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Cloning and Sequencing of Peach Rosette

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Mosaic Virus RNAl
Allan Henry Lammers l
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Botany and Plant
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MSU le An Afflnnettve Action/Equal Opportunity lnetttulonm

CLONING AND SEQUENCING OF PEACH ROSETTE MOSAIC VIRUS RNA]
By

Allan Henry Lammers

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1996

ABSTRACT

CLONING AND SEQUENCING OF PEACH ROSETTE MOSAIC VIRUS RNA 1
By

Allan Henry Lammers

The complete nucleotide sequence of peach rosette mosaic nepovirus (PRMV)
RNA] has been determined. A Michigan grapevine isolate of PRMV was propagated,
purified and cDNA clones representing 99.6% of the RNA] were constructed. cDNA and
direct RNA sequence analyses revealed a RNA species of 7977 nucleotides, excluding a
3’ polyadenylated tail. The 5’- and 3’- untranslated regions are 52 and 1474 nucleotides,
respectively. Computer analysis of PRMV RNA] nucleotide sequence unveiled a single
open reading frame of 6450 nucleotides encoding a 240 kD polyprotein. Analysis of
predicted amino acid sequence of RNA] uncovered amino acid motifs characteristic of a
replicase, a proteinase, an NTP-binding protein and a proteinase cofactor. Order and
identity of these putative proteins are consistent with other nepoviruses. This analysis of
PRMV RNA] further distinguishes the taxonomic subdivisions within the nepovirus
group, confirms subgroup II status of PRMV and lays the groundwork for a pathogen-

derived resistance strategy.

Copyright by
ALLAN HENRY LAMMERS
1996

ACKNOWLEDGEMENTS

I would like to gratefully acknowledge my major professors Dr. Don Ramsdel]
and Dr. Richard Allison as well as my graduate committee member, Dr. Gus de Zoeten. I
thank the members of their labs, including Bil] Schneider, Ann Greene, Fang Gouwei, Dr.
Jeanne Ohrnberger, Min Deng, Deb Rucker, Dr. Jihad Skaf, Dr. Steve Demler, Dr. Pat
Traynor and Christiane Wobus for their friendship and scientific assistance. Thanks
particularly to Jerri Gillett for her kind and helpful assistance, especially with virus
purification. Thanks to Dr. John Halloin for the use of his photographic equipment and to
our department office staff, especially Jacqueline Guyton, for their help. I am indebted to
my family for their love and support, especially my sister Audrey for her skilled technical
assistance. Lastly, I thank my wife and best friend Amy, without whom this work would

not have been possible.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vi
LIST OF FIGURES ............................................................................. vii
CHAPTER 1: LITERATURE REVIEW ........................................................ 1
The Disease ................................................................................. 3
PRMV Transmission ....................................................................... 4
Attempts to Control the Disease ......................................................... 5
1. Control of Vector Populations ................................................. 5
2. Resistance to PRMV among Grapevine Cultivars ........................ 6
3. Toward Engineering Resistance to PRMV .................................. 7
4. Molecular Characterization of PRMV RNA] .............................. 9

CHAPTER 2: CLONING AND SEQUENCING OF
PEACH ROSETTE MOSAIC VIRUS RNA] ............................... 1]
Introduction ................................................................................. 1 1
Materials and Methods .................................................................. 12
Results and Discussion ................................................................... 24
SUMMARY AND CONCLUSIONS ........................................................... 51
APPENDICES ...................................................................................... 54
Appendix A: Table of Subgroup I and Subgroup II Nepoviruses ................. 54
Appendix B: Oligonucleotide Primers ............................................. 58
Appendix C: Cloning and Sequencing of PRMV RNA2 ......................... 59
LIST OF REFERENCES ......................................................................... 67

LIST OF TABLES

Table 1: Amino acid comparison of RNAl-encoded sequences for seven
picorna-like viruses including PRMV ......................................... 47

vi

Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6:
Figure 7:
Figure 8:
Figure 9:

LIST OF FIGURES

Purification of PRMV and Northern analysis ............................... 25
Cloning strategy for PRMV RNA] ........................................... 27
EonII deletion series on PRMV cDNA clone 5-9 .......................... 28
Nucleotide sequence of PRMV RNA] ................................... 29-36
Secondary structure of PRMV RNA 3’ untranslated region ............... 4]
Alignment of nepovirus RNAl-encoded amino acid motifs ............. 46
Genomic organization of PRMV RNA] ..................................... 49
Nepovirus genomic strategies ................................................... 5]
Alignment of PRMV RNA] and 2 3’-terminal sequences ............ 62-65

vii

CHAPTER 1

LITERATURE REVIEW

Nepoviruses are considered a genus within the picornavirus-like supergroup of
plant viruses which includes the potyviruses, comoviruses, and picornaviruses (Goldbach,
et al., 1987). Common features within this supergroup include genomic structure and
organization, as well as nucleotide and amino acid sequence similarity. Nepoviruses have
many unique features not found among other picornavirus-like members. Cadman (1963)
recognized some of these unique aspects; he observed that the nepoviruses formed a
natural group based on particle morphology and their ability to be transmitted by
nematodes. In fact, his acronym nepovirus for a nematode-transmitted polyhedral virus
was one of the first names that the International Committee on Taxonomy of Viruses
(ICTV) approved for a group of plant viruses (Wildy, 1971). The number of definitive
and possible nepoviruses has rapidly increased from eight in 1971 (Harrison et al., 197])
to 26 in 1982 (Matthews, 1982) to the current 36 species (Goldbach et. al, 1995). The
criteria of having a confirmed nematode vector is fulfilled by only 11 of the 36
nepoviruses. The remainder owe their present taxonomic assignment to possession of
other nepovirus characteristics; for example, host range response (infecting vegetables,
small fruits, or fruit trees), and physical and serological behavior (Martelli and Taylor,
1990)

Nepovirus taxonomy is unresolved. In the absence of genomic sequence data,
taxonomic criteria have emphasized physical and serological characteristics. Most
nepoviruses consist of three distinct particle types: a top (T) component consisting of
empty polyhedral capsid proteins; and a middle component (M) and a bottom (B)
component, each containing the identical capsid proteins plus single molecules of RNA2

and RNA], respectively (Martelli and Taylor, 1990). Researchers have attempted to

subdivide nepoviruses using physical and serological criteria. Martelli (1975) proposed a
four-part subdivision based on physical characteristics. Martelli’s scheme depends on the
sedimentation coefficients of RNA2 molecules, however, published particle
sedimentation values and RNA molecular weights are prone to error (F rancki et al.,
1985); F rancki et al. (1985) argued that the nepovirus group should be separated into two
subgroups based on the distinct morphologies of RNA2. Subgroup I would consist of
nepoviruses with RNA2 components smaller than 5.4 kb, while subgroup 11 members
would have RNA2 components greater than 5.4 kb. Since nepoviruses are serologically
unrelated, subgrouping them on this basis alone is insufficient (Francki, et al., 1985).
Until recently, physical and serological data were the only criteria used to separate
the nepoviruses. The nucleotide sequence of many nepoviruses is now available (see
Appendix A). Subgroup I nepoviruses (RNA2 smaller than 5.4 kb) have been well
characterized. In fact, complete nucleotide sequences are known for arabis mosaic virus
(ArMV); grapevine chrome mosaic virus (GCMV); grapevine fanleaf virus (GFLV);
olive latent ringspot virus (OLRSV); raspberry ringspot virus (RSV); and tomato black
ring virus (TBRV) (Loudes et al., 1995; LeGall et al., 1989 and Brault et al., 1989;
Serghini et al., 1990 and Ritzenthaler et al., 1991; Grieco et al., 1995; Blok et al., 1992;
Greif et al., 1988 and Meyer et al., 1986, respectively). Partial nucleotide sequence is
available for tobacco ringspot virus (TRSV) RNA2 (Buckley et al.,1993). Until now, the
only completely sequenced subgroup II nepovirus is tomato ringspot virus (TomRSV).
Partial sequence analysis is available for blueberry leaf mottle nepovirus (BBLMV)
RNA] and RNA2 (Bacher et al., 1994b). Further genomic analysis of subgroup 11

members is needed to confirm subdivision of the nepoviruses.

Based on physical and serological characteristics, PRMV is considered a
subgroup II nepovirus (Ramsdell and Myers, 1974; Harrison and Murant, 1977; Ramsdell
and Myers, 1978; Dias and Cation, 1980). Early molecular characterization of PRMV

focused on the physical properties of the virus. PRMV is unrelated to any nepovirus
serologically, yet it shares many physical similarities with other members of the group. It
consists of 28 nm isometric particles composed of 60 copies of a single capsid protein. A
bipartite, single-stranded, positive-sense RNA genome is separately encapsidated and
both nucleoprotein components are required for infection (Harrison and Murant, 1977).
Mature PRMV proteins are released from two large polyprotein precursors corresponding
to the translation products of RNA] and RNA2, as demonstrated for the nepoviruses
TomRSV, TBRV, GFLV, GCMV, as well as other picorna-like viruses including cowpea
mosaic comovirus (Rott et al., 1991, 1995; Demangeat et al., 1990; Ritzenthaler et al.,
1991; Le Gall et al., 1989; Lomonosoff and Shanks, 1983). Before discussing the
nucleotide sequence analysis of PRMV it is necessary to understand the economic
importance as well as some of the epidemiological aspects of the disease caused by

PRMV.
The Disease

PRMV was first recognized as the cause of a disease of peaches (Prunus persica
L.) in Michigan in 1917 (Klos et al., 1976). Since then it has been reported to infect
highbush blueberry (Vaccinium corymbosum L.) and many cultivars of grapevine (Vitis
Iabrusca L.) (Ramsdell and Gillett, 1981; Ramsdell and Myers, 1974). PRMV infects a
variety of weed species in Michigan; it was detected in 3 of 16 genera of weed species
adjacent to diseased ‘Concord’ vines (Ramsdell and Gillett, 198]). Weeds infected
included curly dock (Rumex crispus L.), Carolina horsenettle (Solanum carolinense L.),
and common dandelion (Taraxacum oflicinale Weber). PRMV is seed-home in dandelion
at a low level (3.6%) (Ramsdell and Myers, 1978). Peaches, highbush blueberry, and

grapevines grown where PRMV is endemic are susceptible to this disease.

PRMV has been reported most often in Michigan, occasionally in southwestern
Ontario (Canada) and once in New York (Ramsdell and Myers, 1974; Stace-Smith and
Ramsdell, 1987). Peach trees infected with PRMV exhibit delayed foliation, chlorotic
mottling and distortion of the early formed leaves, and shortening of the internodes
resulting in an overall rosette appearance to the plant. Typical symptoms in peach also
include chlorosis of the leaves. Chlorotic areas are variable in color intensity and
morphology. Twenty percent of Michigan’s approximately 18,000 acres of highbush
blueberries are produced where PRMV is endemic. Symptoms in blueberry appear as
elongated, crescent-shaped mature leaves and spoon-shaped terminal leaves (Ramsdell
and Gillett, 1981). Common symptoms of PRMV infection in ‘Concord’ grapevine
include leaf deformation, extreme shortening of intemodes, whorling of leaves, a typical
umbrella-like grth habit of the vine, and sometimes death of the plant (Ramsdell and
Myers, 1978). Infection in grapevine also results in delayed dormancy breaking and
uneven bloom, small and uneven berry clusters, and a yield 50-fold lower than uninfected
‘Concord’ vines. Currently, PRMV infection has become a serious problem in over 100
Michigan vineyards (Ramsdell, unpublished). PRMV infection has just recently been
detected in SW Ontario grapevines (Stobbs and Van Schagen, 1996).

PRMV Transmission

The predominant mode of natural transmission of PRMV is via nematodes.
PRMV inoculum is spread by nematodes between vines and from weed hosts to adjacent
vines (Ramsdell and Myers, 1978). Two nematode species, a dagger nematode,
Xiphinema americanum (Cobb), and the needle nematode, Longidorus diadecturus
(Eveleigh and Allen), have been reported as PRMV vectors (Klos et al., 1967; Eveleigh
and Allen, 1982). Another dagger nematode, X. revisi, is a suspected vector of PRMV in
SW Ontario (Stobbs and Van Schagen, 1996). An Ontario population of L. elongatus
(DeMan) transmitted PRMV at a low level (1 plant infected per 46 plants tested) but

investigators attributed this to non-specific retention of the virus (Allen and Ebsary,
1988). Occasional, non-specific transmission of nepoviruses by Longidorus spp. occurs
(Allen, 1986) when unadsorbed ingested particles contaminate the stylet and are
subsequently released into the transmission plant. The potential of Criconemoides Sp. as a
vector of PRMV has yet to be confirmed (Stace-Smith, R. and Ramsdell, DC, 1987).
Electron microscopy of thin sections of nematode vectors has identified virus retention
sites within each of the vector genera. In Longidorus sp., viral particles of RSV
(raspberry ringspot virus) and TBRV adsorbed to the inner surface of the odontostyle. In
Xiphinema sp., the particles of ArMV (arabis mosaic virus), SLRSV (strawberry latent
ringspot virus) are associated with the cuticular lining of the odontophore, the esophagus,
and the esophageal pump (Martelli and Taylor, 1990). The virus retention period within
the Longidorid vector is approximately 3 weeks while PRMV can be retained for up to 11
months in X. americanum. The lengthy virus retention time in X. americanum provides an
excellent over-wintering strategy but complicates efforts to control this disease. The
dagger nematode appears to be the more important nematode vector of PRMV in
Michigan while the Longidorid vector is more important in Ontario (Allen, 1986; Stace-
Smith and Ramsdell, 1987). The preference of one vector species over another in these
two locations is likely related to predominance of local nematode species rather than any

physical differences between vector or viral populations.

Disease Control: 1. Control of Vector Populations

The nematode vectors of PRMV have been found beneath infected grape roots to
a depth of 2.13 meters (Bird and Ramsdell, 1985). The depths at which these
phytoparasitic nematodes persist present a challenge for chemical control of the vector.
Long-term (10 yr.) fallowing of soil fails to prevent GFLV reinfection of grapevines by
X. index (Raski, DJ. et al. 1965). Chemical treatment of the soil using a combined
shallow (20 cm) plus deep (1 m) soil fumigation method provided good control of PRMV

vector populations over an 8-year study period in southwest Michigan (Ramsdell, DC.
and Gillett, J .M., 1983). Virus-free ‘Concord’ vines were introduced into treated soil in
1983 and, to date, have remained healthy. However, the future of chemical control of
nematode-transmitted viral diseases appears ill-fated. The $5000/acre cost of the
combined chemical control strategy described above is prohibitive. Additionally, all
effective fumigants except for D-D (1,3-dichloropropane/dichloropropene mixture), have
been decertified in the US. due to environmental concerns (Ramsdell et al., 1995). This

has led to a search for host resistance to PRMV as a suitable control strategy.

2. Resistance to PRMV among Grapevine Cultivars

The juice grape ‘Concord’ comprises 95% of Michigan's 11,000 acres of
grapevine. ‘Concord’ is highly susceptible to PRMV infection, thus, two different studies
have attempted to find highly resistant rootstock (Ramsdell and Gillett, 1985; Ramsdell et
al., 1995). In the first study, Ramsdell and Gillett (1985) tested the relative susceptibility
of 28 cultivars of American, French hybrid, and European grapevine to PRMV infection.
Groups of five test vines and a single ‘Concord’ control vine, were planted beneath a
mature, PRMV-infected ‘Concord’ source vine. Over a 10-year period, leaf extracts from
these vines were used to mechanically inoculate the herbaceous systemic host
Chenopodium quinoa Willd. or tested by enzyme-linked immunosorbent assay (ELISA).
Very low infection rates were reported for the American cvs. ‘Delaware’ and ‘Niagara’
(0.8% and 1.4%, respectively), while much higher infection rates were reported for cv.
‘Concord’ (35.4%). Ramsdell concluded that although cv. ‘Delaware’ exhibited the
highest resistance level to PRMV, it’s poor, spindly grth made it a less suitable
rootstock than the more robust cv. ‘Niagara’.

In a later study, Ramsdell et al. (1995) compared various scion and rootstock
grapevine cultivars by measuring the effect of PRMV infection on the yield and the

growth of vines. Cultivars tested included ‘Concord’ as well as those that did not show

significant PRMV infection in the earlier study. Results indicated that over a 4 yr period,
the greatest reduction in yield and growth due to PRMV infection occurred in ‘Concord’
(42% and 64%, respectively). PRMV was detected in 5% of ‘Chancellor’ and ‘Couderc
1616’ vines, 7% of ‘Couderc 1202’ and ‘Foch’ vines, 18.2% of ‘Niagara’ and ‘Delaware’
vines, 20% of ‘Teleki 5C’ vines, and more than 50% of the vines of ‘Vignoles’, ‘Teleki
5A’, and ‘Concord’, respectively. The white wine grape cv. ‘Seyval’ remained uninfected
during the study period but unfortunately is not used as a rootstock. Ramsdell et al.
(1995) concluded that cv. ‘C. 1616’ would make a suitable rootstock for the valuable, yet
susceptible, ‘Concord’ and ‘Niagara’ scions. Regrettably, however, very few rootstocks
remain which are not PRMV-susceptible (Ramsdell etal.,1985).

Conventional breeding will continue to have an important role in fiiture
production of PRMV-resistant grapevine. However, resistance is not always available in a
closely associated interfertile relative and/or resistance genes may be tied to undesirable
traits. Further, resistance may be multigenic and difficult to transfer (Grumet, 1995).
Recent advancements in grapevine tissue transformation and genetic engineering of host

resistance provide a plausible alternative for sustainable disease control.

3. Toward Engineering Resistance to PRMV

Molecular approaches have recently been developed for achieving high levels of
virus resistance in a variety of crop systems (review Grumet, 1995). This has proven to be
a highly successful strategy; up to 100% resistance has been obtained. In reports thus far,
the source of genetically engineered virus resistance consists solely of pieces from the
viral genome. Transgenic virus resistance is acquired by introducing a part of a plant
virus genome into the host genome. Various portions of the viral genome have proven
effective, including the capsid protein, movement protein and replicase genes, ribozymes,
and anti-sense RNA. The effectiveness of movement protein, antisense and defective-

interfering RNA, and ribozymes appear to differ among viruses and will not be discussed

further. Capsid protein (CP) and replicase genes have emerged as the most effective
genes for conferring pathogen—derived resistance. Capsid protein-mediated resistance has
been demonstrated for many viruses (Beachy et al., 1990; Fitchen and Beachy, 1993 and
Grumet, 1990, 1995); replicase-mediated resistance is discussed below. In general, CP-
derived protection is limited; the transgenic host is protected from the virus from which
the transgene was derived and a few closely related viruses. CP-derived virus resistance
can be overcome by inoculation with large quantities of virus or naked RNA. The
mechanism of capsid protein-mediated resistance is unknown and may vary among
viruses and viral constructs (Grumet, 1995). Capsid protein-mediated resistance is usually
ephemeral and probably not useful for woody plants.

Replicase-mediated resistance appears to be a more attractive method of
conferring resistance (review Fitchen and Beachy, 1993; Scholthof et al., 1993).
Although the spectrum of protection is narrow and similar to that conferred by the CP
(limited to resistance against the source virus or its immediate relatives), resistance was
not overcome by high quantities of inoculum or naked RNA. The level of resistance
observed in replicase-expressing transgenic lines was greater than that for CP-mediated
resistance (Grumet, 1995). Golemboski et al. (1990) reported that lines expressing a 54
kD replicase protein of tobacco mosaic tobamovirus (TMV) were 100% resistant to TMV
infection. Perhaps more importantly, resistance was retained even at inoculum quantities
up to 1000-fold higher than afforded by the TMV CP gene. Although the mechanism of
replicase-mediated resistance is likewise unknown, its efficiency makes it the preferred
system for pathogen-derived resistance.

Replicase-mediated resistance appears to be an attractive strategy for grapevine.
Successful implementation of this strategy demands that a grapevine transformation
system be available and useful genes be characterized. Various investigators have
successfully transformed grapevine tissue using Agrobacterium-mediated or biolistic

transformation methods (LeGall et al. , 1994; Mauro et al., 1995; Krastanova et al., 1995;

Lupo et al., 1994; Nakano et al., 1994; Martinelli and Mandolino, 1994; Bardonnet et al.,
1994; and Kikkert et al., 1996). A transgene is placed under the direction of a constitutive
promoter, often the 358 transcription promoter of cauliflower mosaic virus. The virus-
derived nucleotide sequence is commonly nested within a cassette, which also contains
one or more selectable marker genes that enables detection of transformed tissue. For
example, if the neomycin-phosphotransferase (NPTII) marker gene is used, transformed
embryonic tissue is selected for kanamycin resistance. Thus, the grapevine transformation
system required for an engineered resistance strategy has been developed.

Protection against PRMV infection in grapevine with a molecular approach such
as replicase-mediated resistance strategy also requires the molecular characterization of
PRMV. Nepoviruses encode their replicase gene on RNA] (Sanfacon, 1995) and for this
reason, we chose to sequence PRMV RNA]. Determination of the entire RNA] sequence
allows for isolation of the PRMV replicase gene. Once isolated, the replicase gene may

be introduced into the grapevine genome to establish PRMV resistance.

4. Molecular Characterization of PRMV RNAl

We have determined the complete nucleotide sequence of PRMV RNA]. A
grapevine isolate of PRMV from southwest Michigan was propagated and purified and
cDNA clones representing 99.6% of RNA] were obtained. cDNA sequence and direct
RNA sequence analyses revealed an RNA species of 7977 nucleotides. The 5’- and 3’-
untranslated regions consist of 52 and 1474 nucleotides, respectively. Computer analysis
of the PRMV RNA] nucleotide sequence unveiled a single long open reading frame of
6450 nucleotides capable of encoding a 240 kD polyprotein. Analysis of the predicted
amino acid sequence of RNA] revealed motifs characteristic of a replicase, a proteinase,
an NTP-binding protein and a proteinase cofactor. The order and identity of these

putative proteins are consistent with other nepoviruses.

10

The molecular characterization of PRMV RNA] presented here is an essential
step for developing a replicase-mediated resistance strategy. Portions of the RNA]
sequence, in conjunction with classical breeding and selection for resistant cultivars, may
provide ‘Concord’ vineyards with PRMV resistance. Obtaining grapevines expressing a
portion of the PRMV RNA] genome will also further our attempt to understand the
mechanism of pathogen-derived resistance.

The PRMV RNA] sequence also serves an important taxonomic role. PRMV
sequence data represents only the second complete sequence of a subgroup II nepovirus
RNA. These RNA] sequence data confirm the subgroup II status of PRMV. Further,
analysis of the PRMV RNA] genome supports Francki’s (1985) bipartite subdivision of
the nepovirus group along with TomRSV. This is the first attempt to separate the
nepovirus group based on genomic sequence information. Sequence homology between
PRMV and TomRSV, as well as similarities in genomic strategy, confirm the relationship
of these subgroup II nepoviruses. Comparison of the PRMV genome to that of TomRSV
and subgroup I nepoviruses highlights many interesting aspects which may add to the

criteria used to distinguish the two nepovirus subgroups.

CHAPTER 2

CLONING AND SEQUENCING OF
PEACH ROSETTE MOSAIC VIRUS RNAl

INTRODUCTION

The complete nucleotide sequence of peach rosette mosaic nepovirus (PRMV)
RNA] has been determined. PRMV has a bipartite, plus sense RNA genome which
contains a 5’-VPg and a 3’-poly(A) tail at the termini. RNA] is 7977 nucleotides
excluding a 3’-polyadenylated tail. The 5’- and 3’- untranslated regions are 52 and 1474
nucleotides, respectively. The nucleotide sequence contains a single long open reading
frame of 6450 nucleotides capable of encoding a 240 kD polyprotein. Analysis of the
nucleotide sequence of RNA] revealed motifs characteristic of a replicase, a proteinase,
an NTP-binding protein and a proteinase cofactor. The order and identity of these
putative proteins are consistent with other nepoviruses, especially subgroup II tomato
ringspot nepovirus (TomRSV).

Francki et al. (1985) proposed taxonomic subdivision of the nepovirus group into
two subgroups based on the size of RNA2. RNA2 is smaller than 5.4 kb in subgroup I
and larger than 5.4 kb in subgroup II. Sequence analysis of the primary and secondary
structure of PRMV RNA] reveals several features characteristic of nepoviruses, such as
nucleotide and amino acid sequence homology, polyprotein expression and overall
genomic organization. Analysis confirms that PRMV belongs to a distinct subgroup of
nepoviruses including TomRSV. With the availability of the complete nucleotide
sequence of another subgroup II nepovirus in TomRSV, comparisons were made between
individual RNA species of this subgroup. Comparison of the length of the 3’-untranslated

region, putative polyprotein processing activity, and the level of sequence homology

1]

12

between nepovirus RNA species strengthens our ability to distinguish between nepovirus
subgroups. PRMV, like TomRSV and partially sequenced subgroup II nepoviruses
BBLMV and CLRV, has a 3’-UTR approximatelyl.5 kb in length while subgroup I 3’-
UTR is less than 0.5 kb. Polyprotein processing of the PRMV polyprotein appears to
closely resemble that of TomRSV and picorna-like viruses such as CPMV, potyviruses
and polio, distinct from subgroup I nepovirus. Overall amino acid sequence identity
between PRMV and TomRSV further confirms PRMV subgroup II status and supports

the subdivision of nepoviruses by F rancki et al. (1985).

MATERIALS AND METHODS
Propagation and Purification of PRMV

Canes of PRMV-infected grapevine (V. labrusca cv. ‘Concord’) were harvested
from a vineyard located at Michigan State University, E. Lansing, MI, in December,
1992. The tissue was ground by mortar and pestle in a 4°C solution of 0.01 M sodium
phosphate buffer, pH 7.5 and rub-inoculated on primary leaves of the herbaceous host
Chenopodium quinoa (Willd.) seedlings dusted with 600 mesh carborundum (Fisher
Scientific, Pittsburgh, PA).

The following purification method for PRMV was adapted from Dias and Allen
(1980). Fifty to 100 g of symptom-bearing C. quinoa leaves were harvested 10 to 14
d.p.i. All subsequent steps were performed at 4°C. The tissue was blended in a
commercial Waring blender for 2 min. in 0.5 M boric acid buffer (12.5 mM sodium
borate, 10 hydrate; 0.5 M boric acid; 0.5% (w/v) ascorbic acid; adjusted to pH 6.5 with
1.0 N sodium hydroxide). Approximately 2 ml of boric acid buffer were used per gram of
tissue. Homogenized tissue was filtered through four-ply cheesecloth into a 500 ml
beaker and chloroform was slowly added to the extract to a final volume of 8.5% and
stirred for 3 min. The solution was centrifuged for 15 min. at 12,000 x g (J2-21

Centrifuge, Beckman Instruments, Inc., Palo Alto, CA) in a No. 30 rotor (Beckman) and

13

the pH of the supernatant was adjusted to 5.3 with 1N HC]. A 30 min. incubation on ice
was followed by 15 min. (12,000 x g) centrifugation. Supernatant was collected in
Beckman Quick-Seal centrifuge tubes (25 mm x 89 mm) and ultra-centrifuged (model
L7-65, Beckman) at 105,000 x g for 4 hr in a Ti50.2 rotor (Beckman). Pellets were
resuspended overnight in 1.8 ml 0.01 M potassium phosphate buffer, pH 7.0.

Linear-logarithmic 0-30% sucrose gradients were prepared and equilibrated at
4°C overnight. Resuspended virus solution was layered onto sucrose gradients, 0.3 ml per
gradient, and centrifuged for 90 min. at 38K RPM (105,000 x g) in a swinging bucket
rotor (Beckman SW 41). A density gradient fractionator (model 185, Instrument
Specialties Co. (ISCO), Lincoln, NB) and absorbance monitor (model UA-5, ISCO) were
used to separate the components of the sucrose column. The absorbance monitor UV
lamp was adjusted to 254 nm wavelength for detection of the virus particles. Other
absorbancy monitor settings included a chart speed of 60 cm/hr and a sensitivity of 2.0;
the baseline was adjusted prior to fractionation. Fifty percent sucrose solution was used to
push the gradient upward at a flow rate of 1.5 ml/min. Fractions corresponding to RNA]-
and RNA2-containing absorbance peaks were collected in approximately 0.5 ml volumes.
Pooled RNA] and RNA2 fractions were diluted in 3 volumes of 0.01 M potassium
phosphate buffer, pH 7.0, and centrifuged for 5 hr at 38K RPM (105,000 x g) in a
Beckman 40 rotor. The pelleted virus was resuspended in 100 u] TE buffer (10 mM Tris-
HC], 1 mM ethyldiethanolamine (EDTA), pH 7.0) and transferred to sterile 1.5 ml
Eppendorf tubes.

Viral RNA was extracted by adding 100 u] of Tris-saturated phenol (1:1 v/v) to
the resuspended virus solution. The mixture was vortexed for 30 sec, and centrifuged in a
bench top centrifuge (Eppendorf 5415C) at 14K RPM for 1 min. The RNA-containing
aqueous layer was transferred to a new 1.5 ml Eppendorf tube and phenol extraction was
repeated twice. Phenol extracted viral RNA was further purified by adding 100 u] of

chloroform (1 :1 v/v). The mixture was vortexed briefly and centrifuged at 14K RPM for

14

15 sec. and the upper aqueous layer containing the RNA was transferred to a new
Eppendorf tube. The RNA was precipitated by adding 1/ 10 volume (10 ul) of 3M sodium
acetate, pH 5.5, and 3 volumes of 100% ethanol (300 pl) and chilled at -80°C for 20 min.
The solution was warmed to room temperature and centrifuged at 14K RPM in a bench
top centrifuge for 15 min. to pellet the RNA. RNA was resuspended in 50 u] water and
RNA concentration was quantified by diluting 5 pl of the RNA solution in 995 u]
distilled water and measuring the optical density with a Beckman spectrophotometer
(model DU-64, Beckman Instruments, Inc., Palo Alto, CA) at A260 m and A280 “m. An
AND/A280 ratio was determined and compared to the published value (Dias and Allen,
1980)

An aliquot (1 ug) of RNA, as determined spectrophotometrically, was
electrophoresed on an agarose gel (0.8% agarose (w/v) dissolved in 1 x TBE: 10X=0.02
M EDTA, 1M Tris base, 1M boric acid, pH 8) to verify RNA concentration and to assess
its quality. A horizontal mini-gel apparatus (Owl Scientific, Inc., Wobum, MA) with a
running buffer consisting of 1 x TBE and 0.1 jig/ml ethidiurn bromide was used for all
agarose gel electrophoresis experiments.

Synthesis and Cloning of PRMV RNA 1 cDNA

Synthesis and cloning of PRMV cDNA utilized a cDNA synthesis kit and a
protocol adapted from manufacturer recommendations (Amersham Corp., Arlington
Heights, IL). Using 1.0 pg purified PRMV RNA template and 1.2 ug oligo (dT)12_18
primer, first strand cDNA synthesis was initiated by avian myeloblastosis virus (AMV)
reverse transcriptase. First strand reaction mixture was incubated at 42°C for 1 hr, then
placed on ice.

Primers for second strand synthesis were generated by nicking the viral RNA
template with E. coli ribonuclease H and subsequent replacement of the RNA strand with
dNTPs by DNA polymerase I. The second strand cDNA mixture (100 u] reaction
volume) was incubated sequentially at 12°C for 1 hr and at 22°C for 1 hr. DNA

15

polymerase I was heat-inactivated at 70°C for 10 min. T4 DNA polymerase was added to
2.5 units per pg of original mRNA template, and the reaction was incubated for 10 min.
at 37°C. Reaction was terminated by the addition of 4 pl of 0.25 M EDTA, pH 8.0, per
100 pl of second strand reaction mixture. Three prime to 5'-exonuclease activity of T4
DNA polymerase ensured blunt-ended termini of duplex DNA fragments which
facilitated blunt end ligation of the cDNA fragments.

Double stranded cDNA product was purified using phenol/chloroform extraction
and ethanol precipitation. The cDNA was extracted with an equal volume of phenol:
chloroform: isoamyl alcohol (25:24:1 v/v/v). The aqueous phase was extracted once with
an equal volume of chloroform (100 pl) and ethanol precipitated with one volume of 4M
ammonium acetate, pH 5.5, and 2.5 volumes of -20°C ethanol. Following 15 min.
precipitation at -80°C the mixture was centrifuged (14K RPM, 15 min.) to pellet the
cDNA. The supernatant was vacuum aspirated and the resultant cDNA pellet was washed
with 100 pl of 2M ammonium acetate, pH 5.5, and 200 pl of -20°C 70% ethanol by
gentle agitation. Washed cDNA was pelleted (5 min. at 14K RPM); the supernatant was
aspirated and the pellet dried for 2 min. in a vacuum. The cDNA pellet was resuspended
in 50 pl of distilled water and purity and concentration of the double stranded cDNA
product was estimated by electrophoresis on a 0.8% agarose gel. This product provided
the 3’-terminal cDNA clone.

To complete cloning of RNA], five additional cDNA clones were primed by
Oligonucleotides designed to complement the desired upstream sequence. The primer
sequences were derived from the 5’-terminal nucleotide sequence of the appropriate 3 ’-
cDNA clone and are listed in Appendix B. Primers were designed to produce overlaps
between adjacent cDNA clones that contained a restriction endonuclease recognition site.
Oligonucleotides were synthesized by the Macromolecular Structure Facility, Department
of Biochemistry, Michigan State University, E. Lansing, MI. Synthesis of PRMV cDNA

16

with upstream primers was similar to that described for oligo (dT)-primed cDNA
synthesis.

Plasmid vector Bluescript KS- (Stratagene, La Jolla, CA) was linearized at the
polylinker EcoRV site. All restriction endonucleases and their appropriate incubation
buffers used in subsequent steps were obtained from Boehringer Mannheim Corporation
(Indianapolis, IN). Linearized plasmid (0.5 pg) was treated with 5 units of calf intestinal
phosphatase (Boehringer Mannheim) to prevent self-ligation of vector termini (Tabor,
1987). All cDNA clones were ligated into the EcoRV site of KS- using a vector/cDNA
ratio of 1:2 with 0.1 pg of vector. T4 DNA ligase (0.1U/pl), 1x T4 DNA ligase buffer
(10x: Tris-HC], 660 mM; MgC12, 50 mM; dithiothreitol, 10mM; ATP, 10mM; pH 7.5)
and dATP (lmM) were added to the vector/insert mixture and incubated overnight at
room temperature (22°C) in a 25 pl reaction volume. Ligation product was used to
transform E. coli DHSa ‘Max Efficiency’ (Life Technologies, Gaithersburg, MD) calcium
chloride competent cells; transformation mixture containing E. coli and the ligation
product was incubated on ice for 30 min., followed by 2 min. of heat shock at 37°C
(Hanahan, 1983). The entire transformation mixture (150 pl) was plated on solid 2xYT
agar media (1.6% w/v tryptone, 1.0% w/v yeast extract, 0.5% w/v sodium chloride and
15% w/v agar). Agar plates were amended with ampicillin to aid in selection of plasmid-
containing colonies (ampicillin, 50 pg/ml); blue/white colony screening of recombinant
plasmids was enabled by the presence of X-gal (0.004% w/v 5-bromo-4-chloro-3-indolyl-
B-D-galactoside, Boehringer Mannheim) and IPTG (20 pg/ml isopropyl-B-D-
thiogalactopyranosid, Boehringer Mannheim) in the agar media.

White E. coli colonies were selected from 2xYT plates and grown in 2 ml of
2xYT liquid with shaking at 37°C (350 RPM). Overnight cultures were collected in 1.5
ml Eppendorf tubes and centrifuged at 14K RPM in a bench top centrifuge to pellet cells
(Lee and Rasheed, 1990). The supernatant was aspirated and resuspended with 100 pl of
solution I (25mM Tris-Cl, pH 8.0; 10 mM EDTA; 50 mM glucose), vortexed thoroughly,

17

and the completely resuspended pellet was held at room temperature (22°C) for 5 min.
Next, 200 pl of freshly made solution I] (0.2 N NaOH, pH 5.5; 1% v/v sodium dodecyl
sulfate) was mixed into the solution by inverting the tube and the mixture was incubated
in an ice-water bath for 5 min. A 150 pl aliquot of 7.5 N ammonium acetate, pH 5.5, was
added and the mixture was placed on ice for 5 min. and centrifuged for 5 min. at 14K
RPM. The plasmid-containing supernatant was transferred to a fresh Eppendorf tube
containing 0.6 volume isopropanol and incubated for 10 min. at room temperature. This
solution was centrifuged at 14K RPM for 10 min. and the supernatant was aspirated. The
pellet was resuspended in 100 pl 2 N ammonium acetate, pH 5.5, and placed on ice for 5
min. Following centrifugation at 14K RPM for 5 min., the supernatant was transferred to
another Eppendorf tube containing 100 p] of isopropanol and held at room temperature
for 10 min. A final 10 min. centrifugation (14K RPM) pelleted the purified plasmid DNA
and the supernatant was aspirated. The pellet was dried in vacuum for 3 min. and
resuspended in 50 pl water. Resuspended, purified plasmid DNA containing cDNA
inserts were size selected by restriction endonuclease analysis.

To determine the size of each cDNA insert, 0.] pg of recombinant plasmid was
linearized with a restriction endonuclease (EcoRI, XbaI or Xhol) with a unique
recognition site within the vector polylinker. In a reaction volume of 20 pl, including 2 pl
manufacturer-supplied 10X incubation buffer, 0.1 pg DNA, 1.0 unit of restriction
endonuclease and water, the DNA template was digested for 3 hr at 37°C. Following
restriction digests, 2 pl of loading dye (30% glycerol, 0.25% bromophenol blue in water)
was added to the digestion mixture. The entire reaction was electrophoresed in a 1X TBE
running buffer with 0.] pg/ml ethidium bromide in a 0.8% agarose gel at 100 volts
(EC452 power supply, E-C Apparatus Corp., St. Petersburg, FL; Horizontal
Electrophoresis System, #Al Owl Scientific, Inc., Cambridge, MA) for approximately 3
hr. The gel was photographed and plasmid sizes were compared with 1 kilobase (kb)

18

double-stranded DNA ladder (Life Technologies, Gaithersburg, MD) and linearized KS-

plasmid. The cDNA clones with the largest inserts were selected for further analysis.

RNAl Origin of cDNA Clones Verified

The RNA] origin of each cDNA clone was confirmed by probing a PRMV
northern blot with a nucleotide sequence unique to each cDNA clone. One pg of purified
PRMV RNA was electrophoresed in a non-denaturing agarose gel (0.8% w/v) and
transferred to nylon membrane (Fisher Scientific, Pittsburgh, PA) according to Brown
(1993). One hundred pmol of Oligonucleotide primer RA42 (5’-
AAATCATCATCGATCTCAAC-3’), complementary to position 483 84857 near the 5’-
terminus of the 3’-most cDNA clone, was labeled with digoxigenin-l l-dUTP according
to manufacturer’s recommendations (3’-Oligonucleotide Tailing Kit, Genius System
version 2.0, Boehringer Mannheim). The RNA] origin of each upstream clone was
confirmed by probing a PRMV RNA blot with the digoxigenin-labeled synthetic
Oligonucleotide primer used for its synthesis. The product of the labeling reaction was an
Oligonucleotide with a 3’-tail containing multiple digoxigenin—l l-dUTP residues which
was then diluted in 10 ml northern pre-hybridization solution (5X SSC, 50% forrnamide
0.02% sodium dodecyl sulfate, 2% (w/v) blocking reagent (Boehringer Mannheim), and
20 mM sodium maleate, pH 7.5) to a concentration of 10 pmol probe/ml. The northern
blot was incubated for 2 hr at 37°C in northern pre-hybridization solution in a sealed
plastic bag. Northern pre-hybridization solution was discarded from the bag and replaced
with the dilute probe solution. At the end of a 6 hr hybridization at 37°C, dilute probe
was decanted and stored at -20°C. The treated membrane was washed twice, 5 min. per
wash, in 2X wash solution (2X SSC containing 0.1% SDS) and subsequently washed
twice with 0.5X wash (0.5X SSC containing 0.1% SDS), 15 min. per wash. All
membrane wasth were performed at room temperature (approximately 22°C). Following

washing, the membrane was incubated in 50 ml maleate buffer (100 mM maleic acid, 150

19

mM NaCl, pH 7.5) for 1 min. Maleate buffer was replaced with 50 ml of northern
blocking solution (10 mM sodium maleate containing 1 % (w/v) blocking reagent) in
which the membrane was incubated for 30 min. One pl (0.75 U) of anti-digoxigenin
alkaline phosphatase Fab fragments (Boehringer Mannheim) was added and the
membrane was incubated in this solution for 30 min. at room temperature. Treated
membrane was washed twice, 15 min. per wash, with 200 ml maleate buffer at room
temperature and equilibrated in 50 ml Genius buffer 3 (100 mM Tris-HCI, 100 mM NaCl,
50 mM MgC12, pH 9.5). The membrane was removed from this solution and placed on
blotter paper (roughly 2X membrane size), covered with approximately 1 ml Lumi-Phos
530 (Boehringer Mannheim), and wrapped in Film Wrap (Gordon Food Service, Grand
Rapids, MI). Treated membranes were exposed to X-OMAT autoradiograph film
(Eastman Kodak Inc., Rochester, NY) for approximately 30 min. Autoradiographs were
developed (model M7B RP X-OMAT Processor, Kodak) and analyzed.

Confirmation of the RNA] origin of each cDNA clone was followed by
nucleotide sequence analysis; the largest of the cDNA clones obtained from cDNA
synthesis with upstream Oligonucleotide primers was sequenced to verify its 3’-overlap
with the preceding clone. The location and nucleotide sequence of primers used for
cloning and northern analysis of each cDNA clone are listed in Appendix B.
Oligonucleotide probe preparation and northern analysis of the five upstream cDNA
clones was similar to that described above.

Exonuclease III Deletion Analysis

Exonuclease III (Boehringer Mannheim, Indianapolis, IN) was used to create a
series of deletions originating at each end of the seven fill] length cDNA inserts
(Henikoff, 1984). Varying the duration of exonuclease III (exo III) treatment and
subsequent treatment with mung bean nuclease (Life Technologies, Gaithersburg, MD)

generated a series of progressively smaller subclones (Maniatis et al., 1982).

20

Restriction endonucleases were used to generate 3'-overhangs adjacent to the
sequencing primer (either universal or reverse) site and 5'-overhangs adjacent to the insert
cDNA. For exo III deletion of the KS-/insert cDNA plus strand, ApaI and XhoI were used
to create 3-’ and 5’-overhangs respectively; Sac] and XhoI were used for similar
treatment of the opposite strand. Exo III treatment produced unidirectional deletions from
5'-overhangs. The linearized DNA was extracted once with buffered phenol, once with
phenol/chloroform/isoamyl alcohol (25/24/1 v/v/v), and ethanol precipitated (see above).
DNA was washed with -20°C 70% ethanol, dried in a vacuum and resuspended in water
for a final concentration of 0.1 pg/ pl.

The temperature and the time of exo III incubation were regulated to obtain a
deletion series on both strands of each cDNA clone (Henikoff, 1984). Exo III activity was
terminated by transferring equal volume aliquots of the digestion mixture to 1X mung
bean nuclease buffer (10X concentration: 0.3M sodium acetate, pH 5.0; 0.5 M sodium
chloride; 10 mM zinc chloride in 50% glycerol) and heating the mixture to 68°C for 15
min. Subsequent treatment with mung bean nuclease ensured blunt-ended termini of the
nested deletion mutants. Exo III-deleted plasmids were re-circularized overnight by
ligation with T4 DNA ligase at 14°C (Slatko et al., 1994). Competent DHSOL E. coli cells
were transformed with the ligation product and screened on 2xYT plates amended with
ampicillin, X-Gal, and IPTG, as described earlier.

Individual E. coli colonies containing exo III-deleted plasmids were selected from
2xYT selection plates and grown overnight in 2xYT with agitation (350 RPM) at 37°C.
Purification of plasmid DNA was similar to that described earlier. Three to 5 pl of the
DNA solution was linearized by restriction enzyme digestion. Restriction endonuclease
ClaI was used to linearize the series of subclones with exo III deletions generated from
the 5'-overhang of Xbal. Endonuclease XbaI was used to linearize eonII mutants
generated from the 5'-overhang of XhoI and reaction conditions for restriction

endonuclease digestion were described earlier. Linearized plasmids were electrophoresed

21

on an agarose gel 0.8% (w/v) and a series of consecutive deletions each differing by 150-
200 bases was selected for sequence analysis. Exo III clones were selected in this manner
to bring the entire cDNA fragment into the sequencing range of either universal or
reverse primers (Boehringer Mannheim) whose respective recognition sites are located
within the Bluescript KS- vector polylinker.

Nucleotide Sequence Analysis of cDNA

The cDNA clones of PRMV RNA] were completely sequenced in both directions
by the dideoxynucleotide chain-termination method of Sanger et al. (1977). DNA
templates sequenced included full length cDNA fragments as well as exo III deletion
subclones. DNA sequencing reactions were primed using either the universal or reverse
primers on the pBluescript KS- vector (Stratagene, La Jolla, CA). DNA sequencing
protocol with Sequenase (Sequenase Version 2.0, United States Biochemical, Cleveland,
OH) was adapted from manufacturer’s recommendations.

For each double stranded DNA template, 2 pg of DNA was combined with 0.]
volume of 2N NaOH and 0.2 volume of 1 mM EDTA, pH 8, in a 30 p] reaction volume.
The reaction was incubated for 30 min. at 37°C and ethanol precipitated using 0.] volume
sodium acetate and 3 volumes -20°C ethanol. Following a 15 min. incubation at -80°C,
the mixture was centrifuged at 14K RPM for 10 min. The pellet was washed in 70%
ethanol, dried and resuspended in 7 pl water.

One pl (0.5 pmol) of either the reverse or forward Oligonucleotide primer, as well
as 2p] of 5X Sequenase buffer (0.2M Tris-HCl, pH 7.5, 0.1 M MgClz, 0.25 M NaCl)
were combined with the denatured DNA solution. The tube containing the primer/DNA
solution was heated to 65°C for 2 min. and slowly cooled to room temperature in a water
bath (approx. 45 min.). Eppendorf tubes containing the annealed mixture were placed on
ice.

Sequenase was added to the DNA/primer hybrid solution in the presence of

deoxynucleotides (dNTPs), dithiothreitol (DTT) and 35$ dATP (Dupont, Boston , MA),

22

for DNA polymerization. Polymerization reaction mixture contained the manufacturer’s
recommended volume of 5X Sequenase labeling mixture, 7.5 pM dGTP, 7.5 pM dCTP,
and 7.5 pM dTTP, 5 pCi 35S-dATP and Sequenase diluted 1:10 (v/v) in accompanying
enzyme dilution buffer. Solution was mixed and incubated at room temperature (22°C)
for 5 min.

Termination reactions were performed in a V-bottom 96-well microwell plate
(VWR Scientific, Batavia, IL). Prior to the addition of the product of the labeled reaction
to 2.5 p] of the termination mixture, supplied dideoxynucleotide termination mixtures
were pre-warmed at 37°C for 2 min. Three and one half p1 of the labeling mixture were
added to each of the four wells in the microwell plate and incubated for 5 min. at 37°C.
Four pl of stop solution (95% forrnamide, 20 mM EDTA, 0.05% bromophenol blue, and
0.05% Xylene Cyanol) were added to each microwell and samples were denatured at 80°
C for 2 min. prior to electrophoresis.

Sequence reactions were electrophoresed on 8% acrylamide gels (41.5 cm x 37.0
cm). An 8% acrylamide gel contained 30 ml 20% acrylamide solution (96.5 g acrylamide,
3.35 g methylene-bis-acrylamide, 233.5 g urea in 500 ml 1X TBE); 45 ml 8 M urea; 75 pl
25% ammonium persulfate; and 75 pl N,N,N',N'-tetramethylethylenediamine (TEMED).
The 8% acrylamide gel polymerized between 0.25 mm spacers (approx. 1hr) and pre-run
in 1X TBE running buffer at 65 watts (W) for 30 min. (Fisher FB650 power supply,
Pittsburgh, PA; SE1500 Sequencer apparatus, Hoeffer, San Francisco, CA). Two and one
half pl of each sequence reaction sample was added per lane between the teeth of a shark
tooth comb (Hoeffer, San Francisco, CA) and electrophoresed at 65 W for approximately
2.5 hr.

The gel was removed from the sequence apparatus, immersed in fixer solution
(15% methanol, 5% acetic acid) for 20 min., transferred to 41.5 cm x 32 cm x 3 mm
chromatography paper (Whatrnan, Hillsboro, OR) and covered with film wrap. The

sequence gel was dried under vacuum at 80°C for 1hr (Vapor Trap, Vacuum Pump,

23

BioRad, San Francisco CA; Slab Gel Dryer, Hoeffer Scientific, San Francisco, CA) and
exposed to 43 cm x 35 cm autoradiograph film (Kodak X-OMAT AR) ovemight in an
autoradiograph cassette (Fisher FBXC 1417). Autoradiographs were developed in an
automatic film developer (Kodak RP X-OMAT Processor, model M78) and analyzed.

RNA Sequence Analysis

Primer extension was used to determine the nucleotide sequence of the 5’
terminus of RNA] after the method of Fang et al. (1995) using viral RNA template and
synthetic Oligonucleotide RA75 (5’-GACCAAATATTCCATCAC-3’) complementary to
RNA] nucleotide position 50-67. The 5’-terminal nucleotide of RNA] was verified using
terminal deoxynucleotidyl transferase (Allison et al., 1988).
Computer-Assisted Genome Analysis

Sequence data were analyzed using Genbank databases and a Genetics Computer
Group (GCG) software package (version 8.1) available through Silicon Graphics, Inc.
computer services at the Department of Biochemistry, Michigan State University, E.
Lansing, MI. The sequence data were assembled and manipulated through the SEQED
program. Restriction endonuclease recognition sites were verified using the MAP,
MAPPLOT, and MAPSORT programs (Devereaux et al., 1984). Sequence comparisons
utilized BESTFIT and GAP GCG programs (Devereaux et al., 1984). Parameters for
BESTFIT and GAP included a gap creation penalty of 5.0 and a gap extension penalty of
0.3. Statistical Significance of alignments was assessed by including a randomization
program (RAN) with GAP and BESTFIT. Ten randomized comparisons were made for
each pair of sequences by repeatedly shuffling one of the sequences and aligning it with
the non-randomized sequence. Similarity between sequences was deemed significant if it
exceeded the mean randomized similarity plus three standard deviations (Doolitle, 1981).
Viral sequences used for comparison to the PRMV RNA] sequence were obtained from

Genbank. Genbank accession numbers for viral sequences are included in Appendix A.

24

Multiple sequence alignments utilized the PILEUP programs (GCG) with a gap weight of
3.0 and a length weight of 0.1. Consensus sequences were generated using files created
by the PILEUP program, followed by analysis with PRETTY (plurality of either 4.0 or
6.0; vote weight and threshold of 1.0, each)(Devereaux et al., 1984). Secondary structure
predictions of proteins used PLOTSTRUCTURE from GCG (Devereaux et al., 1984).
Optimal secondary structures for the 3'-UTR of RNA 1 were predicted with GCG
FOLDRNA (Jaeger et al., 1989). Output files from F OLDRNA were used to plot RNA
secondary structures with GCG SQUIGGLES (Devereaux et al., 1984).

RESULTS AND DISCUSSION
Propagation and Purification of PRMV

Purification of PRMV yielded 0.2-0.3 mg virus/100g infected C. quinoa. A
tracing of a typical sucrose density gradient fractionation for PRMV is shown (F ig.1a)
with top (T), middle (M), and bottom (B) components present in varying quantities. In
similar tracings, Dias and Allen (1980) observed that B component, RNA], frequently
was present in higher proportion than M component, RNA2. An absorbence ratio Ema/E280
of 1.8 for pooled M and B components compared well to published values by Dias and
Allen (1980) which ranged from 1.7 to 1.9.

25

 

(a) (b) (C)

Fig 1. Purification of PRMV and confirmation of the RNA] origin of an oligo (dT)-
derived cDNA clone. The ultraviolet absorbance scanning pattern following 0-30% linear
logarithmic sucrose density gradient centrifugation of purified PRMV is shown (a). The
middle (M) and bottom (B) components were collected separately from top (T)
component (empty capsid protein) and further purified by phenol extraction to liberate
RNA] and RNA2 from components B and M, respectively. In (b), 1 pg of purified
PRMV RNA was electrophoresed on a non-denaturing 0.8% ( w/v) agarose gel, stained
with ethidium bromide and photographed. The RNA gel from (b) was northern blotted to
nylon membrane and probed with digoxigenin-dUTP-labeled Oligonucleotide primer
RA42, the same primer used for PRMV cDNA synthesis of the 3’-terminal cDNA clone.
Probe construction and northern analysis is detailed in the text. A photograph of the
digoxigenin/RA42-probed northern blot is shown (c).

26

Purified PRMV RNA separated electrophoretically as two distinct bands
corresponding to RNA] and RNA2 with estimated sizes of 8 and 7 kb respectively
(F ig.1b) and matched RNA] and 2 sizes predicted by Dias and Allen (1980). PRMV
RNA] and RNA2 sizes are comparable to those of subgroup II nepovirus TomRSV (8214
and 7273 nucleotides, respectively). The RNA2 was substantially larger than the 5.4 kb
cutoff for a subgroup I nepovirus (Sanfacon, 1995). The RNA] origin of each cDNA
clone was confirmed by probing a PRMV RNA northern blot with the digoxigenin-
labeled Oligonucleotide primer used for its synthesis. For example, the Oligonucleotide
complementary to the 5’-terminus of clone 5-9 was used to prime synthesis of cDNA
clone 2-1. Hybridization of this Oligonucleotide to only RNA] (Fig. 1c) evidenced the
RNA] origin of both clones.

cDNA Synthesis and Sequencing of PRMV RNAl

A series of six overlapping cDNA clones was selected for sequencing PRMV
RNA]. The molecular cloning strategy is illustrated in Fig. 2. The cDNA clones are
designated 5-9, 2.1, 50-3.9, 52-4.2, 68-2.90, and 70.20 and contained inserts of the
following sizes, respectively: 3120, 1202, 1408, 1131, 882, 190. Optimal cDNA synthesis
(i.e. largest cDNA product) occurred when the RNA template was heated at 70°C for 1
min, then placed immediately on ice prior to addition of Amersham's first strand cDNA
synthesis reaction components. A series of eonII deletion mutants was created for both
directions in all six full length cDNA clones. At 37°C, nucleotide digestion rates at
susceptible 5'—ends were approximately 250 bases/min. The deletion series for clone 5-9
illustrates the technique (Fig.3). Each exo III subclone chosen for nucleotide sequencing

was 150-300 nucleotides shorter than the preceding clone and together spanned the entire

27

cDNA insert. The cDNA sequence analysis and assembly indicated that collectively,
these inserts represent 99.6% of the PRMV RNA] genome. The sequence of the 5'-
terminal region of RNA] was determined by direct dideoxynucleotide chain termination
sequencing of the genomic RNA template using AMV reverse transcriptase. Direct RNA
sequencing indicated that the S’-tenninal cDNA clone contained all but the 5’-tenninal
44 nucleotides of RNA]. The 5’-tennina] nucleotide (U) was identified with TdTase
treatment. The complete unique nucleotide sequence of the cDNA of PRMV RNA] is
7977 nucleotides (Fig.4). A polyadenylated tract of 30-60 ATP residues is located at the

3'-tenninus of the RNA] nucleotide sequence.

 

 

 

 

 

5']: 1 AAA 3'
TTT 3'
5-9 (3120 bp)
RA42
2.1 (1202 bp)
RA50
50-3.9 (1408 bp)
RA52
52-4.2 (1131 bp)
RA68
68-2.90 (882 bp)
__RA70
70.20 (190 bp)
5’___RA75

primer extension (44 bp)

Fig.2. Cloning strategy for PRMV RNA]. The red rectangle above RNA] represents the
major ORF present in the virion sense. Oligonucleotide primers oligo (dT), RA42, RASO,
RA52, RA68, and RA70 (italicized) were used to generate a consecutive series of RNA]
cDNA clones; respective primer nucleotide sequences are identified in Appendix B. The
primer RA75 was used in primer extension analysis to determine the 5’-terminal 44
nucleotides of RNA]. Overlapping cDNA clones (boldface) are shown with their
respective lengths in base pairs (bp); cDNA clones are positioned relative to RNA].

28

 

Fig.3. Exonuclease III (eonII) digestion of PRMV RNA] cDNA clone 5-9. The 5-9
cDNA contains 3120 unique nucleotides. EonII was used to create a series of single-
stranded nested deletions from full-length 5-9 cDNA insert. Mung bean nuclease
treatment degraded the remaining single strand and ensured blunt-ended termini which
were ligated to re-circularize exo III-treated 5-9 deletion plasmids. Plasmids were
linearized with 5 units of XbaI and subjected to electrophoresis in 0.8% agarose gel.
Linearized deletion mutants are arranged in size on the agarose gels relative to the 1 kb
ladder (on both sides of each gel); the exo III deletion series continues from the upper gel
to the lower gel. Sizes of plasmids ranged from approximately 3 kilobase-pair (kb)
fiagments (little or none of the 5-9 insert remaining) to vector (Bluescript KS-) plus
complete insert (approximately 6 kb, total). From these clones, a series of nested eonII
deletion mutants (approximately 100-300 nucleotides apart in length) was selected for
sequence analysis of the entire 5-9 cDNA.

61

121

181

241

301

361

421

481

541

601

661

721

781

841

901

961

1021

29

TATGAAAAATCACTAATCTATTACCTTCTTAACTATTGCTGTTTCTTTTGTGATGGAATA
M E Y

TTTGGACTATCTTCCTGCGCGAACAAAATGGGTGGCCATAGTGCCAAAAGCTGTCCTGGA
L D Y L P A R T K W V A I V P K A V L E

AGCCACCAGGATAGCTAATGTCCTGCTAGCAAAGCCTGCCAACTTTGCTATTTCTTTTTT
A T R I A N V L L A K P A N F A I S F L

GGCTCAGGGTGCCTCCCTGAAGCCACGTTCTGTAGCTCTGGCGGTTGCAATGGGTTATTG
A Q G A S L K P R S V A L A V A M G Y C

CCACTGGCCCAGAGTTCTGCATCTATACTCCGAAGGAGTTCCCCTAACTTGGGGAGATGC
H W P R V L H L Y S E G V P L T W G D A

ACCACCGGTGCCCCTTTTATTAAGGGCCCTGGCTAAGATGGAATCTGGGCTATATGCCGA
P P V P L L L R A L A K M E S G L Y A D

TGGGAGAGGAACTGGCTTTTTGCCAGTTCAAGAGGCAAGTGCCTCACCTGCGGGCCGCCA
G R G T G F L P V Q E A S A S P A G R Q

GCAAGCCGTCGAAGAGAAAAAGGCTCTTTACAGAGCCAAAGGTGCTGCAGCAACAGCATC
Q A V E E K K A L Y R A K G A A A T A S

GAAAAAGGCTGCTGCTAGAGCAGCCTTGGAAGCCCGCCGTTCCTGTGGCGGACAAGGAAG
K K A A A R A A L E A R R S C G G Q G R

AGCGCCTAAAGTACTGAAAAAGAAGGCCACCAAGCGGGTGGTCACTGCTGCACTGGCAAC
A P K V L K K K A T K R V V T A A L A T

AGTCAAAGAGAGCCAACGCTTGGCTCTATTTTTCCTTTTTCCTCTTCTCTCTTTTCCTCT
V K E S Q R L A L F F L F P L L S F P L

CCCCCTCTCCTCCGTGAAAAGGGGGTTCCTTTTAATCCTCCTCAACGGGAGGATTTTCTT
P L S S V K R G F L L I L L N G R I F F

TCCTCTCCTCCTCCTCCTTTGGTGGCTTTGTAAAAGCCCACTTTCTTATGGGTCCTATTG
P L L L L L W W L C K S P L S Y G S Y C

TGGACCTTGGGCCTCTCTTGGCCCTATTTTAGAAACTGGAGCTCCAGGAGCTCAACGGGC
G P W A S L G P I L E T G A P G A Q R A

ACTTTTTGCCGCTATTCGAAAACTCCCTCTCTCTACTTTTCACGAGAGAGTTCTCTTCCG
L F A A I R K L P L S T F H E R V L F R

GGATACTCAAGTTGCAGTGTCCCAACTTTTCGTTTTGTATCCCTCTGTACATATACTTGG
D T Q V A V S Q L F V L Y P S V H I L G

GGATCTTAATTCTTTTTTCCTTCAGGATTGCCGTGGCATGCGTTTAGCACTGGAAAGTGC
D L N S F F L Q D C R G M R L A L E S A

TCGACGTATTGCAGATGGTATTTCCTCCATTCTTCCTCAGCATCGGGTTGTACATACTTT
R R I A D G I S S I L P Q H R V V H T F

23

43

63

83

103

123

143

163

183

203

223

243

263

283

303

323

343

1081

1141

1201

1261

1321

1381

1441

1501

1561

1621

1681

1741

1801

1861

1921

1981

2041

30

Fig. 4 (cont'd).

TCTTGATGCAGTGAAGAAGGTTGGTTCCTATATTTCAGGAGCTGCCTCTGCAGTTAAAAG
LDAVKKVGSYISGAASAVKS363

TAAAGTTTCTAACTTTACCTCTTCACTCTTTGATTCTATTTTGGACAAATGTAAATATTG
K V S N F T S S L F D S I L D K C K Y C 383

TTTCATGTCCACTTTTTCTCCCTTCTTGGCTTCTCTGCAATCAGCCAAAGCTGAAATTGA
F M S T F S P F L A S L Q S A K A E I E 403

AAAAITTTGGCAAA3ATTGCATGAGTTGGGCTAGGAACTTGTGGAGTAAGGCTCACCTTGC
K F W Q N C M S W A R N L W S K A H L A 423

TCTACAAGCTCTTGGCCTTTATGCCATTTGGGCTTTAGTGTTGACAATCCTTTGTGGGAT
L Q A L G L Y A I W A L V L T I L C G I 443

TGTTTATTTATTAGAATCTCTTTTTATTACTGCGGGGGTAATAGGCTCCCATGGTATTAT
V Y L L E S L F I T A G V I G S H G I I 463

TCTCTCTATTTTTCTTTCCGTGGTTATGGCTGCAGCTGGATTCACTATCTTTACCGTTGG
L S I F L S V V M A A A G F T I F T V G 483

TAAAGAAAGTGCTCAAATGATTCGGACAATGCGCGAGGGTATTCTCATGATGGTAATACC
K E S A Q M I R T M R E G I L M M V I P 503

CGATGATGCCGCTAAGTCGATTGGAGGTAGAACCAGGTACCCAACAGTGCATAGTCTTTT
D D A A K S I G G R T R Y P T V H S L F 523

TGATTTGGCTATGGCACCTGTAAATTTTTTGGAGTCCATTGCTAGTGGACTTTCTCTTTT
D L A M A P V N F L E S I A S G L S L F 543

TTCCACCTCCTCAATTACAGTTTTAGGTAAATTGGGGAATTCTTTGGAAGGTATTCGGAA
s T s s I T v L G K L G N s :L fifiagfi r: R. K 563

AGGCTATAATTGCCTGACCGATTTTATTTCCATTTTCTTTGAGAAGATGGGAGGTCTATG
G Y N C L T D F I S I F F E K M G G L W 583

GGAAGGTATTTCTGGTAAGCAGACCACCTTCTTTCGAGATCTCACCACGGCTGTTAAGAT
E G I S G K Q T T F F R D L T T A V K I 603

TAATATCAGTTCGTGGACCCAGGATGCTCGTCGGTTAATTGAATACCACGAGATGGCTGG
N I S S W T Q D A R R L I E Y H E M A G 623

TACCCTGGATAAGTTCGAGTACGAGAAAGTTCGCCTCTTATTTATCAAGGGAAGAATAGT
T L D K F E Y E K V R L L F I K G R I V 643

CGATACTGCCAATAAGGGCAGGCAATCCCATACCAGCAACCAATTTTTGAGAGTTGTTGG
D T A N K G R Q S H T S N Q F L R V V G 663

TTCTTTGTTGACAGATTTGAGGGAGGTGCGTGCTAAGTGCGCTCGTTCCCTCCGTTTTGA
S L L T D L R E V R A K C A R S L R F D 683

2101

2161

2221

2281

2341

24101

22461

22521

2581

2641

2701

2761

2821

2881

2941

3001

3061

31

Fig. 4 (cont’d).

TGGTTGGCGTCGTCAACCTTTTTGGGTTTATATTTTCGGTGCATCACAGTGTGGTAAGTC
G W R R Q P F W V Y I F G A S Q C G K S

CACTTTAGCCAACTATTTGTGCCCCCTTTTATTGGCACATATGGGTTGGGATGCTCATGA
T L A N Y L C P L L L A H M G W D A H D

CGTCTACTCCAAGGATCCCACAGAAGGATACTGGAGTGGATACTACCAGCAGAAATGTTT
v Y s K D P T E G Y w s G Y Y Q Q K C L

AAAGATGGATGATCTTTCTGCGGTAGTGCCTAAGCAGGTATCTCCTCTTGAGCAACAGCT
K M D D L S A V V P K Q V S P L E Q Q L

CATTCCCCTTATTTCTACGGAGGAGAAAATGGTATCTGCAGCTGAGATTAATGGCAAGGG
I P L I S T E E K M V S A A E I N G K G

AATTCAGTTTTTATCTGAATTGGTCATATCCAGCTCGAATGTGAATGATGCACCTACATC
I Q F L S E L V I S S S N V N D A P T S

GTGTGAGATTCTTGATCCTGAAGCATATCGCCTAAGGAGAAAGGTTCTCTTACGCTGTAG
C E I L D P E A Y R L R R K V L L R C R

ACGTGCAGCGACTTACCAGCATGATGAAGCTGGGAACAAGACTGAGGTAGTTGATGCTGA
R A A T Y Q H D E A G N K T E V V D A E

GGGAAATATTGTGTGTCGACAATATGATCCCAGTGATGCATTAGCTTGCACTGAAGTCAG
G N I V C R Q Y D P S D A L A C T E V S

TGGCTACATGCCAATTCTTGTACTCGGTTCCAGGACCAGCAGGACTGTGGCACCCGCCCA
G Y M P I L V L G S R T S R T V A P A H

CTCCACCATTCCTCTCATTAAGGATGCCATGGATGCGCATTTCTTAGTAGAGGATGCCAA
S T I P L I K D A M D A H F L V E D A K

AAGAGAAGCGTGGGTGCAACAAACAAATATGCACTCGCGAACTGGAGCTGAGGTCTCCAG
R E A W V Q Q T N M H S R T G A E V S S

CTATTTGCAATCCTTAGTGTGTGCACTGGGCTCTTATAAAGCCATTCAGCGCTCTTCCGA
Y L Q S L V C A L G S Y K A I Q R S S D

CGTTTCAGATGCGGGGGAGCGTAAATTTTTGGTAGCTGTTGATGGAACTATTTATTCCAT
V S D A G E R K F L V A V D G T I Y S I

CGATTCTTTAGGTAGGGCGACCAAGGAAGCGGCAGACGCGTACGACAATGTTGAGGCATT
D S L G R A T K E A A D A Y D N V E A L

GGAGTCCACTACCCTTCTGCAATATCGCCTTGATTTTCGACAGGTTAGGGAACATTCCCT

703

723

743

763

783

803

823

843

863

883

903

923

943

963

983

E S T T L L Q Y R L D F R Q V R E H S L 1003

CTTAACCAATGATGGTAGTTTCCATTCCTCTATGGTGAGGGATCTACTAAGGATATCTTG
L T N D G S F H S S M V R D L L R I

S C 1023

3121

3181

3241

3301

3361

3421

3481

3541

3601

3661

3721

3781

3841

3901

3961

4021

4081

32

Fig. 4 (cont'd).

TGAAGAAGCTTGTGTGGTCTCTGTTGATAAAATCAGTAGGGATTCCAAACAACTTCACAG
E E A C V V S V D K I S R D S K Q L H R 1043

GGACTTGTGGAGTGAGTTAAAGCTTGCGAACGATTTTTTTCCGCGTTTCTCAAAAGCTCT
D L W S E L K L A N D F F P R F S K A L 1063

TAACCAACTGCGCGACCAACCACATTTTAAGGTTGATGTGCAGTCAGTTTCCTTCAGCAT
N Q L R D Q P H F K V D V Q S V S F S I 1083

ATGGCTGATTTTAGAGATGCCATTGTTGATAATAGGCAAAAATTCTTCTTTTTTTCAGAG
W L I L E M P L L I I G K N S S F F Q S 1103

CTATCTTTTGGTGGGGGCTTGCATCATGGAGTTTTTTGTCCTTGATAAAACCTTCCTTAG
Y L L V G A C I M E F F V L D K T F L S 1123

TGGATCTGTGGGATTTGGGAGTGCTTTGGCTCTCAAAAACCAATTGGATGTACATAGCTC
G S V G F G S A L A L K N Q L D V H S S 1143

TGTTGCTTCTTCTGGGTCTATTGCAACTCAGTCATATGCACGGAGCATACCAATTGTATG
v A s s G s I A T :§,]E: Y’ A R s I p I v w 1163

 

GGCAAAAGTAGCTCGCTATGCCAATGTCCATTCACAGGTTGAGGAGTCGAGTCATTTCAA
A K v A R Y A N v H s Q v E ”137* s H F N 1183

 

TTTTTTTGAAGATGGCCTGGCGCACCTTTTAGTTAGATTGGTGGGTACTAGTGGTCTTTG
F F E D G L A H L L v R L v G T s G L C 1203

TGAGACTGCTATTTTGTTTGGTTCCAGAGCTATTGCTCTGTGTGCCCATCAGATACGCAT
E T A I L F G S R A I A L C A H Q I R M 1223

GTTCCCAGATCACGACCGGGTTACTGTGCATTATTTGGACAAAGCCCGGATTGCAAAGTG
F P D H D R V T V H Y L D K A R I A K C 1243

CTTTCCTATGACATGGCATTGGGTAAATGCTATTGAGGAAAAAGATACGGAGGTGTGCGT
F P M T W H W V N A I E E K D T E V C V 1263

TTATAGGGACGACCAATTAACGCCTCTCCCTGTCTATCCAGATTCCATTTATCTTAAGGG
Y R D D Q L T P L P V Y P D S I Y L K G 1283

TGAGACACAATTACCGTCTGCAGTTAATATAAATCGAGTTTCCATAAAGAAGCGAAGATA
E T Q L P S A V N I N R V S I K K R R Y 1303

TTATGAGGACGCTTCTTTGACGCCTGATGAACGATTACTGGATGGTGAAAGTCCAATTAT
Y E D A S L T P D E R L L D G E S P I I 1323

ACGTTCGTGGAGTAACGTCGCTGCCTTGAGTACTAGTGTGCAAACAATTTCAAACCCTGC
R S W S N V A A L S T S V Q T I S N P A 1343

ACCTGGTATTGCATACAAGCGTGATTTAAATCGCTACCTGACATCCTCGTATGCTGCGGG
P G I A Y K R D L N R Y L T S S Y A A G 1363

4141

4201

4261

4321

4381

4441

4501

4561

4621

4681

4741

4801

4861

4921

4981

5041

5101

33

Fig. 4 (cont’d).

GGTGCATGATTGTGGTGGTTTAATATCCATTTTGCACCAAGGACGACGCAAGGTTGTGGG
V H D C G G L I S I L H Q G R R K V V G 1383

GTTGCACGTAGCAGGAACTAGAGTTGGACATCTTTTTTCGTCCACTATTAGTTTCTTGCC
L H V A G T R V G H L F S S T I S F L P 1403

ACACGGCAATTTTTCCGATGTTCATTCTCAGGGAGATTTTTTTATACCTGAGGTAGGTGA
H G N F s D v H s ’[[i D F F I P E v G D 1423

   

TCGAGAGGCTGGTTATGAGAAAATAGGATTTATTGATAATTCAGCCAAAGCCCACATACT
R E A G Y E K I G F I D N S A K A H I L 1443

AGTACCACTACCCAATTGGGCAGGGTACCTACTAATTTTGAAACCCCTTCAACTTTTGAT
V P L P N W A G Y L L I L K P L Q L L M 1463

GAGGAGGAGGAAAGAAAATTTCGTCGATGCTGGTGAAACATTTGAAATAAAAGAGCCAGC
R R R K E N F V D A G E T F E I K E P A 1483

AATTCTTTCAAAAAAAGATCCTCGTCTTGAGGATCCTGATTCTTTTGACCCATTGCGGAC
I L S K K D P R L E D P D S F D P L R T 1503

TGGGATGAGCAAATTTGCAAATCCTATGTCTGTACTTGATGAAGCTTTGTTGGAAGCAGT
G M S K F A N P M S V L D E A L L E A V 1523

TTGTGAGGACATTTTTACCACTTGGTATGATGCCCTCCCAGCTGTTACTGACAACCAGGG
C E D I F T T W Y D A L P A V T D N Q G 1543

GAATGTTTCTCGTATTTTATTAGAGAAAACTTCTTTAGATATAGCATTGAATGGAGTTCC
N V S R I L L E K T S L D I A L N G V P 1563

AGGAGATGCTTATCTTGAGCCAATGAAACTTGACACTTCTGAGGGTTATCCCCATTGTGT
G D A Y L E P M K L D T S E G Y P H C V 1583

CAGGCGAGGTCCTGGTGAGAGTGGAAAGCGTCGATTTGTTGAGATCGATGATGATTTCCA
R R G P G E S G K R R F V E I D D D F H 1603

TTTTTCTTTGAAGCCTGATACCGATGTTTTTAAAAACTATCAGGCGCTTTCTGGGACTAT
F S L K P D T D V F K N Y Q A L S G T I 1623

TTCTCAACAAGTCCCAGTCCTCAATTGCGTAGAGTGCTTGAAAGATGAATGTCTCAAGAA
S Q Q V P V L N C V E C L K D E C L K K 1643

AAGGAAAGTGGCTACCCCACGCCTTTTTGATGTGATGCCTTTTGAGCACAATATTCTCTT
R K V A T P R L F D V M P F E H N I L L 1663

GCGGGAATATTTTTTGAATTTTTCCGCTTTTATTCAGGCTAACCGGATTTATCTTTCCGC
R E Y F L N F S A F I Q A N R I Y L S A 1683

TTGTGTTGGAACCAATCCTTATTCTCGAGAGTGGACTACACTCTATGATAGATTAGCAGA
C V G T N P Y S R E W T T L Y D R L A E 1703

5161

5221

5281

5341

5401

S461

5521

5581

5641

5701

5761

5821

5881

5941

6001

6061

6121

34

Fig. 4 (cont’d).

GTATTCCGATACTGGCTTGAACTGTGATTATTCCAAATTTGATGGTTTAATTTCCCATCA
Y S D T G L N C D Y S K F D G L I S H Q 1723

AATATCTCGTGGATGGCTGCAACCATCAACCGTGTTTTTAGAGACGGTGAGGAAGCAAAT
I S R G W L Q P S T V F L E T V R K Q I 1743

TCTGCGCGTAGGAAATCTCCTACTCATGTTCATTGGTCGCCGCTCTATTTGTGGTAGACA
L R V G N L L L M F I G R R S I C G R Q 1763

AGTGTATATGGTTAGGGGCGGTATGCCTTCTGGCTGTGCTTTGACAGTCGTTATAAATAG
V Y M V R G G M P S G C A L T V V I N S 1783

TATTTTTAATGAAATTTTAATTAGGTATGTTTATAGGAAGGTTACACCCGCACCTGCTTG
I F N E I L I R Y V Y R K V T P A P A C 1803

TAATTTTTTTAACAAGTATGTGCGCCTCATGGTGTACGGTGACGACAATCTTCTCACCAT
N F F N K Y V R L M V Y G D D N L L T I 1823

TAAAGAGGAGGTAATTCCTTTCTTTGATGGTCCAGTGATCAAGAGGGAGATGGCTAGTGT
K E E V I P F F D G P V I K R E M A S V 1843

TGGTATCACCATTACGGATGGCACTGACAAGAGTTCATTGACTCTTGAGAGGAAACCTCT
G I T I T D G T D K S S L T L E R K P L 1863

AGCATCTCTTGAATTTTTGAAGAGAGGTTTTAGAGTGCAGGAGAATGGGCTTGTTGTTGC
A S L E F L K R G F R V Q E N G L V V A 1883

CCCTTTAGATAAGACTTCAATGTACACGCGGCTTTTTTATCTACCGCTGGCATTGATGGC
P L D K T S M Y T R L F Y L P L A L M A 1903

ATTTATCCCTGGATATTTTTCGAAGGGAAATGTCAAGAGTTTTTTGGAGGAGATTGTTTT
F I P G Y F S K G N V K S F L E E I V L 1923

GCACCCCAATCACCGCCGAGAATTTTACCGGGTGCGTAATTTTTATGTGAGCAAGGCCCC
H P N H R R E F Y R V R N F Y V S K A P 1943

ACATTGGGGGATATCTTGCCTACATATGGCGCTGCTGTTGATTTTCATTATCGGCAGCAG
H W G I S C L H M A L L L I F I I G S R 1963

ACGACCAATACCCCCTACCAGACGCAACGGCTTTTTGAACGCGTCACATGGAGGGGAACA
R P I P P T R R N G F L N A S H G G E H 1983

TAAAATGATGGCTGGACAGGATTGCCAGACCAGACCATTTGGGGTAACAAGTCGTCTAGC
K M M A G Q D C Q T R P F G V T S R L A 2003

TATCTTGGTAGTAGAACCCAAGTTCCAAGGGGTAGTCAACACTTTATTGTGGCGTGCGGT
I L V V E P K F Q G V V N T L L W R A V 2023

TTCGTCCCTTCGTGGGGGTGAGCGTGGCATTGCATTAAAGTGGAGACTGCCTCTGGAACG
S S L R G G E R G I A L K W R L P L E R 2043

6181

6241

6301

6361

6421

6481

6541

6601

6661

6721

6781

6841

6901

6961

7021

7081

7141

7201

7261

7321

7381

7441

7501

35

Fig. 4 (cont’d).

GGTGTCTTACCTTAACTCAAACGTGGTTAATAGTTTCAGCCTTCACCACGAAACGAGCGA
V S Y L N S N V V N S F S L H H E T S D 2063

CTCTTTTTTGAAGGACTTACATGAGGGATGTCACTTGTATTTAGGTTCGAGATGTACCCT
S F L K D L H E G C H L Y L G S R C T L 2083

TATTACATGGGTGGTGTGCATTGCAGCAGAATTTGCTAAGGCCCAGGGGTTGAGCACATC
I T W V V C I A A E F A K A Q G L S T S 2103

CAGTGTTATAGCTCTGTTTGAGGAGTATAAACCCAGAAAAGGGGGGGATATAGCTCCCCT
S V I A L F E E Y K P R K G G D I A P L 2123

TTTAGCTGAGCGCTCCTATAAGAGGTTCGCTCAAAGACCAATATTTGATATGTCAAGTAT
L A E R S Y K R F A Q R P I F D M S S I 2143

TAAGCAGCATCTAGCTGCTTCCTAAGCGCAGGGGGTCTCTTAGCGCCAGTTTCTAGTCCT
K Q H L A A S * 2150

GTAGGCTAGAGGTCTTGTGGGCCTAACCCACATCCAAGAGGTTGTCATCAATTAGCATTT
TACCTTCGGGTTGAAGATGTGAATGGAAGAGTGATGCCCTTCCAGACCTCTCCTTTGGAG
AACCATGAGTCAACACATGGTCTTGGAGGTCACAGTTCCGATTCTAACTGTGTGCTTTTA
CCAATTTTAAAGAAATGGAAGAGTAGGAGATGCTCTTGTGTGATGAGTGTGTAGATACCT
TCATGTTGCTCATTACAACACATTAATGAATTCATTAATAGTTATGTGTTTGTGGTGGCA
TGTTGGGTGTGTTTATCTATACATGATTTGAAAATCTCAAATGACTAGGGAGAAAGATCC
TGTAGGTGTGGAAATCACCCGCTTTGTTGGAGAGCCAATTCCAACTCTTTGCTACCTTCA
AGAAAGGAGATTGTACTGGTGAAATTCCAGTCCTTATATTTATTGCTTTCTAGGACTTGA
GTCTTTTAGTTTTGCAATCTTGCAGAGTTGCTTTAGTAGATCTGCACGTGAAGTGCGTCA
ACGTTATGGCGTAATAGTGTGTTGTGTCTCCCACACAATAAGTAATGAGACAACGCTGGG
TTAGATCCCGGGAGGGTGGTTCCCTCTGACAACATTTGTGCTTTAGTAGATAAGCACCCT
TTTCTTCCAGTCTTACTGAGGCAGGATATCAAAAGTAGGCTTGCAGATTATAGATTTGTG
GTTAACTGATTAGACTTTGAGTAATTGTAAGAACTATCCATAAGATTATCTTGGATTGTT
TAATACTCTCATGCTTATCAGCTCTTTCCATGAATACTACTGCGATACCGCTGGCGTATT
CTAGTTTTAAAGACGGTATGCTGCTTCCAGCATATAAAAGCAGATATAGTAGCCATAAGC
ATGATGGTTAAGCTAAATTCACCGATGAGTCGGAGGAGCCATCATGTGTACAATAGGGGG

AAGCCCCTATGGCAAATTATCTGTATAGGAGCCCTTTGCTGGGGTTAAAAGCTTAAGGTT

36

7561 TAGTGTAACACAACATTGGGTGTACTCAAGAGCGTGTGGGGTGGCACCCACGTGCTTGGA
7621 TGAGGTCCGGAAATGAATACCGGGGGATAATTAATCCCAGCTCAGGCACTAAGCTGACTT
7681 TCATGGAAGTGTCCATGACGCATTTTAAGGTAGGTTTTAGACATAACCTCCCGGGATGGA
7741 AGTGATTACCATTTCGTTATTCGTTATTAGTTTCTTGCAACTATGATGAGGGGACCACAT
7801 CTTAAGCGATGTTGCTGCATTGCGTACCTATGGTCATCTGGTTAGTTGTCGTATTTTCTT
7861 TTAGCTTTTGTGGCGACAGATGAGGTTTGACTCCTTTTCCTTGACTCTTGACCTAAGTTG

7921 GACACAAAAATATGGTCTTTTGACTTTCAATAGAGTCGATGAAAATGTCTGCATCAC-pO1y(A)

Fig. 4. Nucleotide sequence of the cDNA of PRMV genomic RNA]. The predicted
amino acid sequence of the large ORF of the plus sense (virion sense) RNA is shown
below the nucleotide sequence. Nucleotide and amino acid positions are numbered to the
left and right of the nucleotide sequence, respectively. The termination codon at the 3'
end of the RNA] ORF is marked with an asterisk (*). Binding sites for Oligonucleotides
used in cDNA synthesis of RNA] are highlighted. The predicted polyprotein sequence
was searched for dipeptides E/S, E/G, Q/G, Q/M, and Q/S, which are common proteinase
cleavage recognition sites within como—, poty-, picoma-, and tomato ringspot nepovirus
(TomRSV) polyproteins. By analogy with confirmed dipeptide sites in cowpea mosaic
comovirus B component and putative sites in TomRSV RNA], putative peptide cleavage
sites of PRMV RNAl-encoded polyprotein are identified in gray.

Nepoviruses contain a high U content in their untranslated regions and PRMV
RNA] shares this characteristic (5’-UTR, 46%; 3’-UTR, 32%). These values are more
similar to those reported for TomRSV (44.2% and 31.2%. respectively), than to those of
subgroup I nepoviruses. Subgroup I nepoviruses TBRV, GCMV, GFLV, TRSV, as well
as comovirus CPMV (Lomonosoff and Shanks, 1983), have U content ranging from 40-
48% for both the 5’-UTR and the 3’-UTR (Rott et al., 1991). Interestingly, the 3’-UTR U
content for TomRSV (Sanfacon, 1995) and PRMV approaches the subgroup I level if
only their extreme 3’-tennini are considered (3’-] 10 bp, 44.2% U for TomRSV; 3’-150
bp, 38.4% U for PRMV). Dias and Allen (1980) reported a ribonucleotide composition
(mole percentage) for PRMV RNA] of 23.6 (G), 24.1 (A), 30.9 (U) and 19.9 (C) and

nucleotide sequence analysis of RNA] revealed similar values: 23.8 (G), 24.6 (A), 31.3

37

(U), 20.3 (C). The M, of RNA] as calculated from the nucleotide sequence is 2.6 x 10" as
estimated by PAGE (Dias and Allen, 1980).

Computer analysis of both the plus and minus strands of the genomic RNA]
nucleotide sequence identified several putative open reading frames (ORFs) including a
single large ORF containing 6450 nucleotides. An initiation codon (AUG) was identified
beginning at position 53 and a termination codon at position 6503. Analysis of this ORF
indicated that it is capable of encoding a polypeptide of 2150 amino acids with a
predicted molecular weight of 240 kD (Fig.4). Analysis of the remaining two reading
frames of the plus strand and the three reading frames of the minus strand revealed ORFs
of less than 400 nucleotides.

Analysis of Untranslated Regions

PRMV RNA] 5’- and 3'-UTRs are 53 and 1474 nucleotides, respectively.
Computer prediction of RNA] 3’-UTR secondary structures of the 500 3'-termina]
nucleotides revealed extensive secondary structure including stemloops, bulges, interior
and bifurcation loops (Fig.5). Comparison of nepovirus 3'-UTR nucleotide sequences by
pairwise alignment reveals a low and statistically insignificant nucleotide sequence
identity in this region with a few notable exceptions as follows. Abbreviations of virus
names and references to sequence numeration are identified in Appendix A.

(1) 5'-UUUCUUUU-3' octamer: This octamer was detected at positions 42, 171,
and 7855 of PRMV RNA]. Serghini et a1. (1990) found this octamer at variable distances
from the poly (A) tail in the 3’-UTR of the RNA2 of GCMV, GFLV-F13, and TBRV.
This sequence was also shown to be present once in the RNA2 5’-UTR of GFLV and
SLRSV, respectively, twice in that of TomRSV, and four times in that of TBRV (Kreiah
et al., 1994). Kreiah et al. (1994) also reported that this sequence was present at two
locations in the coding region of RSV RNA2 (positions 2458 and 3478) but not in the

untranslated regions.

38

(2) 5’-GAAAA(A)U-3’: This sequence was first identified by Fuchs et al. (1989)
for GFLV, TBRV, and GCMV, and occupies identical positions at the 5’-terminus of
nepovirus genomic RNAs. All nepovirus RNAs whose entire sequence has been
determined, except for RRSV RNA2, contain this sequence initiating within 6 nucleotides
of the 5’ terminus. The sequence was found in PRMV RNA] at position 4; TomRSV
RNA] and RNA2 (nt 6); TBRV RNA] and RNA2 (nt 3); ArMV RNA2 (nt 3); GCMV
RNA] and RNA2 (nt 4); GFLV RNA] and RNA2 (nt3). Satellite RNA may be associated
with nepovirus infection (Sanfacon 1995). A search for the GAAAA(A)U sequence in
nepovirus satellite RNA revealed that this sequence is located at the 5’-terminus of the
large (>1kb) satRNA but not in that of the small (<0.5kb) satRNA: ArMV 1104 bp
satRNA (lilac isolate) at position 4; CYMV 1165 bp sat RNA at nt 3; TBRV 1375 bp
satRNA (nt 3) and GFLV satRNA (F13 strain) at position 4. It is noteworthy that the
large satRNA molecules have predicted coding regions unlike the small satRNA. The
GAAAAU sequence was also found in variable locations within the coding regions (CR)
and/or the 3’-UTR of several nepoviruses including PRMV (four times in CR at positions
1259, 2365, 4339, and 4454; twice in the 3’-UTR at position 6870 and 7961), TomRSV
RNA] (six times in CR at positions 1571, 2479, 3464, 3698, 4143, and 5363), TomRSV
RNA2 (twice in CR at positions 1467 and 5701), TBRV RNA] (10 times in CR at
positions 1182, 1497, 2008, 3216, 3654, 3726, 4980, 5908, and 6640), TBRV RNA2
(four times in CR at positions 603, 1686, 2335 and 4203), ArMV RNA2U (three times in
CR at positions 2022, 2188, and 3584), GCMV RNA] (9 times in CR at positions 724,
3279, 3555, 5028, 5111, 5603, 5809, 6542, and 6652), GCMV RNA2 (twice in CR at
positions 944 and 4067), GFLV RNA] (seven times in CR at positions 1722, 4218, 5543,
5690, 5813, 5961 and 7064), GFLV RNA2 (once in CR at position 3194). The subgroup
II nepoviruses PRMV, BBLMV, CYMV, and CLRV are distinct from subgroup I
nepoviruses in having the GAAAAU sequence located within the 3’-UTR; the GAAAAU
sequence is present twice in the BBLMV 3’-UTR of both RNA] and RNA2 (positions

39

2418, 2488 in RNA2 and positions 1243 and 1314 of RNA]), and once each in the 3’-
UTR RNA] and RNA2 of CLRV (positions 694 and 720, respectively). PRMV RNA]
and TomRSV RNA] each contain this sequence in seven locations, as identified above.
Although the biological function of this sequence is unknown, the frequency of its
occurrence in the nepovirus genome far exceeds the random probability of its appearance
(1/4°=] in 4096 chances for GAAAAU).

(3) A l7-nucleotide consensus sequence (5’-GGACACAAAAAGAUUUU-3’)
was identified near (but not at) the 3’-UTR of nepoviruses by Fuchs et al. (1989).
Serghini et al. (1990) noted the presence of this sequence in TBRV, GCMV, and GFLV
and Buckley et al. (1993) added ArMV to list of nepoviruses with this sequence. This
sequence was not found in TomRSV, TRSV, or RRSV RNAS (Buckley et al., 1993),
however, a similar sequence was identified near the 3’-termini of PRMV RNA] (14/ 17
nucleotides conserved) starting at position 7920, and also near the 3’-termini of BBLMV
(15/17 nucleotides conserved) as reported by Bacher et al.(1994).

(4) 5’-AAAAGC-3’ or 5’-AAAAAGC-3’ immediately preceding the 3’-poly (A)
tail of nepovirus genomic RNAs was first identified by Rott et al. (1991) for TomRSV,
TBRV and GCMV. This sequence was identified in SLRSV-H RNA2 at a position 3
bases removed from the 3’-terminus and also in RRSV RNA2 commencing at position
3574 (Kreiah et al., 1994). BBLMV RNA] and RNA2 and PRMV RNA] may now be
added to the list of nepoviruses whose 3’-UTR contains this sequence (BBLMV RNA],
position 901; BBLMV RNA2, position 2076). This sequence is found in two locations in
the 3’-UTR of PRMV RNA] commencing at nucleotide 7416 and 7547, respectively, and
positioned 56] and 430 nucleotides from the 3’-terminus, respectively. In the genomic
RNAS of TomRSV, TBRV, GCMV, and SLRSV- H (RNA2 only) this sequence occurs at
the extreme 3’-terminus; as with PRMV RNAl, the AAAAGC sequence in RRSV
(RNA2 only), BBLMV RNA] and RNA2 occurred at variable distances from the 3’-
terminus (354, 1007, 1006, respectively).

40

Three other 3’-UTR nucleotide sequences are conserved among some nepoviruses
but were not detected within the PRMV RNA] sequence. These sequences include a 35
nucleotide region reported for TRSV, TomRSV and RRSV (Buckley et al, 1993); a
stretch of 14 nucleotides identified in SLRSV-H RNA2, GFLV, and ArMV (Kreiah et
al.,1994) and a stretch of 30 nucleotides shared by SLRSV-H RNA2 and TBRV (Kreiah
et al, 1994).

The biological significance of these nucleotide consensus sequences is unknown.
However, it is possible that these nucleotide sequences may be involved in polymerase

recognition or packaging signal functions (Buckley et al, 1993).

41

-
'Q
O

f

.

O
"o
a——-——---—--

Fig. 5. Computer-predicted 3’ UTR secondary structure of the 500 3’-termina]
nucleotides of PRMV RNA] commencing at nucleotide position 7477. Optimal
secondary structures for the 3'-UTR of RNA 1 were predicted with the GCG FOLDRNA
program (Zuker, 1989). Output files from FOLDRNA were used to plot RNA secondary
structures with the GCG SQUIGGLES program.

42

PRMV RNA] Polyprotein Analysis

As expected, the predicted amino acid sequence of the PRMV RNA] polyprotein
shares highest identity with that of subgroup II nepovirus TomRSV (29.8%) and to a
lesser, yet significant, degree with subgroup I nepoviruses (26.7% TBRV, 27.4% GCMV,
and 27.9% GFLV), CPMV B (24.9%). PRMV RNA] amino acid identity with that of
tobacco etch potyvirus (TEV), which also produces a polyprotein is insignificant (Table
1). Predicted RNA] polyprotein sequence was examined for motifs characteristic of a
proteinase cofactor (ProCF), an NTP-binding protein, a viral proteinase and an RNA-
dependent RNA polymerase (RdRp). Alignment of motifs within the polyprotein of
PRMV RNA], TomRSV, GCMV, GFLV, CPMV is shown in Fig. 6. Processing of the
PRMV polyprotein will be described later.
Proteinase Cofactor

A conserved amino acid sequence, F -x27-W-x,,-L-x2,-L-x-E (xn refers to the
number of amino acid residues between conserved residues), is located near the N-
terrninus of the PRMV RNA] polyprotein sequence beginning at amino acid residue 384
(Fig.6a). This region of conserved amino acid residues was previously identified in other
nepovirus and comovirus polyprotein sequences and a proteinase cofactor function was
suggested (Ritzenthaler et al., 1991; Rott et al., 1995). The N-terminal 32K protein of the
CPMV B polyprotein contains this sequence and has been demonstrated to function as a
cofactor for the CPMV 24K proteinase (V os et al., 1988; Peters et al., 1992). PRMV
ProCF amino acid sequence resembles that of TomRSV (24.9% identity) more so than
subgroup I nepoviruses (20.9-22.8%) or CPMV B (16.4%) (Table 1). An N-terminal
consensus sequence detected for TomRSV, TBRV, and GCMV (Rott et al., 1995) was
not found in the predicted amino acid sequence of PRMV RNA].
NTP-Binding Protein

An amino acid motif characteristic of NTP-binding proteins is located

downstream of the PRMV putative proteinase cofactor protein, beginning at amino acid

43

residue 696 (Fig.6b). The highly conserved 'A' and 'B' sites typical of the NTP-binding
protein are G-x4-GKS/T and DD/E, respectively (Gorbalenya and Koonin, 1989;
Gorbalenya et al., 1989). These two sites are thought to be important for anchoring the
replication complex to the lipid membrane (Rott, et al., 1995). PRMV shares the highest
level of amino acid identity with subgroup II TomRSV (27.5%) and lesser identity with
subgroup I nepoviruses (23.0% to 25.4%), CPMV B, and TEV, 21.9% and 14.2%,
respectively (Table 1).
VPg

Nepoviruses, as with many other plant and animal viruses, contain a genome-
]inked protein (V Pg) covalently linked to the 5’-terminus of the genomic RNA (Harrison
and Barker, 1978; Matthews, 1992). Picomaviral VPg molecules are thought to play a
role as a primer for the replication of both plus and minus strand RNA (Matthews, 1992).
A covalently linked VPg at the 5'-ends of PRMV RNAs 1 and 2 was reported (Martelli,
1975). The VPg amino acid sequence is located between the NTP-binding protein and the
proteinase for CPMV and GFLV, 4 kD and 2.9 kD, respectively (Goldbach and
Rezehnan, 1983; Pinck et al., 1991). Tentative location and size of the VPg of TomRSV
(2.7 kD), TBRV (2.3 kD), and GFLV (2.9 kD) have been reported (Rott et al., 1995;
Greif et al., 1988; Ritzenhaler et al., 1991). The putative cleavage pattern of the PRMV
RNA] polyprotein (see below) suggests a 2.9 kD VPg between amino acid positions
1154 and 1179. As with all other nepoviruses, the PRMV VPg is on RNA] between the
NTP-binding protein coding region and the proteinase. Comparison between nepovirus
VPg amino acid sequences revealed no significant identity with the exception of TBRV,

which compared very well with the corresponding VPg sequence in GCMV (76.5%).

Proteinase
Viruses that utilize a polyprotein expression strategy encode a proteolytic enzyme.

A motif characteristic of cysteine proteinases is found in a region beginning at amino acid

44

residue 1219, H-x4o-E-x1M-CG-xs-G-xs-G-x-H-xz-G. The residues H, E, and C (italicized)
form the putative catalytic triad of the proteinase shown in Fig. 6 (Bazan and Fletterick,
1989; Gorbalenya et al., 1989; Hammerle et al., 1991; Dessens and Lomonosoff, 1992;
Margis and Pinck, 1992). The histidine residue (bold face H), is conserved among
PRMV, TomRSV, como-, poty-, and picomavirus proteinases but is replaced by a leucine
in proteases of subgroup I nepoviruses (Rott et al., 1995). Referring to this position as the
“substrate-binding pocket” of the polio 3C proteinase, Bazan and Fletterick (1988)
suggested that the His residue at this position may recognize and hydrogen-bond to the
amino acid residue immediately upstream (-1 position) of the dipeptide cleavage site
before cleaving the polyprotein. The cleavage site specificity of nepovirus subgroup I
proteinases differs from that of picoma-, potyviruses, como-, and subgroup II nepoviruses
TomRSV (Sanfacon et al., 1995), and PRMV. The difference between subgroup I and
subgroup II nepovirus proteinase cleavage site specificity may be due to the replacement
of the His residue with a Leu at the substrate-binding pocket (Bazan and Fletterick, 1988;
Demangeat et al., 1992; Ritzenthaler et al.,l991, Rott et al., 1995). Presence of the His
residue in the PRMV polyprotein suggests that the cleavage sites for maturation of the
PRMV polyprotein may be similar to those of picoma-, poty-, and comoviruses (see
below). Comparison between putative proteinase of PRMV and other members of the
picomavirus superfarnily revealed that PRMV shares a low yet significant level of amino
acid sequence identity with the proteinase of subgroup II TomRSV (27.7%). Proteinase
amino acid sequence of subgroup I nepoviruses and other picoma-like viruses compared
less favorably (19.1-24.3%) (Table 1).
RNA-dependent RNA polymerase

A conserved GDD amino acid motif is characteristic of RNA dependent RNA
polymerases (RdRp) (Argos, 1988). This motif was located in the polyprotein of PRMV
RNA] at amino acid position 1816 (Fig.4). The GDD motif as well as the flanking amino

acids (1710-1821) share extensive sequence identity with other members of the picoma-

45

like species (Fig.6d). Sequence identity between PRMV RdRp amino acid sequence and
other nepoviruses (33%-36%) was higher than that of TEV (22.6%) (Table 1). The
putative active processing site of the RdRp includes a hydrophobic region of 15 amino
acid residues flanking the GDD sequence (Argos, 1988) which is also found in the
putative RdRp of PRMV.

PRMV
TomRSV
TBRV
GCMV
GFLV
CPMV B

PRMV
TomRSV
TBRV
GCMV
GFLV
CPMV
TEV

I’D

PRMV
TomRSV
TBRV
GCMV
GFLV
CPMV B
TEV

(d)
PRMV
TomRSV
TBRV
GCMV
GFLV
CPMV B
TEV

Con

Fig.6. Alignment of the PRMV RNAl-encoded amino acid motifs identifying putative
proteinase cofactor (a), NTP-binding protein (b), proteinase (c), and RNA dependent
RNA polymerase (d) with other picoma-like viruses. Viral abbreviations are defined in
the text. An asterisk (”') designates a plurality of at least four identical amino acids
among the viruses compared. A plurality of at least four hydrophobic amino acids (F, Y,
W, I, L, V, M) is indicated (A) and a plurality of all or all but one amino acid is
highlighted in yellow. Consensus (con) sequence is shown below the aligned sequences.

4

6

F X17 IEKFWQNCMSW X11 L X21 LLE
F X17 IEELWRWSLEW X11 L X21 FAB
F X17 IEVMIKKVKDW X11 L X21 LLE
F X17 VEVLIARVKSW X11 L X21 LIE
F X17 LKKIQEKLSEW X11 L X21 LVE
F X17 LSQLWDKIVQW X11 L X21 LVE
* * i t * * *
F W L L E
WVYIFGASQCGKSTLANY X32 KCLKMDDLS
WVYLYGGPRCGKSLFAQS X32 AICCVDDLS
WIYLFGQRHCGKSNFMAT X31 TFFHVDDLS
WIYLWGPSHCGKSNFMDV X31 TIMEIDDLS
WVYIFGASQSGKTTIANS X33 ACVKVDDFY
TIFFQGKSRTGKSLIMSQ X32 PFVLMDDFA
DFLVRGAVGSGKSTGLPY X71 DFVIIDECH
* * t * +*** * ****
G GKS/T DD/E
"A site" "B site"
H X40 E X101 AGVHGCGGLISILHQGRRKVVGLHVAG
H X46 E X96 NSPEDCGALLVAHLEGGYKIIGMHVAG
H X38 E X86 SRNDDCGMIILCQIKGKMRVVGMLVAG
H X38 E x86 SRNNDCGMLLTCQLSGKMKVVGMLVAG
H X44 E X91 AKKYDCGALAVAVIQGIPKVIAMLSAG
H X35 E X86 TIPEDCGSLVIAHIGGKHKIVGVHVAG
H X34 E X64 TKDGQCGSPLVSTRDG-—FIVGIHSAS
* * 44* + * ********4
H CG G G H/LG
NCDYSKFDGLI X51 PSGCALTVVINS X28 LMVYGDDNLL
NCDYSRFDGLL X49 PSGCALTVIINS X28 LIVYGDDNLI
NCDYSGFDGLL X54 PSGFALTVVVNS X28 LLVYGDDNLI
NCDYSGFDGLL X54 PSGCALTVVMNS X28 LLVYGDDNLI
YCDYKAFDGLI X50 PSGCALTVVLNS X28 LITYGDDNVF
CCDYSSFDGLL X51 PSGFPMTVIVNS X28 LVTYGDDNLI
DADGSQFDSSL X52 NSGQPSTVVDNT X22 YYVNGDDLLI
***** ***** *t* ***+**** *********4
D FD G TV NS/T GDD

Consensus sequence alignment is adapted from Rott et al.(l995).

47

Table 1. Comparison of RNAl-encoded amino acid sequences for seven members of the
picoma-virus supergroup including the nepoviruses PRMV, TomRSV, TBRV, GCMV,
GFLV, and cowpea mosaic comovirus and tobacco etch potyvirus. These viruses employ
a polyprotein strategy for genome expression and mature proteins are post-translationally
cleaved. From the polyprotein precursor (A), mature products include a proteinase
cofactor (B), a nucleotide (NTP)-binding protein (C), a genome-linked protein, or VPg“, a
proteinase (D), and a polymerase (E). Amino acid sequences of individual proteins, as
well as complete polyproteins of each virus were compared using GCG Bestfit or Gap
programs (Devereaux etal., 1984). The amino acid location of each protein relative to the
polyprotein N-terminus is shown at the right of the table. Values are expressed as
percentage amino acid homology and amino acid identity to the right and to the left of the
darkened cells, respectively.

A) RQLX PRMV TomRSV TBRV GCMV GFLV CPMV TEV EQstn
PRMV 1-2150
TomRSV 1-2197
TBRV 1-2266
GCMV l-2253
GFLV 1-2284
CPMV 1-1866
TEV 1-2791
3) P_CE

PRMV 1-559
TomRSV 1-620
TBRV 1-565
GCMV l-460
GFLV 1-417
CPMV 1-326
TEV" **
C) NIB

PRMV 560-1 153
TomRSV 621-1212
TBRV 566-121 1
GCMV 461-1 182
GFLV 417-1217
CPMV 327-919
TEV 1 163-1796
D) ERQ

PRMV 1 179-1413
TomRSV 1237-1465
TBRV 1233-1440
GCMV 1219-1428
GFLV 1241-1460
CPMV 948-1 155
TEV 1850-2279

 

48

Table 1 (cont’d).

E) EQL PRMV TomRSV TBRV GCMV GFLV CPMV TEV RQsit'LQn
PRMV 1414-2150
TomRSV 1466-2197
TBRV 1441-2266
GCMV 1429-2253
GFLV 1461-2284
CPMV 1156-1866
TEV 2280-2791

 

" Comparisons between VPg amino acid sequences are not shown; only TBRV and
GCMV VPg showed significant amino acid identity (76.5%).

* insignificant sequence homology or identity. Significance was assessed by shuffling
one of the pair of sequences being compared repeatedly (10 times) and aligning it with
the non-randomized sequence using GCG (GAP or BESTFIT with randomizing
parameter, Devereaux et al., 1984). Values were deemed significant if they exceeded the
mean randomized comparison plus 3 standard deviations (Doolittle, 1981).

** not present

Processing of the PRMV RNA] Polyprotein

As mentioned in the proteinase section, the histidine residue is conserved in the
putative active site of the proteinase of como-, poty-, picomaviruses and the subgroup II
nepoviruses PRMV and TomRSV but is replaced by a leucine in proteinases of subgroup
I nepoviruses. This suggests that the dipeptide cleavage site specificity of PRMV
proteinase is more similar to the aforementioned viruses than subgroup I viruses TBRV,
GCMV, and GFLV (Hans and Sanfacon, 1995; Grief et al., 1988; LeGall et al., 1989;
Ritzenhaler et al., 1991; Margis et al., 1991, 1994; Hemmer et al., 1995). The known
dipeptide cleavage sites for maturation of polyproteins of como-, poty- and
picomaviruses is E/G, E/S, Q/G, Q/S, and Q/M. (Wellink et al., 1986; Wellink and Van

Karnmen, 1986; Hellen et al., 1989; Palmenberg, 1990). A search for these sites in the

49

PRMV RNAl-encoded polyprotein and subsequent alignment of the proposed cleavage
products to TomRSV and CPMV B revealed a conservation in both order and size of the
putative translation products. Until direct protein sequencing of the mature PRMV
proteins is accomplished, assignment of cleavage sites are tentative. The proposed

genomic strategy for PRMV RNA] is shown (Fig.7).

 

 

 

 

 

 

 

 

E/G Q/S E/S Q/G
Cl I I I
ProCF NTP VPg Pro RdRp
61k 67k 2.9k 26k 83k

Fig. 7. Genomic organization of PRMV RNA]. Large rectangles represent the
polyprotein expressed from the major open reading frame of plus sense RNA].
Conserved amino acid motifs are indicated by uniquely colored boxes positioned at the
relative location of the motif within each protein. Putative proteins encoded by PRMV
RNA] are abbreviated as follows: putative proteinase cofactor (ProCF); NTP-binding
protein (NTP); proteinase (Pro); RNA-dependent RNA polymerase (RdRp) and genome-
linked protein (VPg). Known cleavage sites of CPMV B and putative sites in TomRSV
RNA] aided in identification of potential cleavage sites in PRMV. Picoma-like
proteinase recognition sites are E/G, E/S, Q/G, Q/S, and OM.

Putative RNA] cleavage sites include an E/G dipeptide at positions 559-560
between the putative N-termina] proteinase cofactor and NTP-binding protein. A Q/S and
an E/S site are located between the NTP-binding and putative proteinase at amino acid
positions 1153-1154 and 1178-1179, respectively. The region between these two
cleavage sites is 25 amino acids in length, comparing to 24 or 27 amino acids for
TomRSV (two sites are proposed by Rott et al. (1995) for potential cleavage at the C-
terminus of TomRSV VPg) and 28 amino acids corresponding to the CPMV B-encoded

VPg. A possible cleavage site between the PRMV-encoded RdRp and proteinase is Q/G

50

acid position 1413-1414, which aligns well with the Q/G site in CPMV and the Q/M site
in TomRSV. Corresponding sites in TBRV, GCMV and GFLV are Q/S, Q/I, and R/G,
respectively.

The proposed cleavage sites for the PRMV RNAl-encoded polyprotein result in
mature polypeptide cleavage products which are comparable in size with those proposed
for other sequenced nepoviruses (Fig.8). Sequentially from the N-terrninus of the
polyprotein, putative PRMV protein products from the RNA] polyprotein include a 61
kD proteinase cofactor; 67 kD NTP-binding protein; 2.9 kD VPg; 26kD proteinase and an
83 kD polymerase. Comparable putative proteins in the TomRSV RNAl-encoded
polyprotein are 65 kD, 66kD, 2.7kD, 25 kD, and 82 kD in size, respectively (Rott et al.,
1995). A comparison of the genomic strategies of PRMV RNA] with TomRSV, GFLV
and CPMV, including the location and sizes of mature polypeptides is shown in Fig.8.
Putative cleavage sites for PRMV, TomRSV and GFLV are also included. The known

location and identity of dipeptide cleavage sites for CPMV are shown.

51

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PRMV RNA]
E/G Q/S E/S Q/G
El I I I
ProCF NTP VPg Pro RdRp
61k 67k 2.9k 26k 83k
TomRSV RNA]
Q/ S Q/ S Q/ S Q/ M
E] I I I
ProCF NTP VPg Pro RdRp
65k 66k 2.7k 25k 82k
GFLV RNA]
C/S C/S G/E R/G
Cl I I I
NTP VPg Pro RdRp
45k 85k 2.9k 24k 92k
CPMV B RNA
Q/S Q/S Q/M Q/G
C] I I I
ProCF NTP VPg Pro RdRp
32k 58k 4k 24k 87k

Fig.8. Comparison of the genomic strategy of PRMV RNA] with other members of the
picomavirus superfamily. Large rectangles represent polyproteins expressed from the
major open reading frame of the plus sense RNA and noncoding sequences are
represented by a horizontal line. Conserved amino acid motifs are indicated by uniquely
colored boxes positioned in the relative location of the motif within each protein.
Putative proteins encoded by each genome are abbreviated as follows: putative proteinase
cofactor (ProCF); NTP-binding protein (NTP); proteinase (Pro); RNA-dependent RNA
polymerase (RdRp); and the genome-linked protein (VPg). The known dipeptide
cleavage sites of CPMV B are shown and were used in identifying potential cleavage
sites in TomRSV and in PRMV. Proteinase recognition sites common to poty-, como-,
and picomaviruses include E/G, E/S, Q/G, Q/S, and Q/M.

52

Summary and Conclusions

The complete nucleotide sequence of PRMV RNA] isolated from Michigan
‘Concord’ grapevine has been determined. cDNA clones representing 99.6% of RNA]
were obtained and the cDNA sequence, as well as direct RNA sequencing analysis of the
remaining RNA sequence revealed an RNA Species of 7977 nucleotides, excluding a 3’-
polyadenylated tail of variable length. RNA] 3’-and 5’-untranslated regions are 52 and
1474 nucleotides, respectively. The nucleotide sequence of the genomic RNA] and
RNA2 of all nepoviruses contains a single long open reading frame ORF (Sanfacon,
1995) and is also found in PRMV RNA]. Analysis revealed a single ORF of 6450
nucleotides initiating at nucleotide 53 and terminating at nucleotide 6503. This coding
region encodes 2150 amino acid residues with a coding capacity of 240 kD.

Analysis of the untranslated regions of RNA] revealed several features common
among nepoviruses. PRMV is similar to other subgroup II nepoviruses TomRSV, CLRV,
and BBLMV in having a very long 3’-UTR sequence. The 3’-UTR for these viruses are
1474, 1543, 1500, and 1392 nucleotides, respectively, contrasted with the subgroup I
nepoviruses 3’-UTR which ranges from 198 nucleotides (ArMV) to 301 nucleotides
(TBRV) to 583 nucleotides (TRSV). Evidence demonstrating the precise role of the long
3’-UTR in subgroup II nepoviruses is lacking. Nucleotide sequence identity between
nepovirus 5’- and 3’- UTRs is limited to a few short consensus sequences and it is likely
that some of these conserved sequences have biological significance such as replicase
recognition or packaging signals as they occur at a far higher frequency than that
predicted by random probability.

Comparison of the predicted PRMV RNA] amino acid sequence to that of other
nepoviruses revealed, as expected, that PRMV was most similar to subgroup II TomRSV,
and less so with subgroup I nepoviruses such as TBRV, GFLV, GCMV. Therefore,
bipartite subdivision of the nepovirus group by Francki et al. (1985) is supported by

direct sequence comparison. Analysis of the predicted amino acid sequence of PRMV

53

RNA] uncovered motifs characteristic of a replicase, a proteinase, an NTP-binding
protein and a proteinase cofactor. The order and identity of these motifs within the
PRMV polyprotein aligns well with that of subgroup I nepoviruses and CPMV B, and
especially with that of subgroup II TomRSV (F ig.8). In addition, putative mature proteins
of PRMV share the highest level of amino acid sequence identity with their counterparts
in TomRSV (Table 1).

The cleavage site specificity of subgroup I proteinases differs from that of
subgroup II nepoviruses, perhaps due to a replacement of a Leu residue with a His in the
putative substrate-binding pockets of subgroup II proteinases. Como-, poty-, and animal
picomaviruses, whose well-characterized cysteine proteinases resemble subgroup II
nepovirus proteinases, including those of TomRSV and PRMV, in having a His at this
position, cleave the dipeptides Glu/Gly, Glu/Ser, Gln/Gly, Gln/Ser, and Gln/Met. This
cleavage pattern is distinct from that of subgroup I nepovirus proteinases, whose targets
include Cys/Ala, Cys/Ser, Gly/Glu, Arg/Ala, Arg/Gly and.Lys/Ala (Sanfacon 1995). It is
likely, therefore, that the PRMV proteinase is more related to that of subgroup II
TomRSV and other picoma-like viruses than to that of nepovirus subgroup 1.

Sequence analysis of RNA] confirms PRMV as a member of the subgroup of
nepoviruses including TomRSV and further distinguishes the nepovirus subgroups.
Portions of the RNA] sequence, in conjunction with classical breeding and selection for
resistant cultivars, may provide ‘Concord’ vineyards with PRMV resistance. Obtaining
genetically engineered grapevines expressing a portion of the PRMV RNA] genome will

further our attempt to understand the mechanism of pathogen-derived resistance.

APPENDICES

APPENDIX A

Appendix A

Species of the Nepovirus genus (family Comoviridae) both confirmed (A) and tentative
(B). Viral names and abbreviations are compiled from Goldbach et. al (1995). Nucleotide
sequence information, including Genebank accession numbers, was assembled using the
UW GCG Stringsearch program (Devereaux, J., Haeberli, P., and Smithies, O., 1984).

Nepovirus subgrouping was adapted from Francki et al. (1985).

A. Confinneihlepnximsfinccies l. Subgroup I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Virus Abbreviation Sequence Description GCG Access Code
Arabis mosaic virus ArMV (4 sequences) RNA2 3’ terminal D10086
region (2406 bp) 5/92
capsid protein gene X55460
(1515 bp) 1/91
polyprotein P2-U (3852 X81814
bp) 3/95
polyprotein P2-L (3712 X81815
bp) 3/95
Arabis mosaic virus sArMV (2 sequences) complete satellite M21212
satellite RNA genome (300 bp) 7/89
satellite RNA (1104 bp) D00664
3/91
Arracacha virus A AVA na na
Artichoke Italian latent AILV na na
virus
Cassava American latent CsALV na na
virus
Cacao necrosis virus CN V na na
Crimson clover latent CCLV na na
virus
Cycas necrotic stunt CNSV na na
virus
Grapevine chrome GCMV (2 sequences) RNA2 (4441 bp) 9/93 X15163
mosaic virus
RNA] (7212 bp) 9/93 X15346
Grapevine fanleaf virus GFLV (6 sequences) RNA] (7342 bp) 4/94 D00915
RNA2 (3774 bp) 9/93 X16907
VPg (84 bp) 1/94 838553
RNA2 deletion mutant U11770
(50] bp) 10/94
capsid protein, partial U] 1768
(1515 bp) 10/94
capsid protein, X60775
complete (2305 bp)
10/91
Grapevine fanleaf virus sGFLV (1 sequence) complete sequence D00442
satellite RNA (1114 bp) 3/91

 

54

 

55

Appendix A (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Grapevine Tunisian GTRSV na na
ringspot virus
Mulberry ringspot virus MRV na na
Olive latent ringspot OLRSV (2 sequences) RNA3 (2438 bp) 3/95 X76993
virus
RNA4 (2078 bp) 3/95 X77115
Potato black ringspot PBRSV na na
virus
Raspberry ringspot virus RRSV (1 sequence) RNA2 (3928 bp) 2/93 8460]]
Tobacco ringspot virus TRSV (4 sequences) self-cleavage consensus A13898
region (52 bp) 10/94
mutant D-5] self- A13899
cleavage consensus
region (63 bp) 10/94
complete capsid L09205
protein gene (2018 bp)
9/94
mRNA (360 bp) 12/90 M17439
Tobacco ringspot virus sTRSV (14 sequences) satellite RNA (359 bp) M14879
satellite RNA 7/89
satellite autolytic M31515
junction (7] bp) 12/90
various satellite $63883; $63888;
genomic RNA $63895; $63896;
mutations 863897; S6390];
$63903; $63904;
863907; $63908;
$63910; S6391]
Tomato black ring virus TBRV (8 sequences) RNA] (7362 bp) 10/94 000322
RNA2 strain S (4662 X04062
bp) 9/93
RNA] strain C 3’ X05304
terminus (166 bp) 7/89
RNA2 strain C 3’ X05305
terminus (15] bp) 7/89
RNA 2 strain G 3’ X05306
terminus (100 bp) 7/89
RNA] strain A 3’ X05307
terminus (163 bp) 7/89
RNA2 strain A 3’ X05308
terminus (127 bp)
1 1/87
RNA2 strain ED (4618 X80831
bp) 8/94
Tomato black ring virus sTBRV (7 sequences) sRNA (1375 bp) 9/93 X00978
satellite RNA
isolate C sRNA (1374 D00142; X05689

 

 

bp) 2/91; 7/91

 

 

 

56

Appendix A (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tomato black ring isolate E sRNA (1372 D00143; X05688
satellite RNA (cont’d) hp) 2/9]; 7/91 I
isolate L sRNA (1376 000144; X05687
I bp) 2/91; 7/91 I
SubgrcuullNeuQYinrses
Artichoke yellow AYRV na na
ringspot virus
Blueberry leaf mottle BBLMV (2 sequences) RNA2 3’ terminus U20621
virus (3082 hp) 5/95
RNA] 3’ terminus U20622
(1908 bp) 5/95
Cassava green mottle CGMV na na
virus
Chen'y leaf roll virus CLRV (6 sequences) RNA2 birch isolate 12 863537
3’ terminus (1920 bp)
7/91
RNA] 3’terminus 884124
(1743 bp) 1/94
RNA2 3’terminus 884125
(1805 bp) 1/94
R25 3’ terminus (1182 S84126
bp) 1/94
RNA2 3’ terminus U24694
(1565 bp) 5/95
genomic RNA walnut 234265
isolate (1588 bp) 11/94
Chicory yellow mottle CYMV na na
virus
Chicory yellow mottle sCYMV (4 sequences) T isolate small satellite D00685
virus satellite RNA RNA (457 bp) 6/9]
C isolate large satellite D00686
RNA (1165 bp) 6/91
sRNA S] (457 bp) 7/94 000721
sCYMV sRNA L1 (1145 bp) D00722
12/91
Grapevine Bulgarian GBLV na na
latent virus
Hibiscus latent ringspot HLRV na na
virus
Luceme Australian LALV na na
latent virus
Myrolaban latent MLRSV na na
ringspot virus

 

 

 

 

 

 

57

Appendix A (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Peach rosette mosaic PRMV (1 sequence) RNA] (see Fig.4 na
virus above)
Potato virus U PVU na na
Tomato ringspot virus TomRSV (5 sequences) RNA] (8114 bp) 9/94 L19655
RNA2 (7273 bp) 4/94 D12477
RNA] 3’ non-coding M27936
region (1546 bp) 2/90
RNA2 3’ non-coding M27935
region (1550 bp) 2/90
RNA] 5’ terminus M73822
(1140 bp) 12/91
B. I . 1 I . S .
Arracacha virus B AVB na na
Artichoke vein banding AVBV na na
virus
Cherry rasp leaf virus CRLV na na
Luceme Australian LASV na na
symptomless virus
Rubus Chinese seed- RCSV na na
borne virus
Satsuma dwarf virus SDV na na
Strawberry latent SLRSV (2 sequences) RNA2 (3824 bp) 1/95 X77466
ringspot virus
43K/27K capsid X75165
proteins (2424 bp) 2/95
Strawberry latent sSLRSV (1 sequence) sRN A encoding 36K X69826
ringspot satellite RNA protein (1118 bp) 6/93
TTNV na na

Tomato top necrosis
virus

 

 

 

 

 

 

APPENDIX B

Appendix B

Nucleotide sequence of the Oligonucleotide primers used for cloning and sequencing
PRMV RNA].

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primer Nucleotide Sequence cDNA clone
and binding location on PRMV RNA]

OIIgO (GI-312,18 d(TTT.-ou.18) 5-9 (3.2 kb)
poly (A) tail

RA42 d(AAATCATCATCGATCTCAAC) 2.1 (1.2 kb)
4838-4857

RASO d(ACCACTAGTACCCACCAATC) 50-3.9 (1.4 kb)
3636-3655

RA52 d(CCTTCTGTGGGATCCTTGGAGTAGAC) 52-4.2 (1.1 kb)
2222-2247

RA68 d(GAAATATAGGAACCAACC) 68-2.90 (0.9 kb)
1099-1116

RA70 d(CCCATTGCAACCGCCAGAGCTAC) 70.20 (0.15 kb)
212-234

RA75 d(GTCCAAATATTCCATCAC) RNA sequencing
50-67

 

 

 

 

 

58

 

Appendix C

Appendix C
Cloning and Sequencing of PRMV RNA2

Introduction

Peach rosette mosaic virus (PRMV), a nepovirus, was first recognized as the
cause of a disease of peaches (Prunus persica L.) in Michigan in 1917 (Klos et al., 1976).
Nepoviruses are considered a genus within the picomavirus-like supergroup of plant
viruses which includes the potyviruses, comoviruses, and picomaviruses (Goldbach, et
al., 1987). Common features within this supergroup include genomic structure and
organization, as well as nucleotide and amino acid sequence similarity.

Most nepoviruses, including PRMV consist of three distinct particle types: a top
(T) component consisting of empty polyhedral capsid proteins; and a middle component
(M) and a bottom (B) component. M and B components each contain identical capsid
proteins plus single molecules of RNA2 and RNA], respectively (Martelli and Taylor,
1990). Nepoviruses have been subdivided based on their respective RN2 length. In
subgroup I, RNA2 is less than 5.4 kb. In subgroup II, which includes PRMV and
TomRSV, RNA2 is greater than 5.4kb (F rancki et al., 1985). Nepoviruses have a bipartite
genome with a polyprotein expression strategy (Matthew, 1991)

Many nepovirus subgroup I and II genomic RNAs have been sequenced
completely (see Appendix A) and genomic sequence analysis has provided further criteria
to separate the two subgroups. In subgroup II TomRSV, PRMV, BBLMV, CLRV, for
example, the 3’-untranslated region (UTR) is 1.4 kb or greater compared to 0.5 kb or less
in subgroup I GFLV, GCMV, TBRV. Polyprotein processing in subgroup I and II also
appears to differ: the subgroup II RNAl-encoded proteolytic enzyme functions more

similar to that of como-, poty-, and animal picorna-like viruses than to that of nepovirus

59

60

subgroup 1. Evidence demonstrates that a single amino acid substitution (Leu to His) in
putative subgroup II proteinase substrate-binding pockets may be responsible for
differences in proteolytic activity (Bazan and F letterick, 1988).

PRMV RN A] was sequenced in order to confirm the subgroup II status of PRMV
(chapter 2). RNA] consists of 7977 nucleotides not including its 3’-poly (A) tail. The 5’-
and 3’- untranslated regions consist of 52 and 1474 nucleotides, respectively. Analysis of
the PRMV RNA] nucleotide sequence unveiled a single long open reading frame of 6450
nucleotides capable of encoding a 240 kD polyprotein. Motifs characteristic of a
replicase, a proteinase, an NTP-binding protein and a proteinase cofactor were detected in
RNA] putative amino acid sequence and the order and identity of these putative proteins
are consistent with other nepoviruses. RNA] genomic characteristics confirm PRMV
subgroup 1] status.

The partial nucleotide sequence of PRMV RNA2 was analyzed for nepovirus
features including the presence of extensive sequence homology between the 3’ UTRS of
RNA] and RNA2. RNA2 nucleotide sequence analysis was also performed to confirm
the subgroup II characteristic 3’-UTR (greater than 1.4 kb). Nucleotide sequence analysis
of BBLMV, CLRV, and TomRSV, respectively, indicates that the 3’-terminal 1.4 kb of
the 3’-UTR are nearly identical (e.g. TomRSV RNA] and RNA2 3’-1533 nucleotides
differ at only 3 positions) (Bacher et al., 1994; Scott et al., 1992; Rott et al., 1991 and
Sanfacon, 1995). Although subgroup I nepoviruses show extensive nucleotide sequence
homology among their 3’-UTRs (SO-100%), the extent of homology is limited to a few
hundred nucleotides (Sanfacon, 1995).

Sequence analysis of the 3’-terminal region of RNA2 adds to our understanding of
the PRMV genome and further confirms PRMV subgroup II status.

RNA2 cDNA Cloning and Sequence Analysis
The Michigan ‘Concord’ grapevine PRMV isolate used in RNA] cDNA synthesis

and sequencing was also used for RNA2 cDNA synthesis and sequencing. Materials and

6]

methods including virion and RNA purification, cDNA synthesis, cloning of cDNA into
KS- EcoRV site, exo III deletion of cDNA and nucleotide sequencing of cDNA and exo
III subclones are exactly as described in Chapter 2. Pooled PRMV RNA] and 2 were
used as template for cDNA synthesis.
Results and Discussion

A cDNA clone, 4-2.2, contained a cDNA insert of approximately 4000
nucleotides, as estimated electrophoretically. Nucleotide sequence analysis of 4-2.2
detected two tandem-ligated cDNA fragments whose respective nucleotide sequences
were nearly identical. The 3’-cDNA fragment contained 1501 unique nucleotides
excluding a 3’-poly (A) tail of 41 residues; a 42-residue 3’-poly(A) tail of the upstream
portion (1220 unique nucleotides) separated the two fragments of the hybrid RNA. The
cDNA nucleotide sequences of the two distinct fragments are compared with the 3’-UTR
sequence presented in chapter two (Fig.9).

Nucleotide sequence comparison to RNA] was used to determine the RNA origin
of the two cDNA fragments. GCG BESTFIT and GAP analysis (Devereaux et al. 1984)
were utilized with default gap and length weights of 5.0 and 0.3, respectively. The entire
1220 nucleotides of the upstream fragment of 4-2.2 shared perfect identity with the
corresponding 3’-terminal nucleotides of PRMV RNA]. Upstream and downstream
cDNA fragments were 89.6% identical. Poly (A) tails were not included in the alignment.
Therefore, the origin of the unique downstream segment of cDNA clone 4-2.2 was
assigned to PRMV RNA2. The three potential ORFs of the 3’-terminal 1501 nucleotides
were analyzed for their coding capacity; the longest reading frame consisted of 273

nucleotides with a coding capacity of 91 amino acids (RNA2 cDNA positions 59—332).

(a)
(b)
(c)

(a)
(b)
(c)

(a)
(b)
(c)

(a)
(b)
(c)

(a)
(b)
(c)

(a)
(b)
(c)

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(c)

1
CTCAAAGACC

51
TCCTAAGCGC

TACCQAGCGC

101
AGGTCTTGTG

AGGCITTGTG

151
TTACCTTCGG

TTACCTACGG

201
CTCCTTTGGA

CTCCTTTGGA

251
TCCGATTCTA

TCCGATTCTA

301
GGAGATGCTC

GGAGATGCTI

351

CAACACATTA
CAACACATTA
CAACACACTI

401

GGTGTGTTTA
GGTGTGTTTA
GGTGTGTTTA

451

GATCCTGTAG
GATCCTGTAG
GATCCCITGG

501

T.CTTTGCTA
T.CTTTGCTA
TGCTTTGCTA

AATATTTGAT

..........

AGGGGGTCTT

GGCCTAACCC

GGCCTAACCC

GTTGAAGATG

GTTAAAGATG

GAACCATGAG

GAACCATGAG

ACTGTGTGCT

ACTGTGTGCT

TTGTGTGATG
..GTGTGATG
TTAQGTGGTG

ATGAATTCAT
ATGAATTCAT
ATGAATTCAT

TCTATACATG
TCTATACATG
TTTAQAIATA

GTGTGGAAAT
GTGTGGAAAT
GTGTGIGAIT

CCTTCAAGAA
CCTTCAAGAA
CCTTCAAGAA

62

ATGTCAAGTA

TTAGCGCCAG

ACATCCAAGA

ACITCCAAGA

TGAATGGAAG

AGAATGGAAG

TCAACACA..

TTTACCAATT

AGTGTGTAGA
AGTGTGTAGA
CGCATGTAAA

TAATAGTTAT
TAATAGTTAT
TAATAGTGGT

ATTTGAAAAT
ATTTGAAAAT
ACCCAAAAAT

CACCCGCTTT
CACCCGCTTT
CACCIGCTTA

AGGAGATTGT
AGGAGATTGT
AGGAGATTAT

TTAAGCAGCA

TTTCTAGTCC

GGTTGTCATC

GGAAGTCATC

AGTGATGCCC

AGTAATGCCC

.TGGTCTTGG

TACCTTCATG
TACCTTCATG
TACCTTCATG

GTGTTTGTGG
GTGTTTGTGG
GTGTTTGTGG

CTCAAATGAC
CTCAAATGAC
CTCGAATGAC

GTTGGAGAGC
GTTGGAGAGC
ATTGGAGAGC

ACTGGTGAAA
ACTGGTGAAA
LCTGGTGAAA

50
TCTAGCTGCT

TAACGGTCIT

100
TGTAGGCTAG

0000000000

TGTAGGCTLG

150
AATTAGCATT

AATTAACATT

200
TTCCAGACCT

TTCCAGACCT

250
AGGTCACAGT

AGGTCACAGT

300
TGGAAGAGTA

TGGAGGAGCA

350
TTGCTCATTA
TTGCTCATTA
TTGCTCATTA

400
TGGCATGTTG
TGGCATGTTG
TGGCATGTTG

450
TAGGGAGAAA
TAGGGAGAAA
TAGGGAGAAA

500
CAATTCCAAC
CAATTCCAAC
CAATTCCAAI

550
TTCCAGTCCT
TTCCAGTCCT
TTCCAGAIIT

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(c)

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(C)

(a)
(b)
(C)

(a)
(b)
(C)

551

TATATTTATT
TATATTTATT
TATGTTTATT

601

GAGTTGCTTT
GAGTTGCTTT
GAGTTGCGTT

651

TAGTGTGTTG
TAGTGTGTTG
TAGTGTGTTG

701

ATCCC.GGGA
ATCCC.GGGA
ATCCCGGGGA

751

GCACCCTTTT
GCACCCTTTT
GCACCCTTTT

801

CAGATTATAG
CAGATTATAG
CAGATAACAG

851

CTATCCATAA
CTATCCATAA
CTGTCCATAA

901

CTTTCCATGA
CTTTCCATGA
CTTTCCATGA

951

CGGTATGCTG
CGGTATGCTG
CGGTATGCTG

1001

ATGGTTAAGC
ATGGTTAAGC
GTGGTTIIGI

63

Figure 9 (cont’d).

GCTTTCTAGG
GCTTTCTAGG
GCATTCTAGG

AGTAGATCTG
AGTAGATCTG
AGTAAATCTG

TGTCTCCCAC
TGTCTCCCAC
TGTCTCCCAC

GGGTGGTTCC
GGGTGGTTCC
GGGTGGTTCC

CTTCCAGTCT
CTTCCAGTCT
CTTCCAGTCT

ATTTGTGGTT
ATTTGTGGTT
ATTTATGGTT

GATTATCTTG
GATTATCTTG
LITTGCITTG

ATACTACTGC
ATACTACTGC
ATACTGCTGC

CTTCCAGCAT
CTTCCAGCAT
ITTCCGGCAT

TAAATT....
TAAATT....

ACTTGAGTCT
ACTTGAGTCT
AITTGAGTCT

CACGTGAAGT
CACGTGAAGT
IACGTGAAGT

ACAATAAGTA
ACAATAAGTA
ACIATAAGTA

CTCTGACAAC
CTCTGACAAC
TCCTGIGAAC

TACTGAGGCA
TACTGAGGCA
TACTGAGACQ

AACTGATTAG
AACTGATTAG
AACTGATCIA

GATTGTTTAA
GATTGTTTAA
GACTGTTTAA

GATACCGCTG
GATACCGCTG
GATACCGLTG

ATAAAAGCAG
ATAAAAGCAG
ATAAAAGIGG

....CACCGA
....CACCGA

GAAAATWCATQAA

TTTAGTTTTG
TTTAGTTTTG
TTTAGTTTTG

GCGTCAACGT
GCGTCAACGT
ACGTCAACGT

ATGAGACAAC
ATGAGACAAC
ATGAGACAAC

ATTTGTGCTT
ATTTGTGCTT
ATTTGTGCTT

GGATATCAAA
GGATATCAAA
GAATATCAAA

ACTTTGAGTA
ACTTTGAGTA
GATTTGAGCA

TACTCTCATG
TACTCTCATG
TQCTCTCATC

GCGTATTCTA
GCGTATTCTA
GCGTATTCTA

ATATAGTAGC
ATATAGTAGC
ATATAGTAAC

TGAGTCGGAG
TGAGTCGGAG
TIIGTAGGIG

600
CAATCTTGCA
CAATCTTGCA
CAATCCTGCA

650
TATGGCGTAA
TATGGCGTAA
AATGACGTAA

700
GCTGGGTTAG
GCTGGGTTAG
GCTGGGTTAG

750
TAGTAGATAA
TAGTAGATAA
TAGTAAATAA

800
AGTAGGCTTG
AGTAGGCTTG
AGTAGGCTTG

850
ATTGTAAGAA
ATTGTAAGAA
ATTGTAAGAA

900
CTTATCAGCT
CTTATCAGCT
TTTACCAGCT

950
GTTTTAAAGA
GTTTTAAAGA
GTTTTAAAGA

1000
CATAAGCATG
CATAAGCATG
CGTAAGIATA

1050
GAGCCATCAT
GAGCCATCAT
GAGCCACIAT

(a)
(b)
(C)

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(C)

(a)
(b)
(C)

(a)
(b)
(C)

(a)
(b)
(C)

(a)
(b)
(c)

(a)
(b)
(c)

1051

GTGTACAATA
GTGTACAATA
ATGTAAAATA

1101

TTGCTGGGGT
TTGCTGGGGT
TTGCTGGGGT

1151

TCAAGAGCGT
TCAAGAGCGT
TCAAGAGIGT

1201

AATACCGGGG
AATACCGGGG
AATACCGGGG

1251

GAAGTGTCCA
GAAGTGTCCA
GAAGTGTCCA

1301

ATGGAAGTGA
ATGGAAGTGA
ATGGAQGTGA

1351

ATGAGGGGAC
ATGAGGGGAC
ATGAGGGGAC

1401

ATCTGGTTAG
ATCTGGTTAG
ATCTGATTAA

1451

GTTTGACTCC
GTTTGACTCC
GTTTG1LTCC

64

Figure 9 (cont’d).

GGGGGAAGCC
GGGGGAAGCC
GGGGGAAGCC

TAAAAGCTTA
TAAAAGCTTA
TAAAAGCTTA

GTGGGGTGGC
GTGGGGTGGC
GTAGGGTGGC

GATAATTAAT
GATAATTAAT
GATAATQAAT

TGACGCATTT
TGACGCATTT
TGACGCATTT

TTACCATTTC
TTACCATTTC
TTACCATTTC

CACATCTTAA
CACATCTTAA
CACATITTAA

TTGTCGTATT
TTGTCGTATT
TTGTCGTATT

TTTTCCTTGA
TTTTCCTTGA
TTTTCCTTGI

CCTATGGCAA
CCTATGGCAA
CCTATGGCGA

AGGTTTAGTG
AGGTTTAGTG
AGGTCTAGTG

ACCCACGTGC
ACCCACGTGC
ACCIACATGC

CCCAGCTCAG
CCCAGCTCAG
CCCAGCTCAG

TAAGGTAGGT
TAAGGTAGGT
TAAGGTAGGT

GTTATTCGTT
GTTATTCGTT
GTTATTIGTT

GCGATGTTGC
GCGATGTTGC
GCGATGTTGC

TTCTTTTAGC
TTCTTTTAGC
TTCTTTTAGC

CTCTTGACCT
CTCTTGACCT
CT_._._._._'IGCT

ATTATCTGTA
ATTATCTGTA
ATTATCTGTA

TAACACAACA
TAACACAACA
TAICACAACG

TTGGATGAGG
TTGGATGAGG
TTGGATGAGG

GCACTAAGCT
GCACTAAGCT
ACATTAGGCT

TTTAGACATA
TTTAGACATA
TTTAGACATA

ATTAGTTTCT
ATTAGTTTCT
ATTAGTTTCT

TGCATTGCGT
TGCATTGCGT
TGCATTGCGC

.TTTTGTGGC
.TTTTGTGGC
ITTTTGTGGC

AAGTTGGACA
AAGTTGGACA
AAGTTGGACA

1100
TAGGAGCCCT
TAGGAGCCCT
TAGGAACCCT

1150
TTGGGTGTAC
TTGGGTGTAC
TTGAGTGTAC

1200
TCCGGAAATG
TCCGGAAATG
TCCGGAGATG

1250
GACTTTCATG
GACTTTCATG
GACTTTCATG

1300
ACCTCCCGGG
ACCTCCCGGG
ACCTCCCGGG

1350
TGCAACTATG
TGCAACTATG
TGCAAITATG

1400
ACCTATGGTC
ACCTATGGTC
ACCTATGGTC

1450
GACAGATGAG
GACAGATGAG
GATAGATGAG

1500
CAAAAATATG
CAAAAATATG
CAAAAATITG

65
Figure 9 (cont’d).

1501 1543
(a) GTCTTTTGAC TTTCAATAGA GTCGATGAAA ATGTCTGCAT CAC
(b) GTCTTTTGAC TTTCAATAGA GTCGATGAAA ATGTCTGCAT CAC
(C) T.CTTTTTG“ TTTCAATAGA GTCGATGAAA ATGTCTICAT CAC

Fig.9. Nucleotide sequence of the 3’-1527 nucleotides of PRMV RNA] cDNA (a)
(positions 6450-7977) is compared with the nucleotide sequence from a tandem-ligated
cDNA clone 4-2.2 This clone contained 3’-terminal regions from both RNA] (b) and
RNA2-cDNA (0), respectively. The entire RNA] cDNA nucleotide sequence derived
fi'om the recombinant 4-2.2 cDNA clone consists of 1220 bases which corresponds to the
3’-UTR 3’-terminus in PRMV RNA] and 150] bases from the same region of genomic
RNA2. Nucleotide sequence alignment was generated using PILEUP and GAP (GCG)
with gap and gap length weights of 5.0 and 0.3, respectively (Devereaux et al., 1984).
Nucleotide differences between 3’-termini of RNA] and 2 are underlined. Numbering
above the nucleotide sequence begins relative to position 6450 in PRMV RNA] (Chapter
2 Fig.4). Gaps created in the nucleotide sequence alignment are indicated (...).

Analysis of the RNA2 3’-Terminus

PRMV RN A1 and RNA2 share extensive nucleotide sequence identity in their 3’-
UTRS. All nepoviruses whose 3’-termini have been determined have demonstrated this
characteristic: subgroup II BBLMV (Bacher et al., 1994) CLRV (Scott et al., 1992) and
TomRSV (Rott et al., 199]); and subgroup I nepoviruses GFLV, TBRV, and GCMV
(Sanfacon, 1995). The length of the 3’-UTR distinguishes these two nepovirus subgroups.
PRMV may now be added to the list of subgroup II nepoviruses including BBLMV,
CLRV, and TomRSV who share 3’-UTRs greater than 1.4 kb. This lends fiirther
confirmation to the subgroup 1] status of PRMV.

Researchers have speculated on the significance of the extraordinary length of
subgroup II nepovirus 3’-UTRs (Bacher et al., 1994; Buckley et al., 1993; and Sanfacon

1995). It is possible that replicase recognition sites and packaging signals are contained in

66

this region (Buckley et al., 1993) and are conserved in both genomic RNAs. Although
little significant nucleotide homology exists between nepovirus 3’-UTRs, certain
nucleotide sequences of 8 to 30 nucleotides are conserved (see above). Research is
needed to identify their respective functions. Interestingly, nepovirus nucleotide
consensus sequences detected in RNA] 3’-UTR are found in the RNA2 3’-terminus at the
same position relative to the poly (A) tail. If, in fact, these nucleotide consensus
sequences are important for RNA replication and/or packaging, their identical position in
the RNA] and RNA2 genome would indicate that these functions are carried out in
similar manner for the entire PRMV genome. A comparison of the 3’-terminal 500
nucleotides presented of RNAs 1 and 2 indicated 32 base differences. These substitutions
would destabilize the proposed RNA secondary structure. Our current research is aimed
at determining the effect of the nucleotide differences between RNA] and 2 3’-UTRs on

RNA secondary structures.

LIST OF REFERENCES

LIST OF REFERENCES

Allen, W.R. (1986). Effectiveness of Ontario population of Longidorus diadecturus and
L. breviannulatus as vectors of peach rosette mosaic and tomato black ring viruses.

Canadian Journal of Plant Pathology 8, 49-53.

Allen, W.R., and Ebsary, BA. (1988). Transmission of raspberry ringspot, tomato black
ring, and peach rosette mosaic viruses by an Ontario population of Longidorus elongatus.
Canadian Journal of Plant Pathology 10(1), 1-18.

Allison, R.F., Janda, M., and Ahlquist, P. (1988). Infectious in vitro transcripts from
cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA
components with brome mosaic virus. Journal of Virology 62 (10), 3581-3588.

Argos, P. (1988). A sequence motif in many polymerases. Nucleic Acids Research
16(21), 9909-9916.

Bacher, J ., Warkentin, D., Ramsdell, D., and Hancock, J .F. (1994). Selection versus
recombination: what is maintaining identity in the 3’ termini of blueberry leaf mottle
nepovirus RNA] and RNA2? Journal of General Virology 75, 2133-2138.

Bacher, J ., Warkentin, D., Ramsdell, D., and Hancock, J .F . (1994). Sequence analysis of
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