated with proteasome inhibitors (Fig. 14A, lanes 8, 9, 17 and 18 as compared to lanes
5, 6,14 and 15 and 14B, lanes 2, 3, 8, 9,11,12,17 and 18 as compared to lanes 5, 6,14
and 15). The data may suggest that baculoviruses orchestrate a response between the
proteasome, an eIFZa kinase and its substrate. Such a mechanism has been modeled for
the COP9 signalosome (CSN). Models predict that the CSN may serve as a scaffold for
ubiquitylation and subsequent degradation of specific phosphorylated substrates (Harari-
Steinberg and Chamovitz, 2004). Potentially, the COP9 signalasome could play a
regulatory role in responding to baculovirus infection by providing selective degradation
of phosphorylated proteins including peIF 2015"”. It may be possible to determine if the
CSN has an effect on baculovirus infection by using CSN-specific inhibitors in cell
culture. The CSN is essential for development in Drosophila melanogaster (Freilich et
al., 1999) and appears to direct cell differentiation by orchestrating regulatory cascades.
CSN is structurally similar to the 198 regulatory lid of the proteasome and to eukaryotic
initiation factor 3 (eIF3) (Kim et al., 2001).

Targeting of proteins to the proteasome begins with substrate recognition by an
E3 ubiquitin ligase followed by ubiquitination and delivery to the proteasome. E3 ligases
provide the specificity for targeting of individual proteins to the proteasome. E3 targeting
can occur as a result of endoplasmic reticulum-associated degradation (ERAD),

oxidative stress or the unfolded protein response (UPR) (Crews, 2003). Interestingly, all

72

presently characterized baculoviruses encode a ubiquitin-like protein and several
baculovirus proteins including BmMNPV IAP2, IE2 and PE38 have E3 ubiquitin ligase
activity (Imai et al., 2003). BMNPV IE2, PE38 and the AcMNPV homolog for IAP2 are
all transcribed at early and late stages of infection and are thus, not probable candidates
for mediating late infection cleavage of peIF20ts°'51. However, recent microarray data
indicates that these genes are up-regulated during the late stages of infection (Yarnagishi
et al., 2003) and thus, may differentially ubiquitinate substrates resulting in a possible
change in proteasome activity during this stage of the infection cycle. Evolutionary
conservation of proteins involved with proteasome targeting and activation suggests that
modulation of the proteasome by baculoviruses is important to the replication of the
virus. For example, the ubiquitin ligase activity of IAP3 from OpMNPV is capable of
ubiquitinating pro-apoptotic proteins from Drosophila (Green, Monser, and Clem, 2004).
While ubiquitin has been shown to be a structural part of budded AcMNPV virions
(Guarino, Smith, and Dong, 1995), virus-encoded ubiquitin does not appear to be
required for production of budded virus (Reilly and Guarino, 1996). The Spodoptera
fiugiperda eIF2or sequence (van Oers et al., 2003) has several lysine residues
Surrounding one of the two possible cleavage sites of peIF2a’"5I (GenBank Accession
#AF447395) which are candidates for ubiquitylation. Interestingly, one of these is a
SUMO-like sequence at amino acid K211 (LKAG) which is preceded by a hydrophobic
residue (ABGENT-SUMOpIotTM Prediction). SUMO is a small ubiquitin-related modifier
and shares 18% similarity with ubiquitin, it has been proposed to mediate protein-to-
protein interactions by differential ubiquitination of protein substrates. In yeast,

proliferating cell nuclear antigen (PCNA) is alternatively modified by SUMO and

73

ubiquitin in response to DNA damaging agents (Hoege et al., 2002). Sumolation of
SfeIF2a could be evaluated using immunoprecipitation with eIF201 antibodies followed
by western blots with anti-SUMO antibodies.

Currently, we have no indication as to the number or types of proteins, virus or
host-encoded, that are targeted for proteasome-mediated degradation during permissive

ser5 1

baculovirus infection. Cleavage of peIF2or may not be a specific event but a by-

product of a generalized host response in which many proteins are degraded. However,

“'5 I rather than extensive cleavage into many

we observe a single cleavage of peIF201
short fragments that result from characterized proteasomal degradation (J entsch and
Schlenker, 1995). Moreover, the proteasome may not directly degrade peIFZuS"r51 but
may indirectly mediate cleavage of peIF20ts°Is I through a proteolytic cascade. What
remains to be determined is how a generalized or targeted activation of the proteasome
aids in the baculovirus replication process. Defining the role that the proteasome and
eIF201 phosphorylation plays during baculovirus infection, including its possible
connections to HRF -1, will provide unique insights into the field of virus host range in
invertebrates.
Acknowledgements

The author would like to thank Dr. Paul Friesen for providing vAcIEp35, Dr.
Mathieu Miron for providing antibodies to Dm4E-BP and Dr. Motoko Ikeda for
providing antibodies to polyhedrin. This work was supported by funding from the United

States Department of Education, Graduate Assistance in Areas of National Need

(GAANN) Program.

74

Chapter 3
HSP90 Expression is Enhanced in vAchrf-l-infected
Ld652Y Cells

75

3.1. Introduction
Baculoviruses are large, double-stranded DNA (dsDNA) viruses that primarily
infect lepidopteran species. Baculovirus progeny are produced in a budded or occluded
virus form. The occluded form of the virus is environmentally-stable and consists of
enveloped virions surrounded by a polyhedrin matrix that is encapsulated in an electron-
dense envelope of carbohydrate and protein (Theilmann et al., 2004). The type virus for
Baculoviridae is Autographa caliform'ca Multinucleocapsid nucleopolyhedrovirus
(AcMNPV) which permissively infects Spodoptera fiugiperda 21 (Sf21) cell lines but
not Lymantria dispar 652Y cells (McClintock, Dougherty, and Weiner, 1986). Ld652Y
cells infected with AcMNPV initiate global translation arrest by 9-12 hours post-infection
(h.p.i.) resulting in a non-productive infection (Guzo etal., 1992). In the absence of a full
length, baculovirus apoptotic suppressor, apoptotic programs are also induced in
AcMNPV-infected Ld652Y cells (Du and Thiem, 1997a). Infection with AcMNPV
variants that encode a single additional gene, host range factor-1 (hrf-I), isolated from
Lymantria dispar Multinucleocapsid nucleopolyhedrovirus (LdMNPV), results in a
productive infection in Ld652Y cells (Du and Thiem, 1997b). Phosphorylation of eIFZa
at serine 51 (pelFZaserSI) is the probable mechanism of translation arrest in AcMNPV-
infected Ld652Y cells as eIFZam“ phosphorylation occurs in AcWPV-infected
Ld652Y cells at a time that correlates with the onset of translation arrest (Mecey, William

and Thiem-manuscript in progress). Additionally, translation continues at 48 and 72 h.p.i.

in Ld652Y cells infected with AcMNPV variants that encode a lepidopteran gene for
eIFZa with an alanine substitution at serine 51 under regulation of an immediate-early

and late baculovirus promoter (Mecey-unpublished results). The lepidopteran eIF20t

76

kinase, BeK (Prasad et al., 2003), expression levels appear to be activated at 12 h.p.i. in
AcMNPV-infected Ld652Y cells temporally correlating to phosphorylation of eIFZa and
translation arrest (Mecey-unpublished results) (Fig. 28, Supplementary Data). Proteolytic

ser5 l

cleavage of peIFZoc occurs in Ld652Y and Sf21 cells that are permissively-infected

with baculovirus but not in Ld652Y cells infected with AcMNPV where virus replication

does not occur. Phosphorylation of eIF20t or cleavage of peIF20tschI

appears to be
proteasome dependent as proteasome inhibition with epoxomicin results in a block of
eIF2a phosphorylation, cleavage and permissive virus replication in AcMNPV and
vAchrf-l-infected Sf21 cells and vAchrf-l-infected Ld652Y cells.

The unfolded protein response (UPR) is ancient, with components that are
evolutionarily-conserved from yeast to mammals (Rutkowski and Kaufman, 2004). The
UPR is activated in response to a variety of cellular stresses which result in an
accumulation of unfolded proteins in the endoplasmic reticulum. Hallmarks of the UPR
include, but are not limited to: phosphorylation of eIFZa via the PKR-like endoplasmic
reticulum (ER) resident protein kinase (PERK), an increased synthesis of chaperone
proteins, transcriptional activation of stress-related genes including the GRP78 ER
resident chaperone-BiP, and activation of endoplasmic reticulum associated degradation
(ERAD) via the proteasome (Rutkowski and Kaufman, 2004). In mammalian cells, BiP
binds and regulates the degradation of ERAD substrates (Chillaron and Haas, 2000;
Skowronek, Hendershot, and Haas, 1998). Conversely, deletion of protein components in
the ERAD pathway can result in UPR activation (N g, Spear, and Walter, 2000).

Proteasome inhibition can also result in eIF2a phosphorylation and translation arrest by

the kinase GCN2, an eIFZa kinase that is activated in response to amino acid starvation

77

(Jiang and Wek, 2005). During the UPR, PERK activation can initiate cell cycle arrest
(Brewer and Diehl, 2000). Cis-acting unfolded protein response elements (UPRE) have
been identified in the promoters of ER stress-activated genes as being sufficient for gene
modulation in response to an induced UPR (Mori et al., 1998; Patil, Li, and Walter, 2004;
Yoshida et al., 1998).

In mammalian systems, eIF2a phosphorylation occurring in response to virus
infection is generally mediated by the double-stranded RNA (dsRNA)-activated protein
kinase, PKR. Viruses have evolved numerous mechanisms to overcome recognition by
and the effects of PKR (Gale, Tan, and Katze, 2000; Schneider and Mohr, 2003). Gene
silencing with dsRNA is effective when targeted at a variety of genes in a number of
insect models, including Lepidoptera (Means, Muro, and Clem, 2003) and a PKR
homolog has not been identified in sequenced invertebrate genomes. However, in
shrimp, innate antiviral immunity and a antiviral RNAi interference (RNAi)-like
mechanism are both induced by dsRNA. Double-stranded RNA and baculovirus infection
have been shown to result in activation of the lepidopteran, pathogenesis-related protein,
hemolin (Hirai et al., 2004), indicating that a dsRNA response pathway exists for
lepidoptera. Due to the abundance of viral proteins produced during a permissive virus
infection, the UPR can be initiated during virus infection. The UPR has been shown to be
activated by Japanese Encephalitis Virus (J EV) in fibroblast BHK-12 and neuronal cell
lines resulting in eventual apoptosis but in apoptosis-resistant K562 cells, infection with
JEV does not result in UPR activation (Su, Liao, and Lin, 2002). Human
Cytomegalovirus (HCMV) activates the UPR and then modulates its components in

infected fibroblast cells (Isler, Skalet, and Alwine, 2005). Herpes Simplex Virus 1

78

activates PERK in a mechanism that correlates with increased viral protein synthesis
(Cheng, Peng, and He, 2005).

Heat shock protein 90 (HSP90) acts in a multi-chaperone complex and is known
to interact with proteins involved in signal transduction, transcriptional and cell cycle
regulation (Beere, 2001; Pratt and Toft, 2003; Young, Barral, and Ulrich Hart], 2003).
Inhibition of HSP90 with geldanamycin delays replication of Vaccinia Virus in RK13
cells lines and while HSP90 expression is not enhanced in RK13 cells infected with
Vaccinia virus, its cellular distribution is altered (Hung, Chung, and Chang, 2002).
HSP90 interacts with a number of viral proteins including the reverse transcriptase of
Hepatitis B Virus and Simian Virus 40 T antigen (Hu and Seeger, 1996; Miyata and
Yahara, 2000). Additionally, HSP90 and HSP70 are components of a receptor complex
for dengue virus entry in human cell lines (Reyes-Del Valle et al., 2005). The Drosophila
melanogaster protein, Dpit47, has been identified as an HSP90 co-chaperone that binds
DNA polymerase or, inhibiting its activity (Crevel et al., 2001). HSP90 has over 100
identified client proteins including three eIF2a kinases: GCN2, PKR and the heme-
regulated protein kinase (HRI), to which it binds in concert with HSP70 to prevent
autophosphorylation (Pratt and Tofi, 2003). In response to proteasome inhibition, protein
levels of HSP90 and HSP70 have been shown to increase, while mRNA levels of HSP90
remained unchanged, suggesting possible'translational regulation of the protein (Awasthi
and Wagner, 2005). Heat shock cognate protein 70 (HSC70) is one of few host genes that
is transcriptionally activated in response to baculovirus infection, indicating a role for
chaperone proteins during permissive infection. Preferential synthesis of viral proteins

appears to be regulated at the transcriptional level as an overall decrease of host gene

79

transcription occurs in response to AcMNPV infection in Sf21 cells (Nobiron, O'Reilly,
and Olszewski, 2003). This decreased synthesis of host-encoded mRNA may lead to a
quelled requirement for chaperone proteins and a bypass of a cellular initiated UPR stress
response. Alternatively, chaperone proteins during productive baculovirus infection,
other than HSC70, may be regulated at a translational level thus overcoming the effects
of inhibited host transcription.

Here we investigate regulation of the chaperone proteins HSP90 and HSC7O
during AcMNPV and vAchrf-l infection of Ld652Y cells. We show an increase in levels
of a protein that interacts and immunoprecipitates with antibodies raised against human
PKR that mass spectrometry identifies as HSP90. Inhibition of HSP90 with
geldanamycin results in a block of occluded virus production at 48 h.p.i. in productively-

infected cells and a significant decrease in polyhedrin protein levels.

80

3.2. Materials and Methods
3.2.1. Viruses and cell lines

Sf—21 cells (Vaughn et al., 1977) and Ld652Y cells (Goodwin, Tompkins, and
McCawley, 1978) were maintained in TC100 medium (JRH BioScience, Lenexa, KS)
supplemented with 10% fetal bovine serum and 0.26% tryptose broth. AcMNPV variant
L1 (Lee and Miller, 1978) and vAchrf-l (previously designated vAchPD, Du and
Thiem, 1997) were propagated in St21 cells with titers being determined by plaque assay.
3.2.2. Immunoprecipitation

Cell culture plates (100 mm) were seeded with Ld652Y cells at 2 x 106, cells were
allowed to adhere to the plate surface for at least one hour. Ld652Y cells were infected
with vAchrf-l at an MOI of 10, after a one hour infection, virus was removed and cells
were refed with TC-lOO. At 12 h.p.i., cells were collected, media was removed and
infected cells were lysed in 900 ml of RIPA buffer containing a protease and phosphatase
inhibitor cocktail. Lysates were passed through a 21 gauge needle to shear DNA and
were incubated on ice for 60 minutes. Soluble cellular proteins were then separated from
debris by micrcentrifuging for 10 minutes at 10,000xg in 4°C. The supernatant was
transferred to a sterile eppendorf tube, 25 pg of normal rabbit IgG and 20111 of suspended
(25% v/v) Protein-A-Agarose conjugate (Santa Cruz Biotechnology-#sc-2001) were
added to the supernatant. Whole cell lysate was pre-cleared by rocking at 4°C for 30
minutes then beads were pelleted at 1,450 x g for 30 seconds. The supernatant was
transferred to a sterile eppendorf tube and combined with 10 ug of anti-PKR agarose
conjugate (Santa Cruz Biotechnology-3c #17 02AC) which was then rocked and incubated

overnight at 4°C. The agarose bound antibody and interactive proteins were collected via

81

centrifugation at 1,450 x g for 30 seconds in 4°C. The pellet was rinsed three times with
RIPA buffer, buffer was discarded afier the final wash and the pellet was resuspended in
30 ul of 2X SDS sample buffer. Samples were boiled at 100°C and separated using SDS-
PAGE (Laemmli, 1970) on 12.5% polyacrylamide gels. Proteins were visualized using
Coomassie brilliant blue, destained and excised directly from the gel. The gel band was
excised and hydrolyzed with trypsin according to Shevchenko et al. (1996).

3.2.3. MALDI-TOF-MS

0.5 ul of the digested peptide was mixed with 0.5 ul of saturated of a-cyano-4-
hydroxycinnamic acid in 60% acetonitn'le/O.1% trifluoracetic acid. Mass spectra were
obtained on a Voyager-DE-STR MALDI-TOF mass spectrometer (formerly PerSeptive
Biosystems, Inc., Framingham, MA, now Applied Biosystems) in the linear mode. Peak
lists of the tryptic peptide masses were generated and searched against public databases
using the program Protein Prospector from the University of California, San Francisco
(http:/flarospectorncsfedu).

3.2.4. LC-ESI-MS

Further analysis of the digests and sequence confirmation were performed using a
capillary liquid chromatography system (Waters Corp., Milford, MA) coupled to an
LCQ-Deca ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a
nanospray ionization source. Digests were trapped on a Cap Trap (Michrom
BioRsources, Inc., Auburn, CA) and subsequently separated on a 5 cm x 75 um ID
picofrit column packed with 5 pm ProteoPep C18 material (New Objective, Woburn,

MA). Peptides were eluted with a gradient of 2-95% acetonitrile in 0.1% formic acid.

82

 

Mass spectra of fragmented ions were analyzed using the SEQUEST and MASCOT
programs.

All mass spectrometry analyses were conducted at the Michigan State University Mass
Spectrometry Facility.

3.2.5. Western Blot Analysis

Protein samples were quantified using the DC Reagent System (Bio-Rad #500-

0116), a Lowry-based detergent compatible assay. Protein levels were measured in 96
well ELISA plates at 750 nm using a uQuant microplate reader (Witech AG, Switzerland)
and BiOTCk-KCijor Analysis software package (Fischer Scientific #BT—5270501). Each
sample containing 20 ug of total protein and 10 11] 2X SDS sample buffer was boiled at
98° C for 10 minutes, briefly centrifuged to collect sample and then loaded into a 15%
Iris-glycine polyacrylamide gel. Samples were separated for 2 hours at 110 volts using
SDS-PAGE (Laemmli, 1970) and then transferred to Immobilon-P, PVDF membrane
(Millipore Corp., Bedford, MA) overnight at 4° C in Towbin Buffer (25 mM Tris, 192
mM glycine and 20% ethanol) at 40 volts. Membranes were blocked for 5 hours in 5%
lowfat milk in 1X TBS-Tween (0.2 M NaCl, 66 mM Tris, 0.1% Tween).
Irnmunodetection was performed by incubating blots overnight with 2% lowfat milk in
1 X TBS-Tween and one of the following primary antibodies: anti-eIF 2(1. phosphor-SerSl
serum (Biosource, #44-726G) diluted to 1:3000, anti-KDEL monoclonal antibody
(Calbiochem #420400) diluted to 1:300 or anti-polyhedrin polyclonal (M. Ikeda) diluted
to 1:30,000. The blot was then washed with 2% milk in 1X TBS-Tween and incubated
with horseradish peroxidase conjugated anti-rabbit antibody (Pierce, #31460) or anti-

mouse antibody (Pierce, #31430) diluted to 1:10,000 in 2% lowfat milk in 1X TBS-

83

Tween for 2 hours. Bands were visualized using an ECL detection kit (Amersham-
Pharmacia Biotech, #RPN 2209) and exposure on Classic Blue Sensitive autoradiography
film (Midwest Scientific).
3.3. Results
3.3.1. HSP90 is activated in Ld652Y cells productively-infected with Achrf-l

In searching for an eIF20t kinase that was activated in response to AcMNPV and
vAchrf-l infection in Ld652Y cells, western blots showed an increasing level of a protein
at 12 and 24 h.p.i. that interacted with antibodies raised against human PKR, specifically
in vAchrf-l-infected cells (Fig. 18, Lanes 3 and 7, as compared to Lanes 1, 2, 4-6, 8).
Immunoprecipitation with PKR antibodies and cell lysates from vAchrf-l-infected
Ld652Y cells combined with subsequent MALDI-TOF mass spectrometry analysis
identified four independent peptide matches to Spodoptera frugiperda 90 kDa heat shock
protein (HSP83) (See Table 1). One lane was excised fi'om the IP gel for western blot
transfer to correlate band for excision with the immunoreactive band (Fig. 29,
Supplementary Data). Additional western blots utilizing a commercial HSP90 control
protein and PKR antibodies confirmed that the antibody was cross-reactive (Fig. 18, Lane
10). HSP90 is ubiquitous throughout eukaryotic cells and is identified in many mass
spectrometry analyses. However, considering that the anti-PKR antibody recognized the
H SP90 control protein, it is likely that the actual protein that was immunoprecipitated by

anti-PKR was HSP90.

84

 

is
E :8
. .5 E
12 h.p.I. 24hpl § g
F: E
‘- 1- D.
-= 5 E g ‘3‘ E E g 8 g
2 i s s 2 i s s a a:
nw'a- 2...“; 1'“ "'" “F. . f.-
1 2 3 4 5 6 7 B 9 10

Figure 18. HSP90 levels increase in productively-infected vAchrf-Il infected Ld652Y
cells from 12 to 24 h.p.i. Western blots showing increasing levels of an ~80kD protein in
vAchrf-l infected Ld652Ycells from 12 h.p.i. (lane 3) to 24 h.p.i. (lane 7). Western blots
were probed with anti-PKR polyclonal antibody which interacts with an 84 kDa PKR

fusion protein (lane 9), antibody also interacts with HSP90 control protein of
approximately the same size (lane 10).

85

 

TIC gi Ref. Xcorr Sequence

8. Fruglperda

9.9e5 90“” Heat 4.11 (K)HLEINPDI~ISIVETLR
Shock Proteln

gi 12005809
7,795 gi 12005809 3.93 (K)GWDSEDLPLNISR

 

 

 

3.9e6 gi 12005809 3.30 (K)GWDSEDLPLNISR

 

3.496 91 12005809 329 (K)HLE|NPDHSIVETLR

 

1.6e6 gi 12005809 2.96 (K)HFSVEGQLEFR

 

9.5e5 gi 12005809 2.94 (K)HFSVEGQLEFR

 

2.696 9i 12005809 2.86 (K)SLTNDWEDHLAVK

 

 

 

 

 

 

TIC: Total Ion Chromatogram

Xcorr: Cross-correlative value mm other peptides in database after
MSIMS analysis

gi Ref. Genbank general accession number

Table l. Sequest summary of mass spectrometry analysis for immunoprecipitation with
anti-PKR antibodies in vAchrf-l-infected Ld652Y cells at 12 h.p.i.

3.3.2. Inhibition of HSP90 with geldanamycin inhibits polyhedrin protein synthesis
and occlusion body production in permissively-infected cells at 48 h.p.i. but has little

effect on budded virus prodcution

It was interesting to note a de-novo synthesis of HSP90 in vAchrf-l-infected
Ld652Y cells beginning at a post-infection time that correlates to the onset of translation
arrest in AcMNPV-infected Ld652Y cells. While heat shock proteins are ubiquitous
throughout the cell, increased synthesis of heat shock proteins is a hallmark of the
unfolded protein response that is activated in response to virus infections in systems other

than invertebrates. HSP90 activity can be directly inhibited by the antibiotic

86

geldanamycin which blocks the ATP binding site of HSP90. HSP90 chaperone function
is dependent upon its ATPase activity. To define a role for HSP90 during permissive
baculovirus infection we added 1 uM geldanamycin directly to cell culture media
immediately following the infection of S121 cells with AcMNPV and vAchrf-l. Treated
cells were examined at 3, 12, 24 and 48 h.p.i. using a Nikon TMS inverted phase-contrast
microscope, 48 hour infected cultures were re-treated with geldanamycin at 24 h.p.i. to
guard against effects attributable to degradation of the inhibitor. Surprisingly, virus
occlusion bodies were not seen in permissively infected cells treated with geldanamycin
at 24 h.p.i. and were found at low levels at 48 h.p.i. (Fig. 19, ZB compared to 2A and 3B
compared to 3A). Similar results were found in vAchrf-l -infected Ld652Y cells that were
treated with geldanamycin (data not shown). To determine whether HSP90 inhibition was
completely blocking synthesis of late viral proteins, cell lysates were collected at 24 and
48 from Sf21 cells permissively-infected with AcMNPV and Ld652Y cells infected with
AcMNPV and vAchrf-l. Lysates were screened using western blots with antibodies
raised against the late baculovirus proteins polyhedrin and p39. Blots showed that
geldanamycin neither enabled AcMNPV replication in Ld652Y cells (Fig. 20, Lanes 4
and 10) nor blocked late viral protein synthesis in permissively-infected cells at 48 h.p.i.
(Fig. 20, Lanes 12 and 14). However, in both permissively-infected Sf21 cells at 48 h.p.i.
and Ld652Y cells at 24 and 48 h.p.i., polyhedrin protein synthesis was reduced in those
cells treated with geldanamycin (Fig. 20, Lane 14 as compared to Lane 13 and Fig. 3,
Lanes 6 and 12 as compared to 5 and 11). The other bands found on Fig. 20, lanes 5, 11
and 12 are consistently produced on western blots from productively-infected

invertebrate cells at the very late stages of infection when probed with anti-polyhedrin

87

antibodies. On these blots, by 48 h.p.i., many bands can be seen, ranging in size from 1-
26 kDa, possibly representing degraded fragments of the polyhedrin protein. In mock-
infected samples and at earlier times post-infection, the anti-polyhedrin antibody is not
cross-reactive. Similar results were observed when the same blots were stripped and
reprobed with p39 antibodies (data not shown). To determine whether HSP90 inhibition
had any effect on budded virus production, infected cells were treated with geldanamycin
as previously described and titered via standard plaque assay. Plaque assays showed that
geldanamycin may have a minor inhibitory or slowing effect on permissive virus

replication in infected Ld652Y and Sf21 cells (Figure 21).

88

- .13.“..-

 

Untreated 8121 Geldanamycin-treated
cells at 48 h.p.i. Sf21 cells at 48 h.p.i.

 

Figure 19. Geldanamycin delays or prevents occlusion body formation in
lepidopteran cells permissively-infected with baculovirus. Sf21 cells that are
permissively-infected with either AcMNPV (panel 2A) or vAchrf-l (panel 3A) normally
show production of occlusion bodies by 48 h.p.i. When 801 cells are treated with 1 uM
geldanamycin, occlusion bodies are not visible at 48 h.p.i. in AcMNPV (panel ZB) or
vAchrf-l (panel 3B) —infected cells. (Image Presented in Color)

89

 

24hp| 48hp1
'5 2' g ‘0'
'0' 8 8 ti 0 g Q
3 + + g + + 8
0>->- >. >>
+ee§e .;gessw:
>->- coco coco
§§§§33 §§:§33%%
3335?? 3355??55
:‘zz‘gtgxzz‘g‘gzz
8§%%2§ 08%%22%%
§E<<>> §§<<>><<
..., Polyhedrin
: - -
1 2 3 4 5 6 7 8 91011121314

Figure 20. In cells treated with the HSP90 inhibitor, geldanamycin, polyhedrin
protein levels are reduced in 8121 and Ld652Y cells productively infected with
baculovirus. Polyhedrin protein levels were assayed by using anti-polyhedrin antibodies
Total cell lysates were prepared from Ld652Y cells that were mock-infected or infected
with AcMNPV or vAchrf-l or AcMNPV-infected Sf21 cells. Infected cells were
harvested at 24 and 48 hours post-infection. Proteins from whole cell lysates were
separated on 15% SDS-PAGE gels and analyzed with immunoblotting.

90

 

 

A.

Geldanamycin in Infected Ld652Y cells

1.ooe+os , - . .. . . mAchrM
: - ~‘ ~ flvAchrf-1IGeld.
IAcMNPV

1.00E+07 = EACMNPVIGeld.

rt. n' N: in“

f H.511“ .. 1 ,
<ch’
1 if ‘

M l, U’
‘V .
I“;

R

_ 1‘; ..r"

 

1.00E-I-06

pfulml

.7" a
' 4;:
"e
'3 3‘

1.00E+05

iv
1

55-11%

'2'?

1.00E+04

 

91

Geldanamycin in Infected Sf21 cells

1.00E+08 ,. _ _,' ... “r y .; ElvAchrf-1
~ ’1‘? nvAchrr-1Iceld.
. ‘5: «If? IAcMNPV
1.00307 - 55 ‘ EAcMNPV/Geld.
E
31.00306 .
0.
1.00305
1.00304

 

 

0 h.p.i. 24 h.p.i. 48 h.p.i.

Figure 21. Geldanamycin reduces budded virus production in vAchrf-l-infected
Ld652Y cells. A. vAchrf—l titers are reduced within at least one order of magnitude in
infected Ld652Y cells treated with geldanamycin. B. Geldanamycin does not reduce
vAchrf-l or AcMNPV budded virus production in Sf21 cells. Ld652Y or Sf21 cells were
pre-treated with 1 uM geldanamycin from 1 hour preceding infection to time of
collection. Cells were mock-infected or infected with either AcMNPV or vAchrf-l and
virus was harvested at 24 h.p.i. and 48 h.p.i. Virus was titered on Sf21 cells using a
standard plaque assay (O'Reilly, Miller, and Luckow, 1994). Plates were scored for occ+
plaques at 4 and 6 days post-infection.

ser5]

3.3.3. Proteolytic cleavage of peIFZa precedes and is independent of HSP90

activation in permissively-infected Ld652Y cells

We previously reported that a proteasome-dependent, proteolytic cleavage of peIF20ts°m

occurs by 24 h.p.i. in invertebrate cells permissively-infected with baculovirus (Mecey-

92

manuscript in progress). To determine if that cleavage was dependent upon an increased
synthesis of HSP90; mock, AcMNPV and vAchrf-l-infected Ld652Y and Sf21 cell
lysates were collected at 24 and 48 h.p.i. and western blots were conducted to assay for

peIF20tse'5' cleavage in geldanamycin-treated cells. Western blots show that cleavage of

“'5' occurs in untreated and geldanamycin-treated, permissively-infected cells by

pelF20t
24 h.p.i. (Figure 22, lane 3 compared to lane 6, lane 8 compared to lane 11 and lane 9
compared to lane 12). These data suggest that either HSP90 activation occurs
downstream of proteasome-enabled eIF2a phosphorylation or cleavage of peIF20ts°m in
invertebrate cell lines that are permissively-infected with baculovirus or that HSP90

activation and eIF20t phosphorylation and/or cleavage are events that occur independent

of one another.

Ld652Y Sf21
NO Geld. w/ Geld. No Geld. wl Geld.

5;; 5.; >7 (1).“:
34253112; 2‘:th
gaggeegaéset»

<> <> <>§<§
--...— ...-sun- ...-.- ~37 kDa

d all. -- --zskoa
123456789101112

Figure 22. Proteolytic cleavage of peIFZamfl occurs in Ld652Y and 8121 cells
permissively-infected with vAchrf-l and AcMNPV in the presence of geldanamycin.
Western blots showing proteolytic cleavage of peIF201ser51 in Ld652Y cells infected with
AcMNPV and vAchrf-l that were untreated (lanes 2 and 3) or in the presence of
geldanamycin (lanes 5 and 6) at 24 h.p.i. Proteolytic cleavage of peIF201$ch1 also occurs
in Sf21 cells infected with AcMNPV or vAchrf-l that were untreated (lanes 8 and 9) or
treated with geldanamycin (lanes 11 and 12).

93

 

 

3.4. Discussion

Infection of the Lymantria dispar 652Y cell line with the baculovirus AcMNPV
results in global translation arrest and a non-productive infection (Guzo et al., 1992;
McClintock, Dougherty, and Weiner, 1986). Expression of a single gene, host range
factor-I, isolated from LdMNPV precludes translation arrest in AcMNPV-infected
Ld652Y cells and results in virus replication (Chen et al., 1998; Du and Thiem, 1997b;
Thiem et al., 1996). Apoptosis is enhanced and global translation arrest is initiated during
the late stages of AcMNPV infection in Ld652Y cells (Thiem and Chejanovsky, 2004).
During a productive infection, events correlating to the late phases of virus replication are
DNA replication of the virus, expression of late viral genes and a virus-mediated shut-off
of host transcription.

In this study we identify an increased expression of the chaperone protein HSP90
that occurs in cells permissively-infected with vAchrf-l during the late stages of infection
(Fig. 18). Further, we determine that geldanamycin-mediated inhibition of HSP90 delays
or inhibits the production of the very late baculovirus polyhedrin protein and reduces or
eliminates the production of occluded virus in productively-infected cells at 48 h.p.i., a
time when occluded virus is normally present (Fig. 19 and 21). Inhibition of HSP90 with
geldanamycin has no inhibitory effect on the proteasome-dependent cleavage of
peIF201s¢r5 1, indicating that HSP90 activation occurs independently or downstream of

eIF2a phosphorylation at serine 51 (Fig. 22).

94

Other groups have shown that chaperone proteins may be required during
permissive baculovirus infection. HSC70, also known as BiP, was shown to be
transcriptionally up-regulated in Sf9 cells permissively-infected with AcMNPV
(N obiron, O'Reilly, and Olszewski, 2003). HSP90 was not identified in the same study as
being transcriptionally activated; however, the same group shows the majority of host
transcription being shut-off between 12 and 18 h.p.i. during a permissive infection. The
shut-off of host transcription suggests a potential role for translational regulation of
chaperone transcripts during a permissive infection. Indeed, HSP90 levels increase from
12 to 24 h.p.i. in Ld652Y cells that are permissively-infected with vAchrf-l and HSP90
appears to play a role in the production of late viral proteins and occluded virus (Fig. 19,
20 and 21).

In the non-productive infection of AcMNPV in Ld652Y cells, the virus enters the
nucleus and executes the early stages of virus replication. During these early stages of
infection, host and virus transcription and translation continue (Guzo et al., 1992).
Previously, we have shown that eIF20 phosphorylation at serine 51 occurs during
AcMNPV infection of Ld652Y cells at a time that correlates to the onset of global
translation arrest. Additionally, we have shown that both eIF20t phosphorylation at serine
51 and cleavage of peIF2aser51 are enabled by the proteasome (Mecey-manuscript in
progress). The additional load of viral transcripts produced during the early stages of
infection may result in stress on the protein processing capacity of the endoplasmic
reticulum and its localized chaperone machinery cumulating in the induction of an

unfolded protein response.

95

The unfolded protein response (U PR) has been characterized in C. elegans and
plays a regulatory role in the development of the nematode (Shen et al., 2001). Many
homologous components of the mammalian UPR pathway have been identified and
characterized in other invertebrates (Olsen et al., 1998; Santoyo et al., 1997) including
the lepidopteran, Bombyx mori (Goo et al., 2004). Hallmarks of the unfolded protein
response include eIF20t phosphorylation at serine 51 resulting in a global arrest of cap-
dependent protein synthesis and a diverted pathway resulting in either apoptosis and cell
destruction, or cell recovery as mediated by an increased synthesis of chaperone proteins
and ERAD degradation of unfolded/unprocessed proteins (Ma and Hendershot, 2004;
Rutkowski and Kaufinan, 2004). The proteasome has recently been identified as a key
regulator in the UPR by facilitating ERAD degradation (N g, Spear, and Walter, 2000).

Although our host range system has many of the hallmarks of a UPR stress
response pathway, one must consider that this pathway has not been fully characterized
for Lepidopteran insects and that the comparisons made are based on information
obtained from mammalian systems. Different stress response pathways such as those
resulting from DNA or oxidative damage may also be induced in response to infection.
Investigation into kinase signaling leading to eIF20. phosphorylation in AcMNPV-
infected Ld652Y cells may help to define initiated pathways during non-permissive
infection. Additonal information should also be gathered with regards to the expression

of other chaperone proteins during baculovirus infection.

96

 

 

 

Chapter 4

Summary and Future Directions

 

 

97

 

4.1. Research Summary

a.) Research conducted by other laboratory members show that translation arrests
gradually in AcMNPV-infected Ld652Y cells with a complete arrest of protein synthesis
occm'ring by 13-14 h.p.i. (work conducted by X. Du). In those same infected cells,
polysome profiles show a monosome peak occurring at 9 h.p.i. indicating that translation
arrests at the initiation stage (work conducted by W.A. Williams). Possible effects
occurring from the inhibition of translation elongation were not investigated in this study.
b.) Phosphorylation of eIF20t at serine 51 occurs in Ld652Y cells that are infected with
AcMNPV at 12 h.p.i. In Ld652Y cells that are infected with vAchrf-l, eIF20t appears to
be protected from phosphorylation as compared to mock and AcMNPV-infected cells.
Expression of the proposed AcMNPV-encoded, dominant-negative inhibitor of eIF20t
kinases, PK2, continued throughout the infection but PK2 was insufficient at blocking

phosphorylation of eIF20t in AcMNPV-infected Ld652Y cells at late times post-infection.

$ch 1

c.) Proteolytic cleavage of peIF2a appears to occur in Ld652Y cells permissively-

infected with vAchrf-l and Sf21 cells permissively-infected with AcMNPV and vAchrf-

ser5 1

1. Cleavage of peIF20r. was not induced by stress-inducing agents and was not blocked
with cell permeable protease inhibitors.

(1.) Treatment with the proteasome inhibitor epoxomicin in Sf21 and Ld652Y cell lines
infected with AcMNPV and vAchrf-l or vAchrf-l, respectively, resulted in prevention of

ser5 l

eIF201 phosphorylation at serine 51, a block of peIF20t cleavage and no virus
replication. The proteasome inhibitor, MG132, yielded similar results in vAchrf-l-

infected Ld652Y cells but only reduced virus titers while not blocking peIF201scr51

98

cleavage in infected Sf21 cells. The conflicting result observed for both proteasome
inhibitors may result from a varying potency of each inhibitor or its specific mode of
action. Both results indicate that the proteasome is involved during baculovirus infection.

0.) Heat shock protein 90 expression is enhanced in Ld652Y cells that are permissively-
infected with vAchrf-l. Inhibition of HSP90 with geldanamycin resulted in a delay or
reduction of very late stage baculovirus protein expression and a block of occlusion body

ser5 1

production at 48 h.p.i. Cleavage of pelF201 occurs independently of enhanced HSP90
expression. These results suggest that enhanced expression of HSP90 accompanies

productive baculovirus infection and is integral to the production of occluded virus.

99

 

 

 

 

 

4.2. Future Directions

The current data indicates that proteasome-enabled cleavage of peIFZasm'
accompanies productive virus infection in both Ld652Y and Sf21 cells infected with
vAchrf-l and AcMNPV and vAchrf-l, respectively. Additional data produced in this
study demonstrates that the inhibition of the proteasome with epoxomicin or MG132
blocks phosphorylation of eIF20t at serine 51 and inhibits or reduces the production of
virus progeny in those same infected, lepidopteran cell lines (See Fig. 23). The primary
questions that remain are: is baculovirus replication enabled by the proteolytic cleavage

of peIF20ts¢r5 l and, if so, does HRF-l participate in that process?

100

 

 

 

 

b

eIFZo Phosphorylation No Virus
Blocked Replication

 

 

 

Proteasome
m Infection Activity _—>a <:l
————>
E
Virus Replication peIFZo‘m‘ Proteolytically ' "1
Blocked? Ieaved

1

Virus Replication
Enabled

Figure 23. Proposed model of post-translational modification of eIF20t in response
to baculovirus infection. The current data shows eIF2a becoming phosphorylated in
response to AcMNPV-infection in Ld652Y cells. In Sf21 and Ld652Y cells that are
productively-infected with AcMNPV and vAchrf-l or vAchrf-l, respectively, a (Fig. 23
caption, cont.) proteasome-enabled cleavage of peIF201§cT51 occurs by 24 h.p.i. In those
same infected cells, when treated with proteasome inhibitors, eIF20t is protected from
phosphorylation at serine 51 and virus replication is inhibited. (Image Presented in
Color)

101

ser5 1

Indeed, if cleavage of peIF20t enables baculovirus replication by preventing
global translation arrest, then one can envision two scenarios as being mechanistically
significant. The first and most probable mechanism involves disabling the inhibitory

ser5 1

capabilities of peIF20t on eIF2B, the second involves the proteolytic degradation of
peIF20ts°m coupled to an increased synthesis of eIF201. The nucleotide exchange factor
eIFZB is a limited factor in the cell, phosphorylation of eIF2a at serine 51 results in the
binding and sequestering of eIF2B. This impoundment of eIF2B prevents the exchange of
GDP to GTP required for the formation of an active eIF2 ternary complex resulting in
translation arrest at the initiation stage (See Fig. 24A and 24B). Proteolytic cleavage of
peIF20tse'51 may render two partial eIF20t fragments that are incapable of inhibiting eIF2B
but capable of forming a functional ternary complex with other subunits (See Fig. 24C).
One method to test this hypothesis is to express the predicted partial eIF2a fi'agments
individually from a recombinant AcMNPV in Ld652Y cells and then conduct plaque
assays to determine budded virus titers. Alternatively, engineered plasmid constructs with
truncated forms of eIF2oc could be transfected into Ld652Y cells followed by AcMNPV
infection and plaque assay. While Figure 24C does not reflect it, functional ternary
complex formation could result from interaction with either partial eIF2a component so,
both would have to be expressed and evaluated. If budded virus titers increased as
compared to those produced at 0 hours post-infection, one could conclude that AcMNPV
replication had been enabled by the expression of a truncated eIF20t sequence. It is

possible that phosphorylation of eIF201 at serine 51 occurs as a general response to stress

in lepidopteran insects. While experiments utilizing general stress induction agents in

102

 

 

 

 

both Ld652Y and Sf21 cells did not result in cleavage of peIF2asc'5‘, levels of full-length
peIF20tscr51 may show differential regulation in response to stress (Fig. 12). More
experiments need to be conducted to determine the effects of stress on eIF2or
phosphorylation in lepidopteran cells. Proteasome-enabled degradation of peIF201‘cr5 l
may occur as a means to overcome translation arrest while levels of eIF20t recover. If
budded virus titers do not increase in Ld652Y cells infected with recombinant AcMNPV
expressing either eIF20t truncated protein (vAccleIF201), protein synthesis levels should
be evaluated to determine if global translation arrest occurs. If translation arrest is
prevented in vAccleIF20t-infected Ld652Y cells and a productive infection is not
established, it would indicate that global translation arrest is not the mechanism
preventing AcMNPV replication in that cell line. Other groups have shown that
expression of a C-terminal, truncated form of eIF20t, was not sequestered by eIFZB in
mammalian SAOS-2 cells. Cells expressing this mutant were able to overcome

translational repression induced by PKR even though the truncated eIF201 was

phosphorylated by the kinase (Satoh, et. a1. 1999).

103

 

 

 

 

0; Met
Jo" 12111+ [u

Figure 24. The interaction of eIF20t with eIFZB. A.) The nucleotide exchange factor
eIF2B coordinates the exchange of GDP to GTP to restore functional ternary complex
formation required for the initiation of protein synthesis. B.) When eIF20L is
phosphorylated at serine 51 it binds eIFZB preventing nucleotide exchange, active ternary
complexes can not form and global translation arrest is initiated. C.) A proposed model of
how a cleaved fragment of pelF201§cr51 may interact with eIFZB and still fimction in an
active ternary complex to initiate protein synthesis. (Image Presented in Color)

 

The second proposed mechanism involves the proteasome—enabled cleavage of
peIF2as°r51 as a means to remove the phosphorylated protein from the cell as the cell
recovers from a particular stress. Eukaryotic cells are dependent on eIF20L as a required
component of the initiation of protein synthesis so, this proteolytic degradation would
have to be accompanied by an increased synthesis of eIF20t which is best evaluated at the
transcriptional level using northern blots or real-time PCR. Current data do not suggest
that eIF20t levels increase in productively-infected cells but that is difficult to determine
by observing protein levels as host transcription is being shut-off. In either model as it
relates to vAchrf-l-infected Ld652Y cells, a viral protein may mediate the proteasome-

ser5 1

enabled cleavage of peIF20t to initiate recovery fiom stress resulting from virus

104

infection, thus restoring protein synthesis and possibly enabling permissive virus
infection.

As a part of an initiated stress response pathway, proteasome activity can lead to
to eIF20t phosphorylation at serine 51 (Jiang and Wek, 2005). One can envision a
scenario where once proteasome-dependent eIF2a phosphorylation is initiated in
baculovirus-infected cells, a viral protein, like HRF -1, may mediate the degradation of
peIF2orsc'5‘ by directing proteasome activity (Fig. 25). The question of HRF-l
involvement in enabling baculovirus replication in Lymantria dispar and its correlation to

“'51 are best answered by searching for proteins that

the proteolytic cleavage of peIF201
directly interact with HRF-l . Yeast two-hybrid analysis or immunoprecipitation studies
coupled to mass spectrometry analysis may identify HRF-l interacting proteins and
further provide indications to the level of function for HRF-l. Another protein(s) that
functions in a similar manner to HRF-l may be encoded by the Spodoptera fi-ugiperda
genome thus facilitating AcMNPV replication in Sf21 cells. This would offer an

“'5‘ occurs in Sf21 cells during infection With

explanation of why cleavage of peIF2a
AcMNPV. Alternatively, Ld652Y cells may physiologically differ from 8121 cells to
such an extreme, that a HRF-l-type protein is not required to promote AcMNPV

replication in Sf21 cells.

105

Proteasome } elF2r1 is
ACtIVItY phosphorylated

v

Viral protein (HRF-1?) directs
degradation of pelFZa“r51 via
proteasome activity?

 

Infection 4

Figure 25. Proposed model for HRF-l function in vAchrf-l-infected Ld652Y cells.
AcMNPV-infection of Ld652Y cells results in eIFZa phosphorylation at serine 51 and
global translation arrest. This study shows that proteasome inhibition blocks the
phosphorylation of eIF 201, cleavage of pelF 211W51 and blocks the production of virus
progeny in baculovirus-infected, invertebrate cell lines. In the proposed model, HRF-l

would act downstream of proteasome activation to coordinate cleavage of peIFZaws 1.

106

Appendix A
Supplementary Data

107

eIF4E is available to bind cap substrates in AcMNPV-infected Ld652Y cells.

Protein synthesis can be attenuated through the formation of an inhibitory eIF4E-
4E binding protein (4E-BP) complex. To exclude the possibility that mechanisms other
than eIF20t phosphorylation might be contributing to translation arrest we isolated total
cell lysates from mock, AcMNPV and Achrf-l-infected Ld652Y cells and precipitated
against m7G-bound sepharose (Amersham-Pharmacia). Western blots of precipitated
proteins were probed with Lepidopteran eIF4E antibodies. Western blots show equal
amounts of eIF4E bound to the m7G cap in mock, AcMNPV and Achrf-l infected cells at
3 and 20 hpi (Fig. 15, lanes 1, 2, 3, 4, 5 and 6). This indicates that eIF4E is not prevented

from binding the cap and by inference, that it was not sequestered by 4E-BP.

3hpi 20hpi

 
 

~ AcMNPV

Cap-bound eIF4E

Figure 26. AcMNPV infection in Ld652Y cells does not impair the m7G cap-binding
ability of eIF4E. Total cell lysates from mock-, AcMNPV-, and Achrf-l-infected
Ld652Y cells were immunoprecipitated with m7GTP-bound sepharose. Proteins were
boiled off sepharose beads, subjected to SDS-PAGE and western blotting with
lepidopteran eIF 4E antibodies.

108

 

4E-BP is not over-expressed in AcMNPV-infected Ld652Y cells.

To further investigate the possibility of 4E-BP contributing to global translation
arrest in AcMNPV-infected Ld652Y cells, we probed western blots of total cell lysates
isolated from Mock, AcMNPV and Achrf-l-infected cells at 3, 12 and 20 hpi with
antibodies raised against Drosophila melanogaster 4E-BP (kindly provided by Mathieu
Miron). There was no detectable increase in 4E-BP levels in infected cells over mock-
infected (Fig. 16, lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) suggesting that, unlike
translation arrest induced in Drosophila in response to bacterial infection and wounding
(Bemal and Kimbrell, 2000), over-expression of 4E-BP does not contribute to translation
arrest in AcMNPV-infected Ld652Y cells. These experiments do not rule out possible
changes in 4E-BP phosphorylation. However, the combined eIF4E and 4E-BP data

indicates that translation arrest is not controlled through inhibition of eIF4E.

109

3'
12.
N
A
3'
'2.

AcMNPV
vAch ri-1
VACApk2

E 'r 2!
s a a
E 2 s s E
~‘a’\ 5.-.. ..-“ 4E-BP

...

1234 5678 9101112

Figure 27. 4E-BP is not up-regulated in response to AcMNPV-infection in Ld652Y
cells. Total cell lysates were prepared from Ld652Y cells that were mock-infected or
(Caption continued from p. ) infected with AcMNPV, vAchrf-l or vAcApk2. Lysates
were collected at 12 h and 24 h post-infection. The same blot for each time point was
stripped and reprobed sequentially with antibodies to Drosophila 4E-BP and a-tubulin.
The lepidoteran eIFZa kinase, BeK, is activated in AcMNPV-infected Ld652Y cells.
A novel lepidopteran eIF20 kinase, BeK, was identified in Bombyx mori and
characterized as being activated in response to heat shock and osmotic stress in
transformed Drosophila 82 cells but not in response to immune, endoplasmic reticulum
or oxidative stress. The same group showed that PK2 was capable of reducing BeK
activity suggesting that BeK may act in an anti-viral response (Prasad et al., 2003). Using
western blots and antibodies raised against BeK (P. Brey), we screened infected Ld652Y
cells for BeK induction. Western blots of infected cell lysates show expression of a 65
kDa protein that interacts with anti-BeK antibodies at 12 hours post-infection in

AcMNPV-infected Ld652Y cells (Fig. 14), a time that correlates to eIF20t

phosphorylation in AcMNPV-infected Ld652Y cells. Western blots produced from

110

mock-infected Ld652Y cells or those infected with vAchrf-l from 3-24 hours post-
infection are devoid of any antibody interaction when probed with anti-BeK antibodies
(Fig. 14). An additional band of ~40 kDa is also seen in AcMNPV-infected Ld652Y
cells at 12 h.p.i. Results from this western blot indicates that BeK is expressed at a time

that correlates to eIF20t phosphorylation and global translation arrest in AcMNPV-

infected Ld652Y cells.
Ld652Y
AcMNPV vAchrf-1
+Mi3612244836122448
A "" 3:: BeK
Sf21
AcMNPV vAchrf-1
+Mi36122448361224
B a —..-' ---—-- - ___,__.___.--- BeK

.“ -- .- ...—.--”

Figure 28. The lepidopteran kinase, BeK, is expressed in AcMNPV-infected Ld652Y
cells at a time that correlates to the onset of global translation arrest. Total cell
lysates were collected at 3, 6, 12, 24 and 48 hours post-infection, subjected to SDS-
PAGE and immunoblotted with polyclonal antibodies to BeK.

lll

 

“Qt—J"

4—— PKR Fusion Protein

-r
J.
‘J
.o

i

Exclsed Bands for MS

 

Excised Band .... .
I! I u ’— PKR Mb"
:- ‘ Protein

8' Western Blot from IP Gel Above

Figure 29. Polyacrylamide gel of proteins immunoprecipitated with human PKR
antibodies in vAchrf-l-infected Ld652Y cells. A. Arrows indicate the bands that were
excised for mass spectrometry analysis and co-migration of the PKR control protein.
Infected cell lysates were immunoprecipitated against sepharose bound antibody
overnight, then boiled and separated using SDS-PAGE, gel was stained with Coomassie
blue stain and destained for 5 hours. B. One lane from the IP gel was excised and
transferred to western blot along with the PKR control protein lane. Western blots were
incubated with anti-PKR antibodies; HRP-linked, anti-rabbit secondary antibodies and
bands were detected using ECL detection kit.

112

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