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THESIS

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This is to certify that the
dissertation entitled
Identification of a host protein interacting with

the Marek's disease virus SORFZ protein

presented by

Hsiao-Ching Liu

has been accepted towards fulfillment
of the requirements for

Ph .1) degree in Genetics

 

 

U Ouajor professor \'

Date I’Z/U/ 7F}

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

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6/01 cJCIRC/DatoDuepss-p. 15

 

IDENTIFICATION OF A HOST PROTEIN INTERACTING WITH
THE MAREK'S DISEASE VIRUS SORF2 PROTEIN

by

Hsiao-Ching Liu

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Genetics

1 999

ABSTRACT
IDENTIFICATION OF A HOST PROTEIN INTERACTING WITH
THE MAREK'S DISEASE VIRUS SORF2 PROTEIN
by
Hsiao—Ching Liu
Marek’s disease virus (MDV) is a naturally occurring oncogenic avian
herpesvirus that causes a lymphoproliferative disease in domestic chicken. The genome is
a double stranded linear DNA molecule of approximately 180 kb. Only serotype 1 MDV
has potential pathogenic characteristics, which rapidly induce lymphomatosis and
mononuclear infiltration of various organs. Which gene(s) triggers MD oncogenesis and
by what mechanism is still not understood. In an attempt to study the biological
characteristics of MDV genes, a unique gene, SORF2, came to our attention. An
interesting phenomenon was discovered in the MDV recombinant strain RM] , which
contains a REV LTR insertion upstream of the SORF 2 gene. The unregulated
overexpression of SORF2 due to the LTR promoter was correlated with the loss of MDV
oncogenicity. This finding prompted further investigation of SORF2. Prior to this study,
it was not clear if SORF2 protein is naturally expressed upon infection in a wild type
MDV. To investigate the expression of SORF2, polyclonal antibodies that specifically
recognize SORF2 were generated by immunizing rabbit with purified SORF 2 protein
synthesized in E. coli. A 21kD polypeptide was precipitated with rabbit anti-SORF2
antibodies from MDV infected CEF cells. To directly test if SORF2 is expressed in vivo,
experiments of histochemical staining in MDV infected tissues and induced tumors was

performed. The results demonstrated that SORF2 is expressed in MDV infected tissues

and MD tumors. In a search of potential functional protein partner(s), SORF2 was used as
the bait in yeast two-hybrid screening of a cDNA library constructed from splenic T cells.
The chicken growth hormone structural peptide was found to specifically interact with
SORF2 protein. To corroborate the interaction, the isolated growth hormone cDNA was
used to synthesize corresponding polypeptides in E. coli, which then were assayed for
their ability to interact with SORF2 in vitro. Co-immunoprecipitation of SORF 2 and the
interacting protein, growth hormone, using specific antibodies confirmed the biochemical
evidence for the observed protein-protein interaction. To test the co-localization of both
proteins in vitro and in vivo, IFA double staining examined with confocal microscopy
and gold particles staining examined with transmission electron microscopy were
employed. Results further suggested that these two proteins may interact with each other
since the two co-localized in the cytoplasm. In an attempt to reveal the potential
possibility of cGH to be involved in MD susceptibility, a genetic approach using 272 F2
chickens selected fi'om White Leghorn inbred line 6 x line 7 for the association studies

was performed. A direct association of MD incidence with the cGH gene was not found

(P> 0.05).

ACKNOWLEDGEMENTS

I would like to extend my sincere gratitude to my major advisors, Drs. Jerry
Dodgson and Hans Cheng, for their guidance, support and encouragement in carrying out
this study. I am also very grateful to my advisory committee members, Drs. Sue Conrad,
John F yfe, Robert Silva, and Pat Venta. The advise, guidance, and effort they contributed
to completion of this study were extremely helpful. My appreciation goes to all members
of the ADOL group for their cooperation, assistance, and friendship. A special note of
thanks goes to Dr. Aly Fadly. His encouragement, advice, and support were greatly
appreciated through my graduate school career.

My appreciation goes to my family and friends for their love, understanding, and
support. I am indebted to Drs. Hans Cheng and Jerry Dodgson for all the time and effort
they spent on getting me settled in a career and finishing my Ph.D. studies. I am

extremely lucky to have both of them be my guiding lights. They made all this possible.

iv

TABLE OF CONTENTS

PAGE

LIST OF FIGURES AND TABLES ............................................................................. viii
CHAPTER I: Introduction and Literature Review

Marek's disease ..................................................................................................... 1

Marek's disease virus ............................................................................................ 3

Classification and genome structure ...................................................... 3

Viral attenuation ...................................................................................... 7

Pathology of Marek's disease ............................................................................... 8

Pathogenesis of Marek's disease ........................................................................ 10

Virus-cell interaction .......................................................................................... 12

The regulation of gene expression ..................................................................... 13

Immediate-early genes ........................................................................... 14

Early genes .............................................................................................. 14

Late genes ............................................................................................... 15

Latency associated genes ....................................................................... 16

Immunoresponse to MDV Infection .................................................................. l7

Humoral immunity ................................................................................ l8

Cell-mediated immunity ........................................................................ 18

Non-specific immunities ........................................................................ 19

Vaccines against MD ........................................................................................... 20

TABLE OF CONTENTS (cont'd)

Genetics of Host Resistance to Marek's disease ............................................... 22
MHC genetic resistance ......................................................................... 23
Non-MHC genetic resistance ................................................................ 24

QTL Search and Genome Mapping .................................................................. 25
QTL and genetic markers ..................................................................... 25
Genome mapping ................................................................................... 26
Current status on MD QTL search ...................................................... 28

Specific Aims ....................................................................................................... 28

CHAPTER 11. Identification of Chicken Growth Hormone Interacting with

MDV Sor12
Introduction ......................................................................................................... 3 1
Materials and Methods ....................................................................................... '35
Results .................................................................................................................. 44
The Sorf2 gene encodes a functional polypeptide ............................... 44
Identification of a protein homologue to chicken growth
hormone that interacts with MDV 80er protein ................................ 5]
Interaction of the cGH with Sort? ........................................................ 52
Co-localization of Soer and cGH ......................................................... 57
Discussion ............................................................................................................. 63

CHAPTER III. The Association Between the Chicken Growth Hormone Gene and
Resistance to Marek's Disease

Introduction ......................................................................................................... 68

vi

TABLE OF CONTENTS (cont'd)

Materials and Methods ....................................................................................... 75
Results .................................................................................................................. 8O
Mapping cGH gene ................................................................................ 80
Association of cGH with MD resistance .............................................. 80
Exons comparison between lines 6 and 7 ............................................. 87
Discussion ............................................................................................................. 87

CHAPTER IV. Summary and Future Directions

Summary of results ............................................................................................. 90
Future directions ................................................................................................. 91
REFERENCE LIST ........................................................................................................ 94

vii

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.

Figure 9.

Figure 10.

Figure 11.
Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.

Figure 18.

Figure 19.

Table 1.

LIST OF FIGURES AND TABLES

PAGE
BamHI restriction endonuclease map of serotypel MDV .................... 5
MD QTL on East Lansing chicken genetic map ................................. 3O
Schematic representation of REV LTR inserts in RMl clone ........... 33
Immunoprecipitation analysis of MDV-infected CEF ....................... 45
Sequence of the SORF 2 gene ................................................................ 47
Localization characteristics of Sorf2 on cultured CEF cells .............. 48
Immunohistochemical staining of 80er in neoplastic tissues ............ 50
Summary of yeast two-hybrid library screen ...................................... 52
Sequence of the yeast two-hybrid clones ............................................... 53

Schematic representation of cGH genome structure and cDNA clone

obtained from chicken splenic yeast expression library screen .......... 54
In vitro protein-protein interaction assay ............................................ 56
Co-immunoprecipitation analysis of Sorf2 and cGH ......................... 58
Co-localization of Soer and cGH ......................................................... 59
Immunohistochemical staining of cGH in neoplastic tissues ............. 61

Immunoelectron microscopy of cGH and Sorf2 in neoplastic spleen62
RFLP analysis of East Lansing reference family ................................ 81
Genetic map of linkage group E59 ....................................................... 82
RFLP analysis of cGH in parents of MD resource population .......... 83
PCR-RFLP analysis of MD resource family ........................................ 85

Association analysis for cGH and MD incidence ................................ 86

viii

CHAPTER I

Introduction and Literature Review

Marek’s Disease

Marek’s Disease (MD) is a neurological and T cell lymphoma disease that is
found in domestic chickens; it was first described by Josef Marek (Marek, 1907). The
disease is characterized by a mononuclear infiltration of the peripheral nerves, gonads,
iris, various viscera, muscles, and the skin. Partial or complete paralysis is the common
symptom of MD as a result of the accumulation and proliferation of tumor cells in
peripheral nerves. The relationship of this type of fowl paralysis to other leukotic diseases
found in poultry “.5 once unclear. MD was originally lumped among other leukotic
diseases found in poultry under the term "avian leukosis complex" which was classified
into three forms of lymphomatosis: neural, visceral, and ocular (Jungherr 1939; Jungherr
and Hughes, 1965). Subsequently, based on pathological and field observations, both
Campbell et al. (1956) and Biggs et al. (1961) reported that neural, visceral, and ocular
form of fowl paralysis were distinct—both causally and pathologically—from lymphoid
leukosis. Moreover, the successful experimental transmission of the disease was
accomplished by inoculating day-old birds with blood or lymphoma cells from diseased
birds supporting the evidence for the neural, visceral, and ocular forms of "avian
lymphomatosis" (Sevoian and Chamberlain, 1962; Sevoian et (11., 1962; Biggs and Payne,
1963). Later, Biggs and Payne provided further evidence for disassociating MD from

lymphoid leukosis with respect to their etiological agents (Biggs and Payne, 1963, 1967).

Unlike the avian leukosis virus, the causative agent of MD is highly cell-
associated, infectivity is dramatically reduced when cells are killed or disrupted. In 1967,
it was shown that MD is induced by an avian, oncogenic, cell-associated herpesvirus,
which came to be referred to as the Marek’s Disease Virus (MDV) (Churchill and Biggs,
1967; Nazerian and Burmester, 1968; Solomon et 01., 1968). This virus is quite distinct
from the retroviruses that cause avian leukoses. Prior to the establishment and
application of vaccines, MD generated tremendous economic losses to the poultry
industry. In 1970, annual losses from MD were estimated to exceed $200 million in the
United States alone. Since the 19705, MD has been controlled fairly well through the use
of vaccination. Although vaccination prevents the formation of lymphoma and other
symptoms of MD, it does not, however, prevent infection, reduce replication and the
spread of the virus (Edison et al., 1971, Purchase and Okazaki, 1971). Moreover, even
though commercially available vaccines are used worldwide to protect chickens against
the disease, MD still remains a threat due to increasingly frequent outbreaks of highly
virulent strains of the MDV combined with the incomplete immunity that is elicited by
vaccination (Witter et al., 1980; Schat et a1. , 1981).

Considerable effort has been expended in an effort to develop safer and more
efiecfive vaccines through molecular approaches. Apart from its economic importance,
MD is also of interest to biomedical researchers since it was the first herpes-virus-
induced neoplastic disease to be controlled by vaccination. MD has become a preferred

model, therefore, for the study of viral-induced oncogenesis and atherosclerosis.

The Marek’s Disease Virus
Classification and Genome Structure

Herpesviruses are defined as eukaryotic viruses with single linear double-stranded
DNA genomes ranging from 120 kb to 240 kb. Genomes are replicated and packaged
into icosahedral capsids within the nuclei of infected cells. The capsids range in size
from 100 to 200 nm in diameter and consist of 162 capsomers. The virion is then
embedded in an amorphous protein, the tegument, surrounded by a protein-containing
lipidenvelope (Honess, 1984; Honess and Watson, 1977). More than 100 types of
herpesviruses have been isolated; they are found within a wide range of vertebrates and
each is usually highly restricted in its ability to infect other species, generally a very
limited number. Based on biological properties, herpesviruses are classified into three
subfamilies: alpha-, beta-, and gamma-herpesviruses (Roizrnan et. al, 1992). Although
there is a remarkable variation in biological properties and genomic structure, all
herpesviruses share two basic common features: (1) the lytic infection is characterized
by a highly organized and complex cascade of gene expression: immediate early (IE)
genes are expressed first, followed by early (E) genes and late (L) genes. (2) All
herpesviruses are capable of establishing long term latency in specific cell types in
immunocompetent hosts (Wagner, 1991).

The nuclear capsid of MDV is about 100 nm in diameter and contains 162
capsomers arranged in icosahedral symmetry (Nazerian and Burmester, 1968). The MDV
genome is a double stranded DNA molecule of approximately 160-180 kb with a G/C
content of 46-47% (Lee et al., 1971; Cebrian et al. , 1982). The genome consists of two

unique long and short DNA segments (UL and US), each flanked by terminal and

inverted repeats: terminal repeat long (TRL), inverted repeat long (IRL), inverted repeat
short (IRS), and terminal repeat short (TRS) (F igurel). Like the herpesviruses Epstein-
Barr virus (EBV) and Saimiri, Southern blot hybridization has also revealed the presence
of repetitive internal sequences within the MDV genome at both termini (Cebrian et a1. ,
1982; Ross et al., 1983; Hirai et. al., 1984). Variation in the number of the repeated
sequences results in terminal heterogeneity.

MDV is primarily a cell-associated herpesvirus that has lymphotropic properties
similar to those of gamma herpesviruses. Members of this group, such as EBV in
humans, and MDV in chickens, generally have a restricted host range, and latency is
ofien evident in lymphoid tissue. Viruses of this group are capable of transforming cells
in natural hosts. MDV was re-classified, however, because its molecular structure and
genomic organization more closely resemble alpha herpesviruses, such as Herpes
Simplex Virus (HSV) and Varicella-Zoster Virus (VZV) (Buchmaster et al., 1988;
Brunovskis and Velicer, 1995).

On the basis of agar gel precipitation analysis and viral antibody neutralization,
three serotypes of MDV have been identified. Serotype 1 viruses include the oncogenic
MDVs and their cell-culture-attenuated variants. Serotype 2 viruses include the naturally
occurring non-oncogenic MDVs found in chickens. The non-oncogenic Turkey
Herpesviruses (HV T) found are classified as serotype 3 (Bulow and Biggs, 1975). These
strains share a similar A antigen (Ross etal., 1973; Long et al., 1975; Isfort et al., 1986;
Isfort et al. , 1987), but have group-specific B and C antigens that can be distinguished by
serological methods (Bulow and Biggs, 1975). Tissue isolates fi'om MD tumors can be

used to infect a monolayer of avian fibroblast cells such as chicken embryo fibroblast

 

(CEF) or duck embryo fibroblast (DEF) cells (Churchill and Biggs, 1967; Nazerian et al. ,
1968). The majority of virus particles produced in cell cultures, however, are non-
enveloped. When cultivated in cell cultures, oncogenic serotype 1 viruses grow best in
DEF cells or chicken kidney cell cultures, producing small plaques. Serotype 2 viruses
grow best in CEF cells and produce medium plaques with some large syncytia Serotype
3 viruses (I-IV T) grow best in CEF cells and produce large plaques. Although MDV
infection in chickens is tightly cell-associated, it was found that MDV could replicate
completely in the epithelial cells of feather follicles (Calnek et aI. , 1970).

Serotype 1 isolates can be further categorized into very virulent plus (vv+ MDV),
very virulent (vv MDV), virulent (v MDV), mild (m MDV), and attenuated strains based
on their ability to promote disease in unvaccinated and vaccinated experimental chickens
(Witter, 1997). 648A, a vv+ isolate, cannot be prevented through vaccination with
bivalent vaccine. Md5, Mdl 1, and RBlB strains are classified as vaDV and are
responsible for causing many of the outbreaks in vaccinated chickens, viruses in this class
cannot be prevented in HV T vaccinated chickens. GA, HPRS-16, and JM strains are
classified as vMDV and can cause a high incidence of MD in unvaccinated chickens.

The CU2 strain belongs to the mMDV group. Serial passage in vitro results in the
attenuation of virulent isolates (Churchill et al., 1969; Rispens et al., 1972; Witter, 1982).
Since the 19705, MD vaccines have been produced using non-pathogenic serotype 2
MDV strains (Rispens et al., 1972), and serotype 3 MDV strains (Okazaki et al., 1970),
as well as cell culture-attenuated serotype 1 MDV strains ( Churchill et al. , 1969; Witter,

1982; de Boer et al., 1987).

Viral Attenuation

Attenuated strains of MDV have been produced by repeated passage of an
oncogenic MDV strain through cell cultures and have been used successfully for
vaccination against MD (Nazarian, 1970; Edison et. a1, 1971). Attenuated MDVs are
characterized by loss of tumorigenicity and pathogenicity, increased replication rate in
cultured fibroblasts cells, and alterations in DNA structure. Comparison of viral DNA
structures from pathogenic and nonpathogenic strains of MDV suggest that expansion of
BamHI-D and —H fiagments in nonpathogenic strains is strongly correlated with MDV
attenuation (Silva and Witter, 1985; Fukuchi et al. , 1985). Maotani et al. (1986) reported
that this expansion was due to the amplification of a 132 bp repeat sequence found within
the BamI-H-D and -H fragments. Later, it was discovered that the 132 bp repeats are part
of a transcript containing an open reading fiame (ORF) named Bha which exhibits a
complex pattern of splicing in cells lytically infected with oncogenic MDV strains and in
MDV transformed cell lines. The ORF encodes a transmembrane protein of 63 amino
acids with limited homology in its N- terminus to a mouse T cell lymphoma TLM
oncogene protein. An overexpressed Bha gene prolongs the growth of CEF cells and
enables CEF cells to grow in very low serum concentrations (Peng et al. , 1993). This
region is important because the number of copies of the 132 bp repeat increases following
cell-culture attenuation of serotype 1 viruses, and, thus, can be used as a marker for
indicating decreased tumorigenic capability (Silva, 1992). Moreover, it has been
hypothesized that a crucial gene involved in oncogenicity could be disrupted by the
expansion of the 132 bp repeat region (Ross et al., 1983; Bradley et al., 1989; Chen and

Velicer, 1991). Besides the variation of the 132 bp repeat sequence, there is also a 200

hp deletion in the BamHI-L fragment located in IRL and TRL regions in attenuated
strains (Wilson and Coussens, 1991). Evidence from in vivo studies, however, indicates
that the 200 bp deletion found in attenuated Mdll does not directly affect MDV
oncogenicity.

One approach to identifying putative MD oncogene(s) is to study the mutation
associated with viral attenuation. The classical approach is to inactivate the gene(s) and
then compare the phenotypes of wild-type and mutant viruses. In general, a wild-type
MDV gene can be replaced or disrupted by homologous recombination with a mutant
version carried on a plasmid. This site-specific insertional mutagenesis approach is a
powerful tool that serves to assign the function of a gene. Unfortunately, as MDV is a
highly cell-associated virus, the virus spreads through cell to cell contact but is not
released fiom the cell. Thus, the selection of the recombinant virus is an extremely
inefficient process in MDV mutagenesis study. Therefore, alternative molecular
approaches are needed in order to create attenuated MDV viruses.

Pathology of Marek’s Disease

As stated previously, the MDV is a highly cell-associated herpesvirus. Usually,
enveloped virions enter the cell by conventional absorption and penetration within one
hour following infection. Natural transmission of MD occurs through direct contact
between infected and uninfected chickens, or indirectly through dust-home viral particles
(Calnek et. al, 1970). Clinical signs, including gross lesions, generally start to appear
approximately four to six weeks following infection.

MD is characterized by lymphoid infiltration of various peripheral nerves and

visceral organs, most fi'equently the spleen and the gonads. An association of certain

MDV isolates with ocular lesions and blindness has also been noted. Nerve lesions, such
as enlargement and gray-yellow discoloration, are the most common observations that
have been made with respect to diseased chickens. There are at least two distinct
pathological forms of MD that have been recognized. The classical type of MD
predominantly attacks peripheral nerves and causes cytolytic infections. In contrast, the
acute form of MD involves a form of lymphoproliferation that results in the formation of
tumors in various organs (Calnek and Witter, 1997). Microscopic sections of MD lesions
are characterized by the morphologic heterogeneity of neoplastic lymphoid cells.
Various sizes of lymphocytes, plasma cells, and lymphoblasts have been observed in the
peripheral nerves and/or visceral organs of diseased birds. Lymphomas are developed in
one or more of the following organs: the spleen, the liver, the gonads, the kidney, the
heart, the pancreas, and the proventriculus, as well as muscle and skin tissue.

The lymphoproliferative nature of MD lesions can be easily confused with that of
lymphomas induced by avian leukosis virus (ALV) or reticuloendotheliosis virus (REV).
MD lymphomas are of T-cell origin, whereas ALV and REV mainly cause B cell
lymphomas. These lymphomas can be distinguished by immunocytochemical tests using
antibodies specific to cell surface antigens of B and T lymphocytes. The molecular
mechanism of MDV oncogenesis is quite different from that of ALV. It is hypothesized
that MDV carries an oncogene, whereas the ALV transforms target cells by integration
within or near a cellular oncogene such as c-myc causing the overexpression of its gene

product which initiates oncogenesis.

Pathogenesis of Marek’s Disease

By inoculating susceptible chickens with oncogenic MDV, four different phases
of infection have been established: (1) early cytolytic infection of the lymphoid organs;
(2) a period of latent infection; (3) late cytolytic infection; and (4) the transformation of
T-lymphocytes. In genetically resistant hosts, only the early cytolytic infection followed
by the establishment of lifelong latency has been observed.

The early cytolytic infection phase occurs 3 to 5 days following inoculation and
mostly affects B-lymphocytes plus a few activated T—lymphocytes (Shek et al. , 1983;
Calnek and Spencer, 1985). This productive-restrictive infection can provoke an acute
inflammatory reaction resulting in macrophage and granulocyte infiltration (Payne and
Roszkowski, 1973), which leads to local necrosis. Common consequences of these
events are spleen degeneration and atrophy of the thymus. The severity of this early
cytolytic phase, however, is related to the virulence of the virus.

The infection switches from a cytolytic infection of primarily B-lymphocytes to a
latent infection of predominantly T-lymphocytes at 6-7 days following infection (Shek et
a1. , 1983). This switch is generally associated with the development of an immune
response in the host. Latently infected T-cells can persist for the lifetime of the host.
However, latent infection has also been observed in non-lymphoid cells, such as
’ nonmyelinating Schwann cells and satellite cells in spinal ganglia (Pepose et al. , 1981 ).
The mechanism of MDV persistence in T-cells is unknown.

Progression to a second cytolytic infection has only been observed in genetically
susceptible birds at 2 to 3 weeks following inoculation. In this phase, affected tissues are

not limited to lymphoid organs, but also include epithelial cells in various visceral

10

organs. Cell-free infectious viral particles produced from feather follicle epithelium can
be observed at this stage (Calnek et al., 1970). Focal necrosis and intranuclear inclusions
also occur in the kidney, pancreas, blood vessels, peripheral nerves, and central nervous
system (Payne, 1992).

Lymphoproliferation and the development of T-cell tumors represent the final
stages of MD pathogenesis (Buscaglia and Calnek, 1988; Calnek and Witter, 1997). The
composition of lymphomas, consisting of a mixture of neoplastic, inflammatory, and
immune cells involve almost all visceral organs, skin, muscles, and nerves. This stage
usually occurs at 4 to 6 weeks following inoculation.

Cell transformation is the most salient characteristic of oncogenic MDV strains.
Although the virus initially causes a lytic infection in lymphoid cells, it persists in the
natural host by establishing a life-long, latent infection of T—lymphocytes. There are no
viral antigens that have been detected in latently infected cells. All three serotypes of
MDV can cause latent infection in cells, but only serotype 1 MDV has pathogenic
potential. Although the molecular basis of oncogenic transformation by MDV has not
been established, it has been hypothesized that MDV contains a distinct oncogene(s) in
contrast to chronic transforming RNA oncoviruses, which do not posses an oncogene but
transform cells indirectly by activation of a cellular oncogene (hit-and-run mechanism).
Schat et al. 1991 reported that cellular transformation by the MDV occurs only in a
limited subset of activated T-cells. Because of the rapid onset of tumor formation and the
polyclonal nature of the tumors, it is likely that MDV carries its own oncogene(s). The
final pathogenesis of MD, however, is influenced by many factors, such as virus strain

and the age, sex, and genetic background of the host (Calnek and Witter, 1997).

ll

Virus-Cell Interaction

The MDV is most frequently contracted from the environment via the respiratory
system (Payne, 1985). Once the MDV enters the host, three major virus-cell interactions
have been recognized: (l) productive infection, (2) non-productive latent infection, and
(3) transforming infection (Schat, 1985; Calnek and Witter, 1997). Productive infection
occurs mainly in nonlymphocytes. Two types of productive infection have been
observed: fully productive and productive-restrictive. Fully productive infection by the
MDV has only been observed in feather follicle epithelium (Calnek et al., 1970), which
results in the development of large numbers of enveloped and fully infectious virions.
Nevertheless, a productive-restrictive infection can occur in some lymphoid and
epithelial cells in the host and in most cultured cells, where most of the virions produced
are not enveloped and thus noninfectious. Cell to cell fusion, therefore, becomes the
major virus spreading mechanism in productive-restrictive infections (Calnek and Witter,
1997). In productive infections, the number of replications of viral genome copies per
cell can exceed 1200. In viva productive infection normally leads to the formation of
intranuclear inclusion bodies, cell destruction, and necrosis. In cultured fibroblast cells,
polykaryocytosis is a major component of viral plaques, and it is commonly used as a
marker in biological assays of virus infections.

Latent infection is not a productive type. There are very few copies (about 5) of
the viral genome that have been observed in latently infected lymphocytes,
predominantly in T cells, but also in some B cells. Viral gene expression is highly

limited in latently infected cells. Most translation does not occur, and, normally, no virus

12

or tumor associated antigens can be detected (Calnek et al. , 1981; Shanna, 1981),
although some genes may be transcribed.

A third type of interaction is transforming infection, only found in T lymphocytes
transformed by virulent serotype 1 MDV. Transformed cells contain more copies (5-15)
of the viral genome than latently infected cells (Ross, 1985), and there is more extensive
viral gene expression, occasionally resulting in antigen production (N akaj ima et al. ,
1987; Nakajima et al., 1989). The MDV’s viral DNA in transformed cell lines is highly
methylated, whereas methylation has not been detected in MDV DNA from productively
infected cells (Kanamori et al., 1987). To date, no viral antigens have been found to have
any specific association with transformed cells. Nevertheless, an activated T-cell marker,
Marek’s associated tumor surface antigen (MATSA), is expressed at a higher level in
transformed cells (McCall et a1. , 1987). The majority of these transformed T-cells are
activated T helper CD4+/CD8- cells. Nevertheless, other cell lines that have been
established from experimentally induced lesions are CD4+/CD8+, and CD4-/CD8- T-
cells (Schat et al., 1991). Transformation is only known to occur in T cells. It is also
believed that T cells are susceptible to MDV infection only after activation. The
mechanism that leads from latency to transformation is not well understood. Yet, current
evidence suggests that latent infection is a prerequisite to transformation.

The Regulation of Gene Expression

As with other herpesviruses, MDV gene expression in the lytic infected state is
temporally regulated in a highly organized, cascade fashion (Maray et al., 1988; Schat et
al., 1989). In general, MDV genes have been classified into three kinetic families:

immediate-early genes (IE or on), early genes (E or B), and late (L or 7) genes, based on

13

the requirements for viral protein synthesis or DNA replication (lioness and Roizrnan,
1974)
Immediate-early Genes

IE genes are expressed immediately upon virus entry into host cells and do not
require de nova viral protein synthesis. The expression of IE transcripts is controlled by
enhancer elements located within their promoters. It has been suggested that a virion-
associated transcriptional activator, VP16, mediates MDV IE gene expression (Boussaha
et al., 1996), which itself is produced late in infection (Campbell et al., 1984). VP16
triggers the transcription of the IE genes in HSV, including infected cell peptides (or ICP)
ICPO, ICP4, and ICP27. IE gene transcripts accumulate in the presence of metabolic
inhibitors such as cycloheximide (CHX), a protein synthesis inhibitor. Several MDV IE
_ genes have been identified which are homologous to HSV-l, including ICP4, ICP22, and
ICP27 (Anderson et al., 1992; Ren et al., 1994; Hong and Coussens, 1994; Brunovskis
and Velicer, 1995). An MDV specific 14 kDa phosphoprotein (pp14) encoded by a 1.4
or 1.6 Kb IE transcript has also been reported (Hong and Coussens, 1994). Studies have
demonstrated that MDV ppl4 is detected not only in serotype 1 infected cells, but can
also be found in MDV serotype 1 - transformed lymphoma cell line, MSB-l (Hong et al. ,
1995). However, the basic function of ppl4 is still not clear.
Early Genes

Early genes become activated following IE gene expression and are regulated by
1E gene products. Most B gene products are involved in nucleotide precursor metabolism
and are essential for viral DNA synthesis (Roizrnan and Sears, 1995). E gene products

are identified by their accumulation in the presence of viral DNA synthesis inhibitors

14

such as phosphonoacetic acid (PAA). Several E genes in MDV have been identified,
including thymidine kinase (TK) (Scott et al. , 1989), DNA polymerase (UL30) (Sui et
al., 1995), a unique phosphoprotein with 38 kDa molecular weight (pp3 8) ( Cui et al. ,
1990; Cui et al., 1991; Chen et al., 1992), and a MDV- specific gene, meq, which
encodes a protein with high homology to the fos/jun family of oncogenes (Jones et al.,
1992). The pp38 has been shown to be abundantly expressed in the MDV MSB-l tumor
cell line (Cui et al. , 1991). It has been suggested that the biological function of pp38 is
potentially related to MDV oncogenicity (Calnek and Witter, 1997). The expression of E
genes is responsible for initiating viral DNA synthesis and subsequently for the induction
of late gene expression.
Late Genes

Late gene expression requires both viral protein synthesis and viral DNA
replication (Wagner, 1991). Late genes mainly encode structural proteins essential for
virion, capsid, tegument, and envelop assembly (Roizman and Sears, 1995). Viral
glycoproteins are primarily encoded by L genes whose functions include virus
attachment, penetration, and cell-to-cell fusion (Roizman and Sears, 1995). Since the key
feature of late gene expression is its requirement for activation by viral DNA replication,
L gene transcription will be inhibited in the presence of DNA replication inhibitors.
Based on their dependence on viral DNA replication, L genes can be grouped into 71 and
72 subfamilies. yl gene transcription occurs prior to the initiation of viral DNA synthesis,
and is only minimally affected by inhibitors of DNA synthesis. y2 genes are expressed
late after infection and are not detectable in the presence of effective concentrations of

inhibitors (Roizman and Sears, 1995).

15

L genes in MDV have also been identified, including glycoproteins gB (Ross et
al., 1989), gC (Isfort et al., 1987; Coussens and Velicer, 1988), gD (Brunovskis and
Velicer, 1995), gE (Brunovskis and Velicer, 1995), gH (Scott et al., 1993), gI (Ross et
al., 1991), gK (Ren et al., 1994), and gL (Yoshida et al., 1994). Among these
glycoproteins, a recombinant fowlpox virus (F PV) expressing the MDV gB has shown to
provide some effective protection against MD in vaccinated chickens (Nazerian et al. ,
1992)

Latency Associated Genes

The establishment of latency is a hallmark of herpesviruses. Latency is a
reversible, non-productive infection stage involving a replication of a competent virus.
The development of latency by a virus involves the evasion of the host’s immune
response system and the persistence of a viral genome in latently infected cells. This is
relatively easy for neurotropic herpesviruses, such as HSV, where latency is established
in non-dividing neuron cells. F or lymphotropic herpesviruses, however, this is more
difficult since the virus must latently infect dividing or mitotic cells, such as B or T cells.
In this case, the virus requires a specialized origin of replication to ensure that its genome
is retained in each daughter cell. The mechanism of MDV genome persistence in latently
infected cells is unknown. However, it is clear that CD4+ T-helper cells are the primary
targets of latent infection, although a few B cells may also be involved (Schat et al. ,
1991)

Only a limited set of viral genes is expressed in latently infected cells. The
majority of latency-associated transcripts of the MDV were mapped to the repeats

flanking the unique, long region of the genome by Northem blot analysis (Tillotson et al. ,

l6

1988). The transcripts, however, are cell-type restricted. In the MSBl-transformed cell
line (an expressing cell line) 29 transcripts have been mapped to different regions of the
MDV (Becker et al., 1988). Schat et al. (1988) reported 4 and 7 transcripts in HPl (non-
producing cell line) and CV41 (non-expressing cell line), respectively. Additionally,
most transcripts found in MDV-lymphoblastoid cells are IE genes (Silver et al., 1979;
Schat et al. , 1989), suggesting that IE genes could play a significant role in the
maintenance of latency.

Another important finding is that viral DNA is heavily methylated in these
transformed cell lines. This phenomenon is presumably related to limited gene
expression. In addition to the circular plasmid form in the latent stage, viral DNA has
also been found to be integrated into the host genome in some cell lines (Hughes et al. ,
1980; Kato and Hirai, 1985; Delechuse and Hammerschmidt, 1993). The genome sites
for MDV integration are found to be random, although telomeres appear to be the
preferential targets (Delechuse and Hammerschmidt, 1993). However, the viral
integration is not found to be essential for viral transformation.

Immunoresponse to MDV Infection

The outcome of MDV infection is influenced by the host’s gender, age, genetic
makeup, and immune system response. Male birds are more resistant to MD than
females (Purchase and Biggs, 1967; Cole, 1968) although the reason for this is not
known. Usually older birds are more resistant to a virulent MDV infection (Witter et al. ,
1973). Both humoral and cell-mediated immunity have been found to play a role in

resistance to virus infection.

17

Humoral humanity

Infection of MDV at cytolytic stage primary occur in B lymphocytes results in the
release of viral antigens. Thus, virus-neutralizing antibodies can be detected within 1-2
weeks following MDV infection. These antibodies generally persist throughout the
lifetime of the bird. Because bursectomized birds can survive a MDV infection, it is
presumed that the humoral antibody response may not be essential for resistance to MD
(Shanna and Witter, 1975). Maternal antibodies also reduce the level of MD
development ( Chubb and Churchill, 1968; Ball et al., 1971).

To examine the specific viral antigens induced protective immunity, viral proteins
expressed via fowlpox virus or baculovirus were used as vaccines followed by
challenging birds with oncogenic MDV. Results indicated that only gB has partial effects
for the induction of virus-neutralizing antibodies (Nazarian et. al., 1992). Niikura et al.
(1991) demonstrated that although gC can induce the development of gC specific
antibodies, the antibodies does not have effect in protecting against MD development.
Similar study in unique gene pp38 also demonstrated that it does not induce the
production of protective antibodies (N azarian et al. , 1992).

Cell-Mediated Immunity (CMI)

As a consequence of the production of viral antigens, T cells become activated
and capable of being infected. Since the MDV has a cell-associated nature that CMI has
been strongly suggested to be critical in controlling viral infection and resistance to MD
development (Omar and Schat, 1996). However, due to the lack of in viva evidence, this
suggestion is inconclusive. The CMI responses are directed against viral antigens and

include delayed hypersensitivity (Byerly and Dawe, 1972), in vitro cytotoxicity as

18

measured by plaque reduction, and antibody dependent, cellular cytotoxicity (Powell,
1976; Sharma and Coulson, 1977; Confer and Adldinger, 1980).

Interference of antigen presentation upon virus infection has been suggested to be
one of the potential mechanisms incapacitating the CMI to MDV. However, due to the
limitation that cultured primary cells do not express the MHC class 1 antigen or/and
MDV viral antigen, there is no significant association has been made between any
particular viral antigen and MHC down regulation. Recent studies using chemical
transformed CEF cell line OU2 indicated that MDV early gene pp38 might be involved in
the down regulation of MHC class I glycoproteins via targeting peptide transportation
(Hunt et al.,1999). This result indicated that pp38 may have an association with the
depression of immunity after viral infection. Typically, CMI has been tested by
measuring the ability of mitogens to activate normal peripheral T-lymphocytes to
undergo blast transformation and proliferation. Results indicated that older birds of
resistant chicken lines have lower responses to mitogens (Schat et al. , 1978; Fredericksen
and Gilmour, 1981), although the reciprocal response may occur at a young age
(Fredericksen and Gilmour, 1983; Lee and Bacon, 1983).

Non-Specific Immunities

Macrophages are found to be playing a central role in the regulation of the MDV
infected immune response. It may be involved in resistance by directly limiting virus
replication (Higgins and Calnek, 1976; Haffer et al. , 1979), or by cooperating with
antibodies (Kodama et al. , 1979; Lee, 1979). Gupta et al. (1989) demonstrated that the
use of activated macrophages by injection of thioglycollate broth into the peritoneal

cavity significantly reduced the incidence of MD in challenged birds. Moreover, in vitro

19

studies showed that activation of macrophages can inhibit viral DNA synthesis and cell
proliferation in MD lymphoblastoid cell lines (Lee et al., 1978; Sharma, 1980; Ozaki et
al., 1983). In viva studies suggested that the activity of macrophage among different
resistance chicken flocks is different (Dr. M. Qureshi, personnel communication). These
evidences demonstrated that macrophage act as the first line of defense to MDV
infection.

It is believed that cytokines are involved in regulating MDV gene expression
(V olpini et al. , 1995). It has been reported that the level of interferon, an early response
to MDV infection, is higher in resistant birds (Hong and Sevoian, 1971). Cytokines also
appear to be different in birds infected by various MDV isolates (Shanna, 1989).
Particular cytokines have been associated it is important with the development and
maintenance of latency with the MDV (Buscaglia et al. , 1988; Volpini et al., 1995).

Natural killer (NK) cells are cytotoxic for MD tumor cells and may be involved in
genetic resistance to MD (Powell, 1985; Schat, 1987). There is evidence of increased
NK cell activity after vaccination with the HVT or SB-l viruses (Shanna, 1981; Heller
and Schat, 1985). Moreover, the increasing levels of NK cells in regressive, but not
progressive, tumors could also indicate that NK plays a role with respect to intratumoral
immunity in tumor regression (Sharma, 1983).
Vaccines Against MD

A number of vaccines have been available since 1970 for controlling MD
development in the field. These vaccines consist of attenuated serotype 1 isolates and
serotype 2 or 3 viruses, administered alone or in combination. Recently, recombinant

MD vaccines have also been developed that protect chickens against MD; however, they

20

are not as effective as hoped. The recombinant vaccines were constructed by inserting
immunogenic genes of the MDV, such as gB, in fowl poxvirus or HV T (Nazerian et al.,
1992, Ross et al., 1993). A number of MD vaccines are currently in use. HVT continues
to be an important constituent of the most popular vaccines since it is easily made and
economical in price. Vaccinated chickens become resistant to tumor development and
other symptoms of MD caused by virulent MDV. As stated above, however, vaccination
does not prevent the infection, replication, and spread of the virus (Edison et al., 1971;
Purchase et al. , 1972). The superinfecting oncogenic virus and the vaccine strains are
still able to replicate, and the viral particles shed from feather follicles can still infect
unprotected chickens.

The method for MD vaccine delivery is undergoing a rapid change. Vaccines
were usually administered subcutaneously or intrarnuscularly in newly hatched chickens
in the hatchery (Oei and Boer, 1986). Recently, in ova embryo vaccination technology
has been used in over 80% of commercial broilers in the United States. With this
technology, vaccines are injected into eggs at 17-18 days of embryonation with the use of
multiple-head injection machines that result in lower labor costs and greater precision of
the vaccination (Calnek and Witter, 1997).

Humoral immune responses to tumor-associated antigens have not been detected
in chickens following vaccination. Instead, the number of antibodies increases following
hyperimmunization with lymphoma cells. Thus, vaccinal immunity appears to be based
primarily on cellular responses to viral antigens supplemented by humoral anti-viral and

cellular anti-tumor immune responses.

21

Vaccination efficiency is effected by several factors, including genetic makeup,
age at challenge, stress, and possible infection by other irnmunosuppressive viruses
(Calnek and Witter, 1997). Vaccine failures, however, result mainly from early exposure
to and the emergence of new, more virulent MDV strains. The use of genetically
resistant birds in ongoing research has come to be seen as an especially promising
approach for controlling NH).

The Genetics of Host Resistance to Marek’s Disease

Selection of genetically superior animals through the use of a planned breeding
system has made it possible to influence the genetic makeup, breed (or line) of future
generations. Those chickens that are resistant to MD are those that fail to develop
characteristic MD symptoms upon exposure to oncogenic MDV. Genetic differences in
resistance to MD paralysis have been reported for more than 60 years (Asmundson and
Biely, 1932). Since then, several MD resistant inbred lines have been generated via
selection and mating programs at the Avian Disease and Oncology Laboratory (ADOL).
Of 15 lines developed, White Leghorn lines 6 and 7 proved to be the most interesting
with respect to MD studies. When line 6 chicks are inoculated with the JM strain of
MDV at 1 day of age, less than 3% of the birds develop MD symptoms. In contrast, a
similar inoculation into line 7 birds resulted in a mortality rate over 85%. These lines are
maintained at the ADOL and are over 99% inbred. The level of disease resistance as
measured by mortality among F 1 siblings of a cross between lines 6 and 7 is intermediate
to the parents (~60%); however, the levels of resistance observed in an F2 population

encompass a large spectrum instead of following one gene (two alleles) Mendelian

22

segregation manner. This indicates that there is more than one gene involved in MD
resistance (Stone, 1975).
MHC Genetic Resistance

The major histocompatibility complex (MHC) is a group of closely linked loci
that codes for highly polymorphic cell surface proteins as well as the proteins involved in
the control and development of immune system responses (Klein, 1986). There are two
types of molecules designated, Class I and Class II, respectively, that present
intracellularly processed peptides to T lymphocytes. A distinct feature of many MHC
molecules is their extensive polymorphism. Many MHC gene variants are known to be
more or less associated with disease resistance. The chicken MHC, known as the B
complex, is the best understood mechanism for the development of genetic resistance to
MD. MHC-related resistance functions by reducing the MDV infection level during
latent phase infection, also eliminating the second wave of cytolytic infection.

By measuring the frequency of specific blood groups, it has been observed that
certain B alleles are associated with resistance or susceptibility. Chickens with the B21
allele have been found to be more resistant than those with other B haplotypes (Bacon,
1987; Bacon and Witter, 1992). Other studies have allowed for the relative making of
the other B alleles: moderate resistance, BZ, B6, B14; susceptibility, B1, B3, B5, 313,
815, 819, 827 (Longenecker and Mosmann, 1981). Recent studies indicate that B-
haplotype influences vaccinal immunity and that some haplotypes develop better
protection with vaccines of one serotype than with others (Bacon and Witter, 1994).
Now, instead of directly testing lines for MD resistance, breeders have the advantage of

being able to indirectly select favorable birds by using B blood group antigens. This

23

blood typing system has not been widely adopted, however, primarily because the MHC
has pleiotropic effects. In other words, selecting for alleles that confer resistance to MD
may he disadvantageous for selecting resistance to other diseases or production traits.
Recent studies of a MHC-like, pr-Y haplotype indicated that it might significantly
influence the outcome of infection with MDV (Wakenell et al., 1986). However, a
similar study using an experimental intercross between lines 6 and 7 indicated that the
pr-Y genes do not influence MD resistance (Vallejo et al., 1997). Thus, this suggests
that pr-Y genes may have only a minor effect or that only certain alleles are involved in
MDV resistance.
Non-MHC Genetic Resistance

Recent research suggest that the contribution of resistance to MD by non-MHC
genes may be significantly larger than that of the MHC (Groot and Alhers, 1992).
Studies were done on three commercial White Leghorn lines with different MD
resistance and MHC haplotypes. Two sire lines, heterozygous for MHC haplotype, from

susceptible or resistant lines, were mated to dams, homozygous for MHC haplotype, from

a moderately resistant line. 1359 chicks were produced, scored for MHC haplotype, and
infected with MDV at one day of age. Results indicate that the sire line had a larger
effect than either the maternal line or the parental MHC haplotype. This study suggests
that a higher level of resistance to MD can be derived from non-MHC genes than from
MHC alleles. To further identify non-MHC genes which have an influence on disease
resistance, genetic markers were used to screen for linkage to phenotypic traits that are

associated with MD resistance.

24

QTL Search and Genome Mapping
QT L and Genetic Markers

Animal breeders from around the world are working on developing genetic maps
to help identify specific genes which effect economic traits. These genes are believed to
act in combination to produce continuous or quantitative variations in a trait. These
specific regions or genes are called quantitative trait loci (QTLs). The more we know
about the underlying genetic variation of QTLs, the more we will be able to predict the
responses that will have a major impact on the production, reproduction, and general
health traits of our livestock. We may also be able to produce animals that are designed
for specific environments and increase the level of inheritability of certain desirable
traits. The identification of QTLs could also increase the accuracy of selection for
existing traits and allow for the selection of new traits that are positively associated with
reproduction and health characteristics (Rothschild, 1994).

Unfortunately, our knowledge of the identity of QTL genes, how many QTLs in
combination are responsible for certain variations, as well as individual actions and
interactions, is still very limited. Recently, genetic markers have been used to develop a
better understanding the behavior of QTLs, using marker-QTL linkage. Tanksley et al.
(1982) first reported the linkage between DNA-based markers and QTLs in plants. Two
or more loci are defined as linked if we can observe the non-independent segregation of
alleles at these loci (Botstein et al. , 1980). QTLs can be most easily detected in crosses
between inbred lines with large phenotypic differences. This will provide better evidence

for segregation as well as the fraction of QTLs in the F2 generation (Ellegren, 1993).

25

Genome Mapping

The purpose of gene mapping in breeding programs is to find genetic markers
which are well spaced and which provide broad coverage. QTL detection depends on the
density and polymorphism of DNA-based markers. The development of genetic maps
will also provide the means necessary for a better understanding the evolutionary
relationships between breeding lines. Two types of maps are commonly used: genetic
maps and physical maps.

On a genetic map, the estimation of linkage distance between two markers is
based on the frequency of recombination during the formation of gametes. Genes which
co-segregate during meiosis are said to be linked, and a collection of linked genes on the
same chromosome is then referred to as a “linkage group.” The distance between these
genetic markers is expressed in centiMorgans (cM). The measure of 1 cM is equal to a
recombination rate of 1%. In chicken genome studies, it is estimated that ch is about
equivalent to about 500 Kb. The current consensus concerning the chicken genetic map
contains 1889 loci which describe 50 linkage groups (Groenen et al., 1999). This map
spans 3800 cM, which is considerably larger than previous estimates for the chicken
genome (Levin et al., 1994). The map contains 350 markers within expressed sequences,
201 of which represent identified genes sequences that have significant sequence
identities with known genes.

In contrast, a physical map is a map that displays the position of genes on the
chromosome, usually according to the length of DNA between them. The relation of
genetic distance to physical distance is not uniform, due to the fact that recombination

does not occur evenly along each chromosome or species. On a genetic map, marker

26

 

genes are used as “landmarks” which can then be used to follow the inheritance of
sections of a chromosome; physical maps, on the other hand, mainly provide a direct
measurement of the physical distance along a chromosome.

A large collection of good genetic markers is needed to map QTLs and genes that
determine performance and reproduction traits as well as genetic diseases. In general,
there are two types of markers that are used; they are designated as Type I and Type II.
A type I marker is used within a certain coding region of a specific gene which can be
identified, for example, by means of restriction fiagment length polymorphism (RF LP)
analysis (Fries et al. , 1993). Type 11 markers are anonymous markers that are usually
used for non-coding regions of the genome, such as microsatellite markers. Although a
lot of genetic markers have been described for both human or livestock genomes, many
of the markers which were originally described are only contain two alleles and,
therefore, are less useful in the analysis of disease resistant genes or QT'Ls (Nakamura et
al., 1987). Polyallelic markers, such as microsatellites, are most useful in mapping
QTLs.

When QTLs have been successfully mapped, they could be employed in breeding
programs through the use of "marker-assisted selection." Marker-assisted selection,
selection for favorable QTLs via selection of linked markers, is based on the fact that
QTLs may be located on marker-linked genomic regions (Soller et al. , 1976; Soller and
Beckrnann, 1983; Dentine, 1990; Lande and Thompson, 1990). Dentine (1994) suggests
that the utilization of markers in selection will be most effective for operations with high
selection intensities, multiple offspring from individual matings, and the potential of .

selecting individuals based on numerous progeny, as is the case with poultry and swine.

27

Dentine (1994) points out, however, that the use of linked markers instead of QTL alleles
themselves might complicate selection, since recombination could occur between the
markers and QTLs. Hence, we risk losing the QTLs and increasing the errors that are
made in breeding strategies that stretch over several generations. The distance between
related markers, therefore, will be key to identifying precise QTL positions.
The Current Status Of AID-Q T L Research

It has been demonstrated that a component of MD resistance is associated with
the MHC (B) locus, which encodes Class I and II MHC antigens (Calnek, 1985). But
other loci must also be important since chicken line 6 (the resistant line) and line 7 (the
susceptible line) share the same B locus yet show significantly different levels of
sensitivity to MDV infection. Recently, l4 QTLs have been identified in F2 chickens
from the intercross of inbred line 6 and line 7 that confer resistance to MD (Figure 2),
through the use of biometrical analysis and microsatellite markers (Yonash et al., 1999).
Unfortunately, many of these QTLs were found in large intervals or linked only to a
single marker. So as to better define the location of these QTLs, additional markers need
to be mapped in these regions. The development of recombinant congenic strains (RCS),
using lines 6 and 7, will provide a powerful tool for further refining the location of these
QTLs.
Specific Aims

Since MDV is a herpesvirus that causes malignant T-cell lymphoma in chickens,
increasingly effective management of the disease will require a better understanding of
the molecular basis for viral tumorgenicity, which will in turn provide a valuable

reference for safer vaccine deve10pment. There are two specific goals in this project.

28

One is to identify host protein(s) that interact with the MDV serotype 1 specific
gene, short open reading frame 2 (SORF2) protein. The other is to further evaluate
the potential association of SORF2-interacting protein with resistance to Marek's
disease. Further exploring the interactions between novel virus-host proteins has great

promise for advancing our current level of knowledge concerning the molecular biology

of MDV.

29

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Figure 2. MD QTL on East Lansing chicken genetic map. Bold
and unbold circles represent significant and suggestive QTL,

respectively. QTL=quantitative trait loci.

30

CHAPTER H

Identification of Chicken Growth Hormone Interacting with MDV SORF2

Introduction

Marek’s disease virus (MDV) is a naturally occurring oncogenic avian
herpesvirus that causes lymphoproliferation and induces neoplastic disease in chickens.
A key goal of research on Marek’s disease is to identify the gene(s) in MDV that promote
oncogenesis. One approach to accomplish this goal is to investigate the genes which
function in viral replication, attenuation, latency, and transformation.

Current sequence information suggests that MDV is colinear with the HSV and
VZV genome (Buckmaster et al., 1988). A number of HSV-homologous genes have
been identified and investigated. These genes include gB, gD, gE, g1, gH, ICP4, TK,
VP16, and ICP27 (Ross et al., 1991; Ren et al., 1994; Brunovskis and Velicer, 1995;
Koptidesova et al., 1995; Boussaha et al., 1996). However, there are also novel MDV
genes including short open reading frame 1 (SORFl), 2 (SORF2), and 3 (SORF3) which
are located within the MDV US region. The functions of these remain unknown.

The gene SORF2 is of special interest because a retrovirus LTR region integrated
upstream of the SORF2 gene, which yielded attenuated oncogenicity in the MDV
recombinant strain RMl (Witter et al., 1997). Isfort et al. (1992) have shown that co-
infection of the retrovirus reticuloendotheliosis virus (REV), and MDV in cultured cells
results in integration of the REV LTR into MDV genome. It was observed that the stable
integration of the LTR predominantly occurred at the junctions of the unique and repeat
regions (Jones et al., 1993). In fact, sequence data of the junction of the UL, TRL, and

IRL indicated a region of high homology to the LTR region of REV (Isfort et al., 1992).

31

In addition, this is the region that shows significant heterogeneity among MDV strains.
The incorporated LTR sequence may regulate the transcription of genes around the repeat
regions that may lead to phenotypic changes of MDV. The RMl clone is derived from
the JM strain of MDV through REV insertional mutagenesis. It contains REV proviral
LTR sequences inserted at the junction of the internal short repeat and unique short
regions of the genome (Figure 3) presumably due to homologous recombination.

The RM] clone appeared attenuated for oncogenicity but retained other in viva
properties of virulent viruses including thymic and bursal atrophy, early
irnmunosuppression, early cytolytic infection followed by effrcient replication, and
contact spread. Those characteristics are normally absent in attenuated strains (Witter et
al., 1997). In addition, chickens vaccinated with RMl clones were protected against
challenge with virulent MDV, and levels of protection exceeded those of other attenuated
serotype 1 vaccine virus (Witter et al., 1997). Molecular analysis indicated that RMl
overexpressed a 3 .2 Kb transcript initiated from the LTR promoter which extended across
the coding sequences of SORF2, US1, and USlO, terminating after the USlO-proximal
poly (A) signal in the US region (Jones et al., 1996). There is no evidence to indicate
that the 132 bp repeat region contributed to the attenuation of RM] oncogenicity since
expansion of the 132 bp repeat was not observed. No other gross alteration of the RM 1
strain genome was detected when compared to that of the oncogenic JM parent virus.
Genes expressed from the inserted LTR promoter might potentially be responsible for
altered biological properties of RMl MDV. This phenomenon prompted investigation
into the biological role of SORF 2 during MD oncogenesis. Since the SORF2 is an MDV

unique gene and one of the overexpressed genes of RMl strain. Detailed studies on the

32

 

Figure 3. Schematic representation of REV LTR inserts in RMl
clone. REV=reticuloendotheliosis virus; LTR=long terminal repeat;
SORF2=short open reading fiame 2; USl=unique short 1;
USlO=unique short 10.

33

biological characteristics of RMl and genes affected by LTR transcriptional activation
are necessary for a better understanding of MDV oncogenesis.

SORF2 is a MDV serotype l-unique gene presumably encoding a l79-amino-acid
protein. However, the actual existence of the SORF2 gene product in a wild type virus
infected cells has yet to be confirmed. A comparison of its predicted amino acid
sequence with those of other herpesvirus genes demonstrates that SORF2 might contain
three recognizable domains with homology to human cytomegalovirus US22, UL36, and
human herpesvirus 6 EPLF 3. The detailed function of these genes remains unknown.
However, studies suggest an association with transactivation ability (Tomley et al., 1988;
Stasiak and Mocarski, 1992; Kashanchi et al., 1994; Nicholas and Martin, 1994).
Deletion experiments performed in the MDV US region demonstrated that the SORF2
gene is dispensable for growth in cell culture as well as for in viva growth (Purcell et al.,
1994; Purcell et al., 1995). In an attempt to delineate the role of SORF2 during MD
oncogenesis, the yeast two-hybrid system was used to screen for host protein(s)
interacting with the SORF2 protein. Identification of protein(s) interacting with SORF2
may provide a significant insight into the function of this novel gene.

The yeast two-hybrid system (Fields and Song, 1989) has been used successfully
to detect protein-protein interactions of various species. The yeast two-hybrid system
relies on the modular nature of eukaryotic site-specific transcriptional activators to
generate a transcriptional signal from the interaction of a protein (as bait) fused to a
DNA-binding domain with another protein (as prey) fused to a transcription activation
domain. Interaction between the bait protein and a partner polypeptide encoded by a

member of a cDNA library will be detected by activation of the selective marker gene

34

and the reporter gene in the appropriate yeast strain. It is a powerful genetic screening
tool since the two-hybrid system detects protein-protein interactions occurring directly in
vivo.

In this chapter of my thesis, I have shown that the SORF2 protein is expressed in
infected cells both in vitro and in viva. In search of potential functional protein partner(s)
SORF2 was used as the bait in yeast two-hybrid screening. Chicken growth hormone as
isolated from a cDNA library constructed from splenic T cells was found to interact
specifically with SORF 2 protein. In order to corroborate the interaction detected in the
yeast two-hybrid system, the isolated growth hormone cDNA was used to synthesize
corresponding polypeptides in E. caIi, which then were assayed for their ability to
interact with SORF2 in vitro. Co-immunoprecipitation of SORF2 and the interacting
protein, grth hormone, using specific antibodies confirmed the biochemical evidence
for the observed protein-protein interaction. Results gathered from both in viva and in
vitro co-localization assays also provided further evidence for our observation.

Materials and Methods
Cells and Virus Stock

Chicken embryo fibroblasts (CEF) cells were grown in Leibowitz-McCoy
medium (GIBCO, BRL), supplemented with 4% calf serum (growth medium) or 1% calf
serum (maintenance medium). Primary or secondary CEF cultures were prepared as
described (Silva and Lee, 1984) and infected with the following cell-associated serotype
1 MDVs: pathogenic Mdl 1, JM102, Md5, 648A, CU2, 584; cell cultured-attenuated

JM102, Md] 1; naturally attenuated CVI988.

35

Sequence Comparison

Total cellular DNA was extracted from various MDV serotype 1 infected CEF
cells by standard methods (Sambrook et al., 1989). SORF2 specific primers (Operon
Technologies) were designed to amplify the SORF 2 gene from each strain by polymerase
chain reaction (PCR). The primers were as followed: forward, 5’ ATG CAG CGC CAA
ACC GGA CAT, and reverse, 5’ CTA ATG TAC TAG TTG CTC TAT. PCR was
carried out on a MJ Research Thermal Cycler with the following cycles: 94°C, 2 min; 30
cycles of 30 sec at 94°C, 30 sec at annealing temperature 55°C and 30 sec at 72°
followed with a 5 min extension at 72°C. Each PCR reaction mixture contained 25 ng of
cellular DNA, 200 pM of each dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgC12, 1.0 U Taq DNA polymerase (Life Technologies, Inc.). PCR products were
cloned into the TA cloning vector for sequencing. Sequencing analysis was done by an
ABI 377 automatic DNA sequencer. Sequence comparison was done using the
DNASTAR program (DNASTAR, Inc.).
Generating Antibodies Specific to SORF 2 Protein

In order to overexpress the SORF2 protein, the PCR product of SORF2 with
restriction sites for BamHI and Hind III incorporated into the 5’ end of forward and
reverse primers, respectively, was cloned into a pET28a expression vector (N ovagen,
Inc.) at its BamHI and Hind 111 sites. A positive pET-SORF 2 recombinant was then
sequenced to confirm it contained the correct reading frame. The host strain E. coli BL21
used for cloning and expression was prepared by standard procedures (Sambrook et al.,
1989). The expression and purification of SORF2 recombinant protein were according to

the manufacturer’s procedure (N ovagen, Inc.). Briefly, pET-SORF2 recombinant protein

36

expression was induced with 0.4 mM isopropylthio-B-D—galactopyranoside (IPTG) for 5
hours at 30°C with 250rpm shaking when OD600 reached 0.6-1.0 measured by
spectrophotometer. The cells were harvested when OD6oo reached 2.5-3.0 by
centrifugation at 5000 xg for 5 min at 4°C. The pellet was washed with 50 ml phosphate
buffered saline (PBS), and resuspended in 20 ml PBS. The cell suspension was
sonicated, and the cell debris was removed by centrifugation. For purification of pET-
SORF2 recombinant protein, a NiZ+-afi'rrrity His-Bind Resin column was used to bind a
stretch of 6 consecutive histidine residules expressed from the pET28a vector. The
supernatant was applied to the Ni2+-His column pre-equilibrated with the binding buffer
(5 mM irnidazole, 500 mM NaCl, 20 mM Tris-HCl pH 7.9) plus 6 M urea, the column
was further washed with washing buffer (60 mM irnidazole, 500 mM NaCl, 20 mM Tris-
HCl pH 7.9). The target protein was recovered by elution with 1 M imidazole.
Following dialysis against water overnight, total proteins were purified by preparative
sodium dodecyl sulfate (SDS)-8% polyacrylamide gels and used as antigen to prepare
polyclonal antisera by immunizing rabbits (Bio Syntheses, Inc.).
Yeast Two-Hybrid Screening

Two-hybrid library screening is based on the MATCHMAKER LexA system
(Clontech Laboratories, Inc). The yeast reporter host strain Saccharamyces cerevisiae
EGY48 was grown in YPD (yeast extract/pepton/dextrose) or appropriate selection
medium to maintain plasmids. Yeast transformation was done by the lithium acetate
method as recommended by the manufacturer.

SORF2 was inserted in-frame into the pBD-Lex A vector downstream of the

binding domain between its BamI-II and Natl restriction endonuclease multiple cloning

37

sites. The insert subcloned into pBD-LexA was derived from PCR amplification with the
BamHI, Natl nucleotide sequences sites incorporated into the 5 ’ end of forward and
reverse primers, respectively. A chicken cDNA library (kindly provided by Dr. Robin
Morgan) obtained from splenic T cells was fused with the pB42AD vector. Plasmid
p8op-lacZ was used for integrating the LacZ reporter gene into host strain EGY48
chromosome via transformation to create the EGY48 (p80p-lacZ) strain.

SORF2 in the pBD-LexA vector was used as a bait to screen the chicken spleen
cDNA library in the yeast expression vector system. The protein-protein interaction
screen was performed with a sequential transformation procedure. The yeast reporter
strain, EGY48 (p8op-lacZ) was first transformed with the bait, pBD-SORF 2. Then 100
pg of library plasmid DNA was introduced into the yeast strain expressing the pBD-
SORF 2 hybrid protein. Approximately 1.5 x 106 yeast transformants were selected on
seventy 15 cm plates with synthetic dropout medium without histidine, tryptophan, and
uridine (-His, -Trp, -Ura). After a 15-day incubation at 30°C, all clones were scraped and
replated at high density on -—Leu SD induction medium (SD/Gal/Raf//-His/-Leu/-Trp/-
Ura) plus 5-hromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal) for an additional
15 days. The resulting blue colored transformants were streaked onto SD/-His/-Trp/-Ura
synthetic medium plates for B-galactosidase (B-gal) assays. For the B-gal assay, yeast
transformants were incubated for 4 days at 30°C, then transferred to Whatrnan #1 filter
paper (Whatrnan, Inc.). The filter was immersed in liquid nitrogen for 15 sec, thawed at
room temperature, and then placed on top of another Whatrnan #1 filter presoaked in Z

buffer (60 mM NazHPO4, 40 mM NaHzPO4, 10 mM KCl, 2 mM MgSO4, pH 7.0) with

38

0.27% B-mercaptoethanol and X-gal enzyme substrate at 0.75 mg/ml. The filters were

incubated at 30°C for at least 2 hours or until blue color developed.
Cloning of Interacting Proteins

The plasmids from identified positive clones were isolated by standard procedures
(Sambrook et al., 1989), then transformed into E. cali KC8 competent cells by
electroporation. Transformed cells were plated onto M9/-Trp plates (M9 medium
contains 100mM NazHPO4, SOmM KH2P04, 50m M NaCl, 10 mM NH4C1, 20 mM
MgSO4, 1% glucose, and 10 mM CaClz, pH 7.4) as recommended by the manufacturer.
Plasmids then were isolated and subjected to nucleotide sequencing using the pB42AD
sequencing primer provided with the screening kit. Sequence data were used for
homology searching with the BLAST program.
In vitro Binding Assay

In order to confirm the interaction detected fiom the yeast two-hybrid system, a
glutathione S-transferase (GST) fusion protein of structural peptide of GH was generated
by inserting it in frame into the pGEX-3X vector (Pharrnacia Biotech, Inc.). The GST-
GH plasmid was expressed in the E. cali BL21 DE3 strain (Gibco BRL), induced with
IPTG, and a cell pellet was prepared as described previously. For purification of the
GST-GH fusion protein, Triton X-100 was added into a 20 ml sonicated E. cali cell
suspension (1% final concentration in PBS), and gently mixed for 30 min. The cell
debris was removed by centrifuging at 12,000 g for 10 min at 4°C. One ml PBS-
equilibrated 50% slurry of Glutathione Sepharose 4B (Pharrnacia Biotech Inc.) was added
to the supernatant and incubated for 30 nrin with gentle agitation at room temperature.

The sepharose was washed with PBS and collected by centrifugation several times. The

39

GST-GH fusion protein (or GST protein alone as a negative control partner) was then
used for the in vitro binding assay without eluting from the sepharose beads.

In vitro synthesis of protein was done by the Single Tube Protein System 3
(STP3) (Novagen). pET-SORFZ plasmid containing the entire SORF 2 coding sequence
was used as a template for a coupled T7 -directed in vitro transcription-translation
reaction according to the manufacturer’s instructions. During the incubation with rabbit
reticulocyte lysates, 4O uCi [35S]-methionine (New England Nuclear, Life Science
Products) was added to the mixture. Incubation was at 30°C for 30 min. The reaction
product (2 ul) was analyzed by 8% SDS-polyacrylamide gel electrophoresis (SDS-
PAGE), while 10 ul of the sample was used for the in vitro protein binding assay.

Both the GST protein control and the GST-GH fusion protein were incubated with
[35S]-methionine labeled pET-SORF2 protein translated in vitro at room temperature with
mild agitation for 30 min, followed by several washes with PBS. The supernatants from
each wash were collected for further analysis. Bound protein was eluted with elution
buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0). All the samples were
subjected to 8% SDS-PAGE analysis.

Immunoprecipitation and Co-Immunoprecipitation

MDV infected and mock infected CEF cells were labeled with 100 uCi [35$]-
methionine at 72 hours post infection for 4 hours. Then cultured cells were washed twice
with cold PBS, and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1%
Triton X-100, 0.1% SDS, and 25 mM PMSF). Cytoplasm extract was obtained after
centrifugation then incubated with normal rabbit serum and protein A-sepharose for 1 hr.

Supematants were obtained by centrifugation and then gently mixed with rabbit anti-

40

SORF2 antiserum or pro-absorbed anti-SORF 2 antiserum and protein A-sepharose for 1
hr. Following serial washing with PBS, precipitates were resuspended in 2X
electrophoresis sample buffer (10 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 10% B-
mecaptoethanol, and 0.006% phenol red), boiled for 5 min and subjected to
electrophoresis. The pre-absorbed antiserum was prepared by incubating anti-SORF2
antiserum with recombinant pET-SORF2 protein synthesized in E. coli at 4°C with gentle
agitation overnight. For co-immunoprecipitation, GH cDNA was digested from pB42AD
vector DNA and cloned into eukaryotic expression vector pcDNA3 using EcoRI and
Xhal cloning sites. Cell lysates from the MDV infected or mock infected cells were
obtained and prepared as described above except replacing RIPA buffer with a mild lysis
buffer (containing 142.5 mM KCl, 5 mM MgC12, 10 mM HEPES pH 7.5, 1 mM EGTA,
0.2% NP-40, and protease inhibitors). Cell lysates were incubated with in vitro translated
structural GH peptide, synthesized from pcDNA3-GH with or without [3 5 S] labeling, and
10% (v/v) protein A sepharose. After addition of either rabbit anti-SORF2 polyclonal
antibodies or mouse anti-GH monoclonal antibodies (provided by Dr. Luc Gerghman,
University of Texas), immunocomplexes were precipitated with protein A-sepharose.
Precipitates were washed extensively and analyzed by SDS-PAGE.
Transient Expression of truncated cGH

The pcDNA3 -GH construct was prepared for transfecting into MDV infected or
mock-infected CEF cells. Cells were seeded on coverslips carried by 35 mm culture
plates. Transfection was carried out using M199 medium (Gibco, BRL) pH 7.2 and the
calcium phosphate procedure. Briefly, the transfection mixture contained 3 ug plasmid

DNA in 0.125 M calcium chloride solution, to which an equal amount of 2X Hepes

41

solution (140 mM NaCl, 1.5 mM NazPO4, 50 mM Hepes pH 7.5) was gently added. The
mixture was held at room temperature for 30 min before adding to culture plates. Cells
were shocked with 15% glycerol solution for 5 min after 4 hr of incubation in
transfection mixture. After removing the glycerol, cells were washed with PBS twice,
then re-fed with a normal CEF culture medium.

Indirect Immunofluorescence Assay (IFA)

Samples taken from MDV infected cells, mock-infected cells, and pcDNA3-GH
transfected cells were subjected to IF A studies. Cells were originally seeded on
coverslips and fixed with ice cold acetone for 5 nrin and then air-dried. IF A was
performed as follows: samples were incubated with anti-SORF2 antiserum, pre-absorbed
anti-SORF2 antisertun, or anti-GH antiserum diluted 1:1000 in PBS for 30 min at room
temperature. Cells were then washed with PBS for 15 min. The secondary antibodies,
either goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgG or goat anti-
mouse Texas Red conjugated IgG (Kirkegaard & Perry Laboratories), were diluted
1:1000 in PBS and incubated with samples for an additional 30 min. The coverslips were
then rinsed extensively and sealed with 50% glycerol in PBS. All the samples were
viewed with the laser scanning confocal microscope (Carl Zeiss, Inc.) in the photographic
facility, Michigan State University. The Adobe photoshop program (Adobe Systems,
Inc.) was used to process the images.

Immunohistochemical Staining

Various MDV serotype 1 infected birds with observed tissue lesions, organ

enlargement, or tumor development were obtained fi'om the Avian Disease and Oncology

Laboratory (ADOL). Immunostaining of tissues for SORF2 or cGH was performed using

42

the biotinylated horseradish peroxidase complex (ABC) system (Vector Laboratories).
Sections were incubated sequentially with blocking serum, antibodies, and washing
buffers according to the manufacturer’s instruction. Briefly, unstained forrnalin-fixed,
paraffm-embedded sections of tissues mounted on microscope slides were deparaffinized
by immersion in xylene, followed by rehydration in graded ethanol solutions of
decreasing concentration. The slides were then immersed in methanol containing 2.5%
H202 for 10 min at room temperature to inactivate endogenous peroxidases. The slides
were rinsed in PBS 3 times for 2 rrrin each, followed by incubation in protease solution
for 10 min and another rinse with PBS. To minimize nonspecific background staining,
the sections were incubated in a serum blocking solution for 10 nrin. The sections were
then stained with primary antibodies (either cGH or SORF2) diluted 1:1000 in PBS for
45 min, followed by rinsing with PBS, then incubated with biotinylated secondary
antibodies (either biotinylated anti-rabbit IgG or anti-mouse IgG) for 10 min and further
rinsing with PBS. Color development was done with a peroxidase system in which Nova
Red was used as the chromogen. Negative controls consisted of staining non-infected
tissues, as well as leaving out the primary antibody. The positive control consisted of
staining sections of pituitary gland for cGH, as well as staining sections of RM] infected-
tissues for SORF 2. Finally, slides were rinsed in distilled water and counterstained with
hematoxylin for 2 min and examined microscopically.
Immunoelectron Microscopy Analysis

Immunogold labeling was carried out on ultrathin cryosections. Initially,
neoplastic spleen tissue was fixed in formalin and put into a paraffm block. Routine

histological stains were obtained from sections. The block then was deparafflnized and

43

placed in glutaraldehyde and embedded on Ni-grids (Electron Microscopy Science, Inc.).
One micron thin sections were obtained using a LKB Ultrarnictrome, followed by
sequential incubation with blocking serum, antibodies, and washing buffers, as suggested
by the manufacturer. The secondary antibodies used for SORF 2 were the goat anti-rabbit
IgG conjugated with 40 nm gold particles, whereas for cGH were the goat anti-mouse
IgG conjugated with 20 nm gold particles (Ted Pella, Inc.). Finally, the sections were
stained with uranyl acetate and lead citrate and examined with a Philips 301 Electron
Microscope at the Division of Pathology, Michigan State University. Sections of CEF
cells were prepared by the same procedures and used as negative controls.
Results

The SORF 2 Gene Encodes a Functional Polypeptide

In order to generate polyclonal antibodies specific to SORF2, the polypeptide
corresponding to the full-length SORF2 protein was produced in E. cali as a histidine-
tagged protein using the pET28a vector system (N ovagen, Inc.). The polypeptide was
purified using Ni2+-chromatography and injected into rabbits to induce the production of
anti-SORF 2 antibodies. Rabbit antiserum were collected and used to monitor protein
expression encoded by the SORF 2 gene in vitro and in viva. To test the specificity of
rabbit anti-SORF 2 polyclonal antibodies, immunoprecipitation analyses of MDV-infected
CEF cells with the rabbit anti-SORF2 antibodies or anti-SORF2 antibodies pre-absorbed
with purified SORF2 protein were performed. Results in Figure 4 indicated that a major
band corresponding to a polypeptide with molecular weight 21 kDa was precipitated with

anti-SORF2 polyclonal antibodies in MDV-infected CEFs. Presumably it was SORF 2

Anti- Pre-
SORF2 absorbed
Ab Ab

>
D
E
+
in
m
U

CEF
CEF + MDV

 

Figure 4. Immunoprecipitation analysis of MDV-infected CEF.
Left two lanes=Rabbit anti-SORF 2 polyclonal antibodies were used
in an immunoprecipitation assay with MDV-infected CEF cells
labeled with 35S-methionine. A 21 kD polypeptide (indicated by the
arrow) was preciptated and visualized by radioautography. Right
two lanes=the same as before except the anti-SORF2 antibody was
preabsorbed with recombinant SORF2 protein.

45

protein expressed upon MDV infection. To confirm this, immunoprecipitation analysis
with the anti-SORF2 polyclonal antibodies pre-absorbed with purified recombinant pET-
SORF2 protein was carried out. Results in Figure 4 shown that the 21kDa peptide was
not detected in infected cells while using pre-absorbed antiserum. Immunofluorescent
assay was performed in MDV infected cells using pre-absorbed antiserum indicated a
negative result (data not shown) suggested that the obtained anti-SORF2 polyclonal
antibodies were specific to SORF 2. Taken together, we concluded that the major 21kDa
peptide seen in the result of immunoprecipitation were mostly likely to be MDV SORF 2
products.

The SORF2 gene is a unique MDV serotype 1 gene which is presumed to encode
a 179 amino acid protein (z21 kD). DNA sequence comparison among 9 different strains
including Mdl 1/14 (fourteen cell-culture passages), Mdl 1/78, JM102/l3, JM102/60,
Md5/15, 648A/21, 584/14, and CVI988/20 did not indicate there was any variability in
SORF2 sequence (Figure 5). This result clearly suggests that SORF2 is conserved among
various strains and stable while passing in cultured cells.

In order to study the localization and expression pattern of SORF2 in MDV
infected cells during plaque development, immunofluorescent labeling of SORF2 was
performed at various time points after virus inoculation. Results were monitored by
confocal microscopy. The confocal images in Figure 6 demonstrate that SORF2 was
found in both the cytoplasm and nucleus at early stages post infection. However, it was
localized only in the cytoplasm once plaques start appearing. This was an important
observation since SORF 2 was suggested to have homology to transactivation domains of

other genes from herpesviruses. In theory, transactivation function would normally be

46

41

81

121

161

201

241

281

321

361

401

441

481

521

ATGCAGCGCC

CCGGTTTGGA

TGGCAGAGAT

AATGAGCCCA

AGGGGATTCG

ACTGCCCTTT

GGGGGTTTGG

TGTTAGAGTA

TAAGATGGAA

CTGGCAACGG

GGGGAGAGGT

GTTAGCCACG

AAATCTTTAG

TAGAGCAAGT

AAACCGGACA

ATCGCAGGGG

GGCAAAGATG

TTTTGATTCC

TGAATTGGTC

GAAAAGTGTC

AGGCCAGCTT

TTGTGAAGCA

ATAGTAGGCA

GGATGCAATT

ATATGCCTAT

AGTTTTTCCG

GGAGGGAGGT

AGTACATTAG

TATGGAAGAC

ACCGAGAATG

GATTGTTACA

GTCTACCATC

CGAAAATTCC

CCGATTTTTG

TCATAAAGGG

CTTTATTTAC

TTGTAGACGA

ACTCATTCTT

GAAGAAGATA

AATTCCTTGA

TTACCATTGT

Figure 5. Sequence of the SORF2 gene.

47

AAAAAGAGAA

CTTTTTCAGA

TGAAGGAATT

GCAGATCTCG

GTGGTCGTCT

TCTGAGAATT

CAGGAGGAGC

CACAACCTGT

TGTGCCATGT

GTTGCCGAGG

CTCTGCATAA

AATTGGAGTG

GGAGAATATA

 

Figure 6. Localization characteristics of SORF2 on cultured CEF
cells. A=mocked infected CEFs stained with SORF2 antibody.
B=MDV-infected CEF cells stained with SORF2 antibody. C=Same
as B except prior to MDV plaque formation. D=Same as B except at
higher magnification of the MDV plaque.

48

observed in the nucleus. However, our finding suggested that at least late in infection,
the bulk of MDV SORF2 remains outside of the nucleus.

To study further the possible expression of MDV SORF2 in infected tissues,
histochemical examinations of neoplastic organs from a diseased chicken were compared
to atrophied thymus from RMl infected chickens, as well as tissues from negative birds.
Moderate to strong immunolabeling for SORF2 was found among all tissues examined.
The MDV-free bird tissues or omission of primary antibody both showed no positive
staining for SORF2. As judged by anti-SORF2 staining distribution, vagus nerve and
feather follicle (about 2% of cells expressed) exhibited fewer SORF2- expressing cells
than MDV-infected spleen, thymus, and bursa (about 15% of cells expressed). Kidney
and ovary showed intermediate level of expression. Sections obtained from RM]-
infected thymus, as predicted, showed a higher level of staining even though a depletion
of lymphoid cells was noticed. The observation of a high amount of SORF 2 expression
in RMl-infected thymus confirmed the earlier evidence that SORF2 mRN A was
overexpressed in RMl strain infections (Jones et al., 1996). Tissues examined were
taken at 4 weeks post inoculation, and example results are shown in Figure 7.

Microscopy of irnmunoreactive sections revealed the expressed SORF2 proteins
were deposited mainly in the cytoplasm (Figure 7). These observations corroborated our
earlier in vitro finding, namely, that SORF2 was expressed in cytoplasm of CEF cells.
Taken together, we conclude that the SORF2 gene expresses a polypeptide corresponding

to 21 kD in cultured cell CEFs, as well as in infected birds.

49

 

Figure 7. Immunohistochemical staining of SORF2 in neoplastic
tissues. A=MDV-induced tumor from kidney. B=MDV-induced
tumor fi'om thymus. Magnification in both is 20X.

50

Identification of a Protein Homologue to Chicken Growth Hormone that Interacts with
MDVSORFZ Protein

In a screen to identify functional polypeptides that interact with the MDV SORF 2
protein, a chicken cDNA library derived from splenic T cells mRN A in the pB42AD
yeast expression vector was employed. The results are summarized in Figure 8.
Approximately 1.5 x 106 yeast transformants containing pBD-SORFZ, the bait,
transfected with library plasmid DNA were plated on SD/Gal/Rafl-UrN-His/Iew-Trp
plus X-gal plates. All transformants grown on this induction medium were LEU2
positive clones. After 15 d of incubation at 30°C, 7 LacZ positive colonies were isolated.
Among these, only 2 were confirmed to be positive upon B-gal assay and were processed
for firrther studies. After selecting against the pBD-SORF2 bait plasmid using M9/-Trp
medium, the prey plasmids were amplified in E. cali cells and subjected to nucleotide
sequence analysis. A database search and sequence alignment demonstrated in Figure 9
identified both sequences as corresponding to that of chicken growth hormone with an
open reading We coding for 216 amino acids (Tanaka et al., 1992). These two
independent clones were identical. Interestingly, both lacked the signal polypeptide and
the first amino acid of the structural polypeptide. The start codon ATG from the signal
peptide was retained on both clones (Figure 10) indicating that they might be artifact
cDNA clones perhaps due to mRNA secondary structure caused the skipping of this
missed region while coping the mRNA. However, this suggestion needs to be confirmed

by further experiments.

51

100 ug cDNA derived from chicken spleen

Transform yeast
Plate on SD/-Ura/-His/-Trp

1.5 x 106 clones

Plate on SD/Gal/Rafl-UrN-Hisl-Trp/Ieu
plus X-GAL

1.7 x 104 clones

Blue colonies
with X-GAL

7 clones

Plate on SD/-Ura/-His/-Trp
plus B-galactosidase assay

2 clones

Figure 8. Summary of yeast two-hybrid library screen.

52

l ggaggccgtt caagcaacac ctgagcaact CTCCCGGCAG GNE:ggctcc
51 aggctcgtgg ttttctcctc tcctcatcgc tgtggtcacg ctgggactgc
101 cgcaggaagc tgctgccacc TTCCCTGCCA TGCCCCTCTC CAACCTGTTT
151 GCCAACGCTG TGCTGAGGGC TCAGCACCTC CACCTCCTGG CTGCTGAGAC
201 ATACAAAGAG TTCGAACGCA CCTATATTCC GGAGGACCAG AGGTACACCA
251 ACAAAAACTC CCAGGCTGCG TTTTGTTACT CAGAAACCAT CCCAGCTCCC
301 ACGGGGAAGG ATGACGCCCA GCAGAAGTCA GACATGGAGC TGCCTCGGTT
351 TTCACTGGTT CTCATCCAGT CCTGGCTCAC CCCCGTGCAA TACCTAAGCA
401 AGGTGTTCAC GAACAACTTG GTTTTTGGCA CCTCAGACAG AGTGTTTGAG
451 AAACTAAAGG ACCTGGAAGA AGGGATCCAA GCCCTGATGA GGGAGCTGGA
501 GGACCGCAGC CCGCGGGGCC CGCAGCTCCT CAGACCCACC TACGACAAGT
551 TCGACATCCA CCTGCGCAAC GAGGACGCCC TGCTGAAGAA CTACGGCCTG
601 CTGTCCTGCT TCAAGAAGGA TCTGCACAAG GTGGAGACCT ACCTGAAGGT
651 GATGAAGTGC CGGCGCTTCG GAGAGAGCAA CTGCACCATC TGAGGCCCCG
701 TGCTGCGCCA TGGCTGACGG CCCTGTCCCC CCCCCCCCCT CCTCCCCGTC
751 ACCAAAAACA CGAGGAATAA ACCC

Figure 9. Sequence of the yeast two-hybrid clones. Comparison of the
sequences for growth hormone (GH) clones recovered in the yeast two-

hybrid screen to the published cDNA sequence. The bases in capital letters
are common to both sequences while those in small letters are found only in
the published GH sequence. The ATG start codon at position 43 is boxed.
The signal peptide, which is missing in the yeast two-hybrid clones, spans
bases 46 to 120.

53

DNA (3.9 Kb)

  

I
.I
O
O
I
I
O
I
O
I
a
O
O
O
C
C
o
O
O
O
O
O
I
I.
.0

Ida mg n I. -- mRNA (774 bases)

AUG Signal
El nun-r Yeasttw
AU hybrid clone

Figure 10. Schematic representation of cGH genome structure and cDNA
clone obtained from chicken splenic yeast expression library screen. Each
box represents an exon. The shaded region represents the signal peptide.

54

Interaction of the cGH with SORF2

Experiments were performed to confirm direct binding of cGH to SORF 2 as
suggested by results of yeast two-hybrid screening. An in vitro protein binding assay was
performed using GST-fusion proteins. The GST-fusion protein, with or without the
added structural GH polypeptide (designed as sGH) identified from yeast two-hybrid,
was incubated with [3SS]-methionine-labeled SORF2 protein translated in vitro. After
extensive washing, bound protein and [3SS]-SORF2 were eluted with 100 mM
glutathione. Aliquots of the protein from the supernatants, washes and eluants were
resolved on SDS-PAGE and exposured to X-ray film. The results shown in Figure 11
demonstrated that, while SORF2 protein was not retained by GST protein alone, SORF2
can be retained by GST-GH fusion protein presumably due to the presence of GH. This
result is in agreement with the result of the yeast two-hybrid system assay and indicates
that the interaction between SORF2 and sGH is a direct and specific protein-protein
interaction without other intermediary factors (e.g., yeast proteins) involved.

The in vitro binding assay provided the biochemical evidence for the
hypothesized SORF2-cGH interaction. To further characterize the possibility of
interaction between native forms of the two proteins, co-immunoprecipitation using
either SORF2-specific or cGH-specific antibodies was carried out. Previous evidence
showed that cGH is not expressed in cultured CEF cells (data not shown), therefore, in
vitro translated sGH protein was used in this study. The fact that SORF 2 and sGH have
similar molecular weights complicates the analysis. Alternative [3 5 S] labeling of SORF2
or sGH was used to overcome this problem. MDV-infected CEF cell lysates were mixed

with [35S]-labeled sGH translated in vitro. Proteins were precipitated with anti-SORF2

55

GST-GH GST only
S W2 W4 W6 E S W2W4W6 E

 

 

~

    

Figure 11. In vitro protein-protein interaction assay. Labeled SORF2
was added to beads bound with GST or GST fused to GH.
S=supematant or unbound material; W2, W4 and W6=wash two, four,
and six, respectively; E=elution. The arrow indicates the location of
SORF2.

56

polyclonal antibodies then subjected to SDS-PAGE analysis. Under the same
precipitation conditions, [35S]-labeled MDV infected cell lysates were incubated with in
vitro translated unlabeled GH peptides, then precipitated with anti-GH monoclonal
antibodies. Results (Figure 12) indicate that SORF2-specific antibodies were capable of
co-precipitating sGH peptides only when the SORF2 proteins (fi'om cell lysates) were
present. Likewise, SORF2 proteins were co-precipitated by cGH-specific antibodies
together with sGH. Both co-precipitations showed equal efficiency. Taken together, the
above findings demonstrate that the observed protein interaction was specific and
efficient in vitro. It thus seems likely SORF2 and sGH interact with each other in viva.
Co-Localization of SORF 2 and cGH

To validate the SORF2-cGH interaction, we examined whether these two proteins
are capable of co-localizing within cells. Co-localization was determined using a double
immunofluorescence staining approach. As described previously, cGH is not detectable
in CEF cells. Therefore, transient expression of sGH was induced by introducing a
eukaryotic expression construct pcDNA3-GH into cultured cells. The pcDNA3-GH
plasmid DNA was transfected into MDV-infected CEFs 2 days post infection. Protein
expression was detected by IFA assay at 24 hours after transfection. In order to localize
the proteins, F lTC-labeled goat anti-rabbit IgG conjugate and Texas Red-labeled goat
anti-mouse IgG were used, followed by visualization with confocal microscropy. As
noted previously, we observed that SORF2 was expressed in both the nucleus and
cytoplasm before plaque formation. It was evident that transiently expressed sGH co-
localized with SORF2 in the cytoplasm (Figure 13). Another observation was both

SORF 2 and sGH were found mainly in the cell cytoplasm afier plaques appeared (data

57

SORF2 Ab GH Ab

GH labeled cells labeled
CEF +MDV CEF +MDV
.. 28 kD

 

Figure 12. Co-immunoprecipitation analysis of SORF2 and cGH.
Lysates fi'om uninfected and MDV-infected cells were precipitated
with antibody to SORF2 or GH. In the case of SORF2 antibody, in
vitro labeled GH was added. For the GH antibody, unlabeled in
vitro translated GH was added to labeled cell lysates.

58

 

SORF2 GH Co-Iocalization

Figure 13. Co-localization of SORF2 and cGH. MDV-infected CEF
cells were co-transfected with pcDNA3-GH plasmid. Protein
expressions were detected by immunofluorescence assay at 24 hours
after transfection. SORF 2 and GH are labeled with FlTC-labeled and
Texas Red-labeled antibody, respectively.

59

not shown). This result suggested that SORF2 and variant GH, if it ever exist, or secreted
structural GH which lacks the signal peptide are possible to interact if they present at the
same biological space.

cGH is a hormone secreted primarily from the pituitary gland. Previously, it has
been suggested that cGH may also be expressed in immune-responding tissues (reviewed
by Harvey and Hull, 1997). To confirm this observation, histochemical staining of
various normal and neoplastic tissues was performed. It was obvious that deposited cGH
proteins were detected in MDV-infected neoplastic spleen, thymus, ovary, kidney, and
liver, indicating that cGH is not solely a pituitary gland secreted protein and therefore
could potentially interact with SORF2. Partial results are demonstrated in Figure 14.
Positive staining for cGH in the bursa, spleen, thymus tissues (data not shown) of MDV
negative birds was also observed.

To identify colocalization in viva, double staining for cGH and SORF 2 was
performed by staining for GH antigen using 20-nm gold particles as markers followed by
staining for SORF 2 using 40-nm gold labels as markers. Samples were taken from
MDV-infected neoplastic spleen since the cDNA library used for yeast two-hybrid
screening was derived from spleen. The negative controls used included mock-infected
CEFs as well as the omission of primary antibodies in neoplastic spleen during the
staining process. Results were examined by transmission electron microscopy and are
presented in Figure 15. Not surprisingly, cytoplasmic reactions were observed in MDV
induced neoplastic spleen cells for cGH and SORF2, although staining was occasionally,
but rarely, observed in the nucleus as well. These in viva observations support the in

vitro observations that cGH and SORF2 proteins may co-localize in the same subcellular

60

 

Figure 14. Immunohistochemical staining of cGH in neoplastic
tissues. A=MDV-induced tumor from spleen. B=MDV-induced
tumor from thymus. Magnification in both is 20X.

61

      
 

-. :X .
o 7. -.-.o ll ”f

1 “a; 115.1%

Figure 15. Immunoelectron microscopy of cGH and SORF2 in
neoplastic spleen. A=system control where the SORF2 and GH
antibodies were omitted. B=same as A except SORF2 and GH
antibody were added. SORF2 and GH are labeled with 40 nm and
20 nm gold particles, respectively. The magnification is 57,000X.

62

compartments, but specific compartments could not be identified. Negative control
staining confirmed the specificity of the labeling observed. This further confirmed the
positive staining results were reliable.

Discussion

Previous studies suggested that overexpression of the SORF2 gene in a
recombinant MDV clone, RMl , possibly results in the attenuation of viral oncogenicity
(Jones et al., 1996; Witter et al., 1997). Prior to our studies, it was not clear whether
SORF2 was expressed in a natural infection. Here, we have demonstrated that serotype 1
viruses contain a functional SORF2 gene encoding a 21 kD polypeptide.

Sequence comparison among various strains including oncogenic, cell culture
attenuated, and naturally non-oncogenic strains indicated that there is no difference in
SORF2 sequence among those strains tested. FA and immunohistochemical staining
further confirmed that SORF2 is expressed in lytically infected culture cells and in
infected or neoplastic tissues. Taken together, we propose that normal expression of
SORF 2 itself is not directly associated with oncogenicity since SORF 2 protein is detected
in attenuated strains as well. Moreover, the failure to detect SORF2 in FA assay of tumor
derived cell lines MSBl, and RP] (data not shown) suggested that SORF 2 might be
involved in viral replication or latency/activation. Jones et al. (1996) reported that the
viral replication and/or spread is as least as rapid in RMl as in the fully virulent JM
parent virus. Apparently, overexpression of SORF2 does not impair the replicative
ability of the virus. However, deletion of the gene results in decreased viral plaque-
fonning units (Dr. Mark Parcell, personal communication). Thus, it remains possible that

SORF2 is involved in viral replication but is not essential.

63

Cytoplasmic localization of SORF 2, however, could suggest an alternative
hypothesis as to biological role. A yeast two-hybrid screen was utilized to identify
proteins that might interact with SORF 2. The cDNA library used was derived from
splenic T cells activated by the mitogen concanavalin A to mimic an immune response
stage after virus infection. Two clones of the chicken growth hormone gene were
isolated by this method. Further experiments were done to examine the interactions of
cGH with SORF2.

It was demonstrated that SORF 2 protein could directly and specifically bind to a
GST-GH fusion protein without other intermediary factors. In addition to in vitro
binding evidence, co-immunoprecipitation was used to confirm this interaction. Results
demonstrated that either sGH or SORF 2 protein could be precipitated by antibody to the
other protein when sGH protein translated in vitro was added to MDV-infected cell
lysates. cGH is a pituitary hormone, and the cGH gene contains an open reading fiame
encoding a signal polypeptide (4 kD) which functions in protein translocation joined to a
structural polypeptide (22 kD) involved in cGH signalling. The cDNA library used for
the yeast expression system was constructed from splenic T cells. Might immune cells
themselves be a source of GH? Recently, considerable effort has been expended in
evaluating the interactions of the endocrine and the immune system. Numerous such
interactions have been described between these two systems (reviewed by Auemharnmer
and Strasburger, 1995). Studies of human growth hormone (hGH) have shown it to play
an important role in the cellular and humoral immune system. Moreover, hGH peptides
have been shown to be synthesized and secreted by immunocompetent cells (Kao et al.,

1992; Lytras et al., 1993), with hGH receptors also being present on these cells (Barnard

et al., 1985; Badolato et al., 1994). Similarly, our studies in the chicken as well as those
of other groups, demonstrated the detection of cGH cDNA in spleen, thymus, and bursa
(Render et al., 1995), indicating there is a possible association between GH and the
immune response.

The cDNA clones isolated from the yeast two-hybrid screen encoded an altered
form of cGH (Figure 8), lacking the signal polypeptide and the first amino acid of the
structural polypeptide, indicating that the cDNA clones obtained derived from a 5’-
truncated cDNA or arose from alternative initiation or splicing of cGH mRNA. The
signal polypeptide of cGH consists of Exon 1 and part of Exon 2 (Figure 10). Sequence
data fiom our clones lack the entire signal polypeptide, as would be required for the cGH
polypeptide to be expressed intracellularly so that it could function in the two-hybrid
assay. There were no consensus splice sites or obvious alternate promoters flanking the 5 ’
end of the cGH coding sequence in the two clones. This finding suggests that the
deduced cGH may be a product of a 5’ truncated cDNA clone, which was selected by the
requirements of the two-hybrid assay system. However, the observation of human GH
mRNA derived by alternative splicing (McCarthy and Phillips, 1998) indicates that
alternative splicing or initiation of transcription of a cGH mRN A cannot be ruled out.

One major characteristic of yeast two-hybrid screening is that the protein
interaction must occur inside the nucleus. Any cGH cDNA in the two-hybrid library that
retain a functional signal sequence would likely result in secretion of the hybrid protein
and would therefore fail to be detected. The signal polypeptide functions in the secretion
of cGH, and it is cleaved during the secretion process. To test if the cDNA library used

contained complete cGH cDNA, PCR reactions were performed using primers upstream

65

of the ATG start codon (forward 5’-TGA GCA ACT CTC CCG GCA GGA) and
downstream of the signal peptide sequence (reverse 5’-TGG GAG CTG GGA TGG T'I‘T
ATG). As predicted, two fiagments of different sizes were amplified. Sequence analysis
confirmed that one fi'agrnent represented the complete cGH mRN A form, the other
fragment represented the altered form. This result confirmed that the complete cGH
cDNA was present in the library, however, but did not interact with SORF2 in the yeast
two-hybrid system as predicted. Whether the truncated cGH cDNA was generated in
cDNA synthesis or derived from a different cGH mRNA isofonn in the cell remains to be
determined.

Independent immunohistochemical staining experiments demonstrated that cGH
proteins exist in chicken immune cells (Render et al., 1995). It is now well established
that the activity and proliferation of lymphoid cells and lymphoid organs are stimulated
by growth hormone (reviewed by Gelato, 1993; Weijent and Blalock, 1995). Moreover,
cellular localization of growth hormone receptors and binding proteins in immune tissues
has been described (Hull et al., 1996). Our findings fiuther support the association
between GH and immune system. IFA co-localize truncated cGH and SORF2 in vitro,
EM study using irnmune-gold particles co-localized both proteins in viva. However,
further elucidation of the biological function(s) of the interaction is essential to reveal the
role of SORF2, if any, during MD oncogenisis.

An early observation indicated that rapid viral replication was observed in the
RM] strain (Witter et al., 1997). T-cell activation is a prerequisite for MDV infection
and cell transformation. The rapid replicative phenotype of RM] strain, presumably due

to unregulated viral gene expression, may limit the life span of infected lymphocytes

66

resulting in rapid cell death. The rapid cell death may limit resources available to the
infecting virus, therefore diminishing the T cell activation response. During the normal
viral cytolytic infection that SORF2 is possibly released from lytic cells. Thus, SORF 2
and circulated GH are possible to interact if they happen to locate at the same place.
Whether the RM] caused phenomenon was due to the overexpression of SORF2 genes
resulting undesired SORF2-cGH interaction remains unknown. Further study will be
required to examine this hypothesis.

A ninnber of questions concerning the role of MDV transformation in the natural
host have yet to be addressed. The identification of viral gene products and the
elucidation of their fimction will substantially strengthen our understanding of specific
virus-host interaction. Cell transformation is one of the defining characteristics of
oncogenic MDV. It consists of a complex and highly regulated process beginning with
the specific recognition between viral and host proteins, which leads to tumor formation
in certain organs. The identification of the SORF2-cGH binding might provide a

valuable example of one such interaction.

67

CHAPTER III
The Association Between the Chicken Growth Hormone Gene and Resistance

To Marek’s Disease

Introduction

Marek’s disease (MD) is a lymphoproliferative disease of chickens that affects
peripheral nerves and most visceral organs. Diseased chickens may present varying
severity of symptoms, ranging from chronic peripheral nerve neuropathy, characterized
by demyelination and partial or complete paralysis, to an acute cancerous disease in
which multiple visceral lymphomas can be observed (Payne, 1985; Calnek and Witter,
1997). The causative agent of MD is a cell-associated herpesvirus named Marek’s
disease virus (MDV). MDV can interact differently with different types of cells resulting
in productive infection, latent infection, or cell transformation. To date, MD is fairly well
controlled by vaccines. However, MD vaccines control rather than eliminate losses from
MD because they do not block MDV infection and replication. It becomes obvious that
genetic resistance is needed to complement vaccinal protection.

One major goal in studying genetic bases for resistance and immunity to Marek’ 3
disease is to identify and characterize physiologic factors that regulate or affect optimal
immune system function, development, and regulation. A better understanding of the
function and structure of the individual factors will further genetic improvement of
chickens. The MHC, or B locus, of chickens is involved in the control of disease
resistance and immune responsiveness. The MHC genes in chicken are organized in two

genetically independent polymorphic gene clusters. The B complex and the recently

68

discovered pr-Y clusters both contain class I and class 11 genes which are believed to be
involved in mediating cell-cell interactions and antigen presentation in the immune
response (Miller et al., 1996). The B complex and pr-Y genes are on the same
microchromosome but are genetically unlinked (Fillon et al. , 1996; Miller et al., 1996).
Although the MHC is thought to play a central role in MD resistance; genetic studies
indicate that the contribution of resistance to MD by non-MHC genes may also be
significant (Groot and Alhers, 1992; Yonash et al., 1999).

Recently, advances in understanding chicken non-MHC loci involved in MD
resistance have been made using ADOL inbred lines 6 and 7. Line 6 is very resistant to
MD, but line 7 is very susceptible. They both share the generally resistant MHC B2
haplotype. Hence, it is believed that the response to MDV in line 6 and line 7 is
governed by non-MHC susceptibility genes. Comparison studies of these two inbred
lines have been focused on their virological and irnmurrlogical characteristics related to
resistance to MD.

Growth of MDV in cell cultures derived fiom either of the two lines of chicken is
similar (Shama and Purchase, 1974), whereas adsorption of MDV to spleen cells as well
as growth of the virus in chicken embryos or chickens is greater in line 7 than in line 6
(Lee et al. , 1981; Powell et al., 1982). Gross pathological changes in the lymphoid
organs during cytolytic infection are more severe and virus replication is greater in line 7
than in line 6 birds (Lee et al., 1981). When line 6 chicks that have undergone
thymectomy receive a transplantation of thymus fragments fiom line 7 birds, they show a
decrease in resistance to MD in comparison with normal line 6. These results suggest that

the lymphocytes of line 7 birds may be more susceptible to transformation (Powell et al. ,

69

1982, 1986). Another possibility is that line 7 lymphocytes may have more receptors for
MDV, or line 6 have fewer target cells (lymphoid cells) with the MDV receptor on their
surface (Powell et al., 1982). Moreover, studies also demonstrated that line 6 chickens
have a higher cellular immunity against MDV-infected cells and tumor cells than line 7
chickens (Lee et al., 1981). Given what we know so far, it is proposed that the
mechanism of MD resistance observed in line 6 chickens is operating mainly at the level
of target cells of viral infection, as well as the immune response to viral antigens and
tumor antigens (Lee et al., 1981).

Another differential immune response has been observed in macrophages. The
function of macrophages as part of the immune system is complicated. They play
important roles in immune monitoring, enhancing or inducing immune responses, and
under certain conditions they also inhibit the immune response of the host. In MD
resistance, macrophages on one hand inhibit the growth of virus or tumor cells but on the
other hand, they lower the responsiveness of normal lymphocytes to mitogens. In
general, there is no correlation between resistance to MD and various chicken lines with
different macrophage activity except in lines 6 and 7. In vivo studies point out that the
phagocytosis index of normal macrophages originating from line 6 is significant higher
than of those from line 7. Upon infection, both lines develop increased phagocytosis and
inhibition of MDV plaque formation; however, the effect is significantly higher in line 7.
The number of target cells against MDV is also found to be more in line 7 (Gallatin and
Longenecker, 1981). It is suspected that MDV itself or MDV related antigens may take
part in the mechanism of macrophage activation in line 7 chickens (Powell et al. , 1983).

Studies of macrophage-produced cytokines demonstrated that line 6 macrophages (LPS

70

stimulated culture supernatant fractions) produced greater T'NF-like factor, and lower
nitrite activity than line 7 macrophages (Dr. M. Qureshi, personal communication). This
observation may help explain the basis of intrinsic immune response differences between
these two lines.

The use of genetic markers coupled with a pedigreed reference population make it
possible to identify genome regions associated with disease resistance. This genetic
approach requires reference/resource families making use of diverse crosses to produce at
least three generation families demonstrating a measurable variation in quantitativetraits
among F2 individuals. Great efforts have been made towards identifying quantitative
trait loci (QTL) affecting MD susceptibility using co-segregation analysis in F2
intercross chickens bred from line 6 and line 7 (Vallejo et al., 1997; Yonash et al., 1999).
This 6x7-reference family offers the opportunity to identify non-MHC QTLs affecting
several components of MD susceptibility. Recent results from our studies suggest that
chicken growth hormone (cGH) gene could be a candidate for MD QTL.

It is becoming more apparent that the endocrine system plays a significant role in
the regulation of the immune system and cell transformation. Previous studies using a
yeast two-hybrid analysis identified cGH as a cellular protein that specifically binds to
the MDV SORF2 gene product. To further examine the relationship between the cGH
locus and MD incidence, the 6x7 F2 cross was employed to test for co-segregation of the
cGH gene and MD resistance.

Many studies demonstrate that cGH exerts a wide range of cell- and tissue-
specific responses, including regulation of lipid, nitrogen and carbohydrate metabolism,

as well as induction of cell growth and differentiation. These effects are mediated by the

71

binding of cGH to its cell surface receptor (GI-IR), activation of downstream cellular
signal transduction events and altered expression of specific genes in target cells.
Moreover, there is considerable evidence for the importance of GH in the maintenance,
control, and modulation of the immune system (reviewed by Harvey and Hull, 1997). It
is therefore possible that cGH may play an important role in MD oncogenesis, especially
as it interacts with the MDV unique gene product SORF2.

Indeed, immune function is impaired in GH deficiency and can be restored by
exogenous GH therapy (Khansari and Gustad, 1991; Corpas et al., 1993; Johnson et al. ,
1993). Immunostimulatory effects of GH include increased lymphocyte proliferation
(Yoshida et al. , 1992; Murphy et al. , 1993) and the production of cytokines
(Chandratilleke et al. , 1994; Kappel et al. , 1994), immunoglobulins (Kimata and
Yoshida, 1994), and other immune factors (Murphy et al., 1992). Immune cells and
immune tissues thus are likely to be target-sites for GH action, especially as the GHR is
structurally homologous with cytokine receptors (Wlodawer et al. , 1993). Hemopoietic
and lymphoid actions of exogenous GH include increased thymic size, thymocyte
proliferation and differentiation, proliferation of thymic epithelial cells and their secretion
of thymulin, activation and proliferation of lymphocytes, increased production of
cytokines, and the activation of monocytes, macrophages, phagocytosis, the generation of
superoxide anions, and the intravascular migration of immune cells (Aurenhammer and
Strasburger, 1995).

Immune tissues are not only GH target tissues, but also sites of GH synthesis. It
is well established that GH is derived from the pituitary gland via systemic circulation.

Classically, it has been considered to be an endocrine hormone with actions distant from

72

its site of production. Pituitary somatotrophs are primarily responsible for the synthesis
of GH within the pituitary gland, although GH synthesis also occurs within prolactin and
thyrotropin secreting cells (Harvey, 1995). It is suspected that a large portion of GH
circulates through binding to a protein similar in structure to target-cell GHR. Free GH
in the circulation degrades rapidly and only has a half-life of 20-30 minutes.

It is now known that GH gene expression is not only restricted to the pituitary
gland. GH irnmunoreactivity is found widespread in many tissues, and mRNA analysis
suggests almost ubiquitous expression of the GH gene or closely related genes. In vivo
studies showed that GH mRN A is present in human and rat peripheral blood leukocytes
and in the spleen, thymus, and bone marrow, in which about 10% of mononuclear cells
are GH irnmunoreactive (Weigent et al., 1988; Weigent and Blalock, 1991). The
translation of the GH transcript in immune tissues is indicated by the incorporation of 3H-
laheled amino acids into irnmunoreactive GH. Studies in chicken GH irnmunoreactivity
in the spleen, thymus, and bursa also detected GH concentrations at 10%, 2%, and 1%,
respectively, of that in the pituitary gland (Render et al. , 1995). Furthermore, the Pit-1
gene, a regulatory factor for growth hormone, was originally thought to be a pituitary
specific factor required for somatotroph proliferation and GH gene expression. It has
been found in pituitary thyrotrophs and lactotrophs, the immune system, and the placenta.
Most of these tissues also possess GHR that is responsive to GH stimulation. It is
therefore believed that GH may have paracrine or autocrine actions within some of its
sites of synthesis (reviewed by Harvey and Hull, 1997).

Stimulation of GH synthesis or release in immune tissues is likely to be closely

regulated by autocrine, paracrine, or endocrine actions of GH-releasing hormone

73

(GHRH) produced in those cells (Guarcello et al., 1991). The locally produced GH
appears to exert an autocrine effect. This is supported by the study of insulin-like growth
factor I (IGF-I) production. IGF-I is a mitogenic peptide structurally related to proinsulin
and lGF-II which mediates many of the growth-promoting effects of GH in postnatal
animals (F roesch et al. , 1985). There is compelling evidence that IGF-I production is
regulated by both insulin and GH and that both hormones interact in vivo to maintain
normal IGF-I concentration. Evidence has been presented that IGF-I production by
leukocytes is reduced due to the lack of GH function by the presence of GH antibodies
(Baxter et al., 1991). Similarly, a block in GH synthesis by addition of GH antisense
oligonucleotides reduced the proliferation of rat lymphocytes in vitro (Wei gent et al. ,
1991). These studies point out that some immune responses are mediated in an autocrine
manner. However, the relative importance of endogenous and exogenous GH to
lymphocyte functioning remains to be elucidated.

Studies showed that the production of GH in rat by leukocytes from spleen,
thymus, and the peritonetun is increased during immune challenge, as provoked by
intraperitoneal injections of lipopolysaccharide (LPS), and F reund’s Complete Adj uvant
(Baxter et al. , 1991). The expression of GH from myeloid cell lines is similarly increased
in actively proliferating cells (Costoya et al., 1996). It is also known that the GHR gene
is expressed in immune tissues. Other studies showed that labeled GH and GHR
irnmunoreactivity can be detected on the surface of circulating immune cells and
immortalized lymphocyte cell lines (Arrenbrecht, 1974; Ban et al., 1991; Badolato et al. ,
1994). These receptors are present in subpopulations of B- and T-lymphocytes and

natural killer cells and are most abundant in B cells of mammals. In contrast, in avians

74

the splenic, thynric, and bursal GHR/GH binding protein (GHBP) immunoreactivity is
largely associated with macrophages and other large mononuclear nonlymphoid cells and
is particularly present in the nucleus (Calduch-Giner et al. , 1995). In the chicken, it is
found that GHR/GHBP immunoreactivity can be detected in thymic, medullary, antigen-
presenting interdigitating, and ellipsoid-associated cells. In all cases, it is present in both
nuclear and cytoplasmic compartments (Hull et al., 1996). The finding of widespread
distribution of GHR/GHBP immunoreactivity indirectly suggests a possible role for GH
in regulating immune function.

Clearly, GH exerts some marked effects on immune responding tissues. It
stimulates the proliferation of immunocompetent cells and modulates humoral and
cellular immune function. As discussed in Chapter II, cGH has been found to be capable
of interacting with MDV SORF 2 protein. The importance of cGH in MD remains to be
revealed. In this chapter, a candidate gene analysis was performed testing the cGH gene
as a possible contributor to the variation in MD susceptibility among ADOL 6x7
intercross F2 birds.

Materials and Methods
Experimental Animals

A reference pedigree was established at the Avian Disease and Oncology
Laboratory (ADOL), East Lansing for the purpose of genome mapping. It is comprised
of 400+ backcross (BC) progeny using an inbred White Leghorn line and an inbred Red
Jungle Fowl line as grandparents. Initially, one breed-crossed F 1 male was backcrossed
with several inbred White Leghorn females to generate BC progeny, of which 52 BC

animals have been used for establishing genetic linkage groups and genome mapping.

75

For QTL identification purposes, inbred lines 6 and 7 were used as a
grandparental generation to produce more than 300 fully pedigreed F2 intercross
chickens. The F2 intercross chickens, along with grandparental lines and F1 control
birds, were produced in five separate hatches (i.e., ~72 chickens per hatch) for the

evaluation of MD susceptibility.

Assessment of [MD Susceptibility

The F2 intercross chickens generated fiom 6x7 were randomly assigned into six
isolators within each batch (i.e., 16 birds per isolator). The rearing and monitoring of
chickens was conducted using standard poultry research conditions. At 1 week of age,
the chickens were challenged intra-abdominally with 2000 pfil (plaque formation unit) of
MDV JM102 strain virus. The infected birds were died or survived for additional 10
weeks and then were sacrificed by C02 inhalation and necropsied. Nerves and other
organs were examined for gross and/or microscopic MDV lesions and tumor
development. Blood was obtained from each bird during the experiment and was used
for DNA purification.

Several components of MD susceptibility were measured before termination and
at termination. Viremia (V IR) is the MDV concentration at 2 weeks after MDV
challenge (i.e., number of virus pfu per 106 peripheral blood cells; Witter et al., 1969).
Tissue (T18) is the number of different tissue or organs showing gross tumors or lesions
at necropsy, including vagus, brachial or sciatic nerves, heart, gonad, spleen, bursa, lung,
and thymus viscera. Survival (SUR) is the number of days fiom the chicken MDV

challenge to death. Disease (DIS) is the overall phenotypic assessment of each chicken

76

for MD susceptibility judged by either presence (noted as susceptible) or absence (noted
as resistant) of any gross lesion. Tumor index (TUM) is an index developed using the
following scoring system: 0=alive, healthy, absent of tumors; 1=single neural lesion;
2=single visceral lesion or multiple neural lesions or microscopic lesions; 3=multiple
nemal lesions; 4=single visceral plus multiple neural lesions or multiple visceral plus
single neural lesions; 5=multiple visceral plus multiple neural lesion at necropsy or dead
with gross lesions. MD index (MDI) is an empirical pooled index that was developed to
assign weights to each MD trait based on consensus suggestions from experienced MD
pathologists and poultry veterinarians. It was estimated using standardized MD data and

the following expression:

MDI = log10{[(VIR*15)+(TIS*25)+(DIS*25)+(SUR"10)+(TUM*25)]+100}
RFLPAnalysis

A 550 hp cGH fragment was excised from the pcDNA-GH plasmid using BamHI
and HindlII restriction enzymes. The isolated fiagment was random labeled with 32P-
dCTP using an Oligolabelling kit (Pharrnacia, Inc.). The DNA samples were prepared
either from blood cells or from -70°C frozen spleens. Afier digestion of 15 ug of each
DNA sample with the restriction enzyme Mspl or Sacl in a 37°C water bath for 18 hours,
the DNA fragments were separated via electrophoresis in 0.8% agarose gels and
transferred onto charged nylon membranes. The blots were prehybridized in a mixture
containing 10% dextran sulfate, 0.5% SDS, 50 mM NazHPO4, 5X Denhart solution, and
0.5 M NaCl. Membranes were hybridized with the 32P-labelled GH probe for 16-20

hours at 65°C. Final washes were at 65°C in 0.7 X SSC, 0.5% SDS for 15-20 min.

Autoradiography was canied out overnight at -80°C using Kodak XAR-S film and

77

Cronex Lighting Plus intensifying screens. Fragments were measured using a 1 Kb
Ladder (Gibco, BRL) as standard.

For PCR-RFLP, primers for PCR were as follows: GH For 5’-ACC TGG AAG
AAG GGA TCC AAG; GH Rev 5’-GGC CGT CGT GGA GCT GTG AGC. Each PCR
mixture contained 50 ng of genomic DNA, 200 uM of each dNTP, 4 ul of 10 X PCR
buffer, 1.5 mM MgC12, and 0.2 U Taq DNA polymerase (Life Technologies, Inc.). A
total of 10 pmole of each primer was added to a final volume of 40 ul. PCR was carried
out on a MJ Research Thermal Cycler with the following cycles: 94°C, 3 min; 30 cycles
of 30 sec at 94°C, 30 sec at annealing temperature 55°C and 30 sec at 72°C followed with
a 5 min extension at 72°C. A total of 20 ul of the amplified product was mixed with 2.5
pl 10 X digestion buffer A, 1 U SacI restriction enzyme (Perkin Elmer, Inc.) in a final
volume of 25 ul reaction mixture. A one hour 37°C incubation was carried out followed
by mixing reaction mixture with the loading buffer and separating on 1% agarose gels;
results were visualized after etlridium bromide staining.
Linkage Analysis

The East Lansing genetic maps were developed previously by multipoint analysis
of the genotypes on the 52 BC family using the program MAPMAKER/EXP, version 3.0
(Lander et al., 1987; Lincoln et al., 1992). The data gathered from the cGH genotyping
was used to localize the cGH gene. Linkage groups were determined by the results of
pairwise comparisons (two-point analysis) with a minimum LOD linkage (loglO of odds)
score of 3.0 for statistical acceptance of linkage and with a recombination fiaction of 0
=0.32. Following this, three-point analyses were performed for each linkage group

comparing three consecutive markers at a time. A LOD value of 3.0 was again used as

78

the linkage criteria for triples, while the multi-point analysis generated the most likely

loci order and genetic distances expressed in centiMorgans.

Statistical Analysis

F or association analysis, the components of MD susceptibility were assessed for
significant deviation among 272 F2 generated from lines 6 x 7 with various cGH
genotypes. The factors of hatch, isolator, sex, and weight of chicken were used as
covariates to correct the measurements in a nested design using the GLM procedure (SAS
1988). A single point analysis using one-way AN OVA with each genotype class
considered as a treatment was performed. Simple associations between the F2 progeny
GH genotypes and MD susceptibility values were assessed using analysis of variance
(ANOVA; SAS 1988). The tests of significance were conducted using the AN OVA F-

test.

DNA Sequences Comparison

PCR primers were designed from the intron regions that will amplify fiagment
across each cGH exon. PCR reactions were carried out using inbred lines 6 and 7 DNA
as templates separately. The reaction conditions were the same as described previously.
Primers for each exon are as follows: Exon 1, For 5’-AAC CAG GCA GGA AAA TCA,
Rev 5’-TCA GCC CAC CAC AGC ACA; Exon 2, For 5’-CAC TGC TCT CCA CCC
TGT, Rev 5’-ATC ACC TCT CCT CCC CTA; Exon 3, 5’-CCG GGA AAG AGT GAG
GAA, Rev, 5’-GCC AGC AGC CCC TCG CTC; Exon 4, 5’- GCC AGC AGC CCC
TCG CTC Rev, 5’-CCC TCC TCC CTC CCC TTT. Each PCR product was cloned into
TA cloning vectors then transformed into E. cali competent cells provided by the

manufacturer (Invitrogen, Inc.). DNA was then isolated and subjected to DNA sequence

79

analysis using Dye terminator reactions (Perkins Elmer, Inc.) by 377 AB] automatic
DNA sequencers. Sequence comparisons were carried out using the DNASTAR
program.
Results

Mapping cGH Gene

In order to position the cGH gene on the East Lansing chicken genetic map,
linkage analysis was carried out using 52 BC birds. DNA polymorphisms for the cGH
gene were obtained from East Lansing reference family using RFLP analysis. Two
alleles of 1.3 Kb and 1.1 Kb were detected using the restriction enzyme Mspl. Allelic
segregation analysis agreed with 1:1 Mendelian segregation of the two alleles. Partial
data are shown in Figure 16. The genotypes of each of the individual birds were scored
for linkage group identification, in which 1130 marker loci had been mapped previously.
Linkage analyses demonstrated that the cGH gene belonged to a linkage group E59. The
linear order of this linkage group is ACLY-LEIOOl6-AD00376-GCT0022-ROS0315
(ROS0071)-COL1A1a-COL1A1b-cGH-LEIO350 (Figure 17). The length of the linkage
group was estimated at 58.50M (sex averaged), and the genetic distance between each
locus was estimated as shown in Figure 17.

Association of cGH with MD Disease Resistance

In order to obtain individual genotypes for F2 population, inbred lines 6 and 7
were tested for DNA polymorphisms using the RFLP technique. DNA polymorphisms
for the cGH gene were detected using restriction enzyme SacI which gave rise to two
alleles of 4 kb and 4.4 kb (Figure 18). In the original RFLP test, a 4kb fiagment was
denoted as the A allele observed in line 6, and a 4.4 kb fragment was denoted as the B

allele obtained from line 7. Using the published cGH gene sequence (Tanaka et al. ,

80

BC progeny

 

1.3 kb

1.1kb

 

Figure 16. RFLP analysis of East Lansing reference family. The
polymorphism was obtained using Mspl restriction enzyme and
probed with cGH.

81

0-0 Ti ACLY

3-9 W LE10016

19-3 W ADL0376

28.9 “'GCT0022
30.9 "' ROSO315 ROSOO71

.i—l

37-0 ' COLlAla
39-2 ._. COLlAlb

52.5 “' GH

 

 

58-5 _. LEI0350

Figure 17. Genetic map of linkage group E59. Markers or genes are
shown to the right while the distance in cM is on the left.

82

. 4— 4.4 kb
- 4— 4.0 kb

Figure 18. RFLP analysis of GH in parents of MD
resource population. Polymorphism was obtained by using
SacI restriction enzyme and probed with cGH.

83

1992), a PCR technique was designed to amplify a le fragment of intron 4 which
covers this predicted Sacl polymorphic site. All F2 birds were genotyped for the cGH
polymorphism using the PCR-RFLP technique. The polymorphic restriction site for Sacl
was identified at position 3633-363 8. The amplified DNA digested with Sacl yields a
single le fragment representing the allele from line 7, or produces two fragments of
360 and 640 hp representing the allele from line 6. Partial results of PCR-RF LP in intron
4 are presented in Figure 19. Allele fiequencies in the F2 averaged 0.48 for the A allele
and 0.53 for the B allele. Genotype frequencies were estimated as 0.24 for AA, 0.48 for
AB, and 0.28 for BB, about the 1:2:1 ratio expected.

The cGH polymorphisms typed in the F2 6x7 intercross were used to assess the
possible association with disease resistance to MD. The traits measured on the F2 birds
were virenria at 2 weeks after MDV inoculation, number of tissues showing gross tumors
or lesions at necropsy, number of days which birds survived from MDV infection to
death, tumor index scores from 0 to 5, and MD index (MDI) after adjustment of VIR,
TlS, DIS, SUR, and TUM measurements. No statistically significant association between
the cGH polymorphic alleles and the variation in incidence traits among the F2 was
revealed (Table 1). Maximum F ratios, standard errors and average mean of each
genotypic class for each MD incidence were also presented in Table l. The overall
phenotypic assessment of MD susceptibility (DIS) for each individual bird did not
indicate any statistical significance (P>0.05) after using chi-square analysis. Thus, we
conclude that no major effect of the cGH locus on the disease incidence of MD could be

detected in the White Leghorn line 6x7 intercross F2 population.

F 2 progeny

 

67F112345678910

0.64 kb
0.34 kb

 

Figure 19. PCR-RFLP analysis of MD resource family.
Polymorphism were obtained by digestion of PCR
product with SacI restriction enzyme followed by
resolution with agarose gel electrophoresis.

85

 

Tablel. Association analysis for cGH and MD incidence. Mean i standard error for
MD incidence according to cGH genotypes in lines 6 x 7 intercross F2. The F-statistic

and p- values for tests of differences among genotypic classes are also given.

 

Traitsa Genotypes of cGH among F2

A/A

 

(n=66) P"
VIR 96.00i 11.23 102.21i8.00 8344:1037 0.36
ns 12310.16 1.271 0.11 0.65
SUR 445412.19 45.91:].58 47.681205 0.58
TUM 1 0510.13 1.055009 0.931012 0.73
MDI 1.65:0.09 1.64:0.07 0.65

 

‘VIR= MDV plaque formation unit(pfu) in 1 x 106 peripheral white blood cells at 2
weeks post-challenge; TIS= number of different tissues with MD lesions in diseased
chicken; SUR= number of days from chicken MDV challenge to death; TUM = MD

tumor index; MDI = MD index.

bp-values 0.05 and S 0.01 are considered as suggestive and significant, respectively.

86

Exons Comparison between Line 6 and Line 7

PCR fragments covering each exon of cGH were cloned into TA cloning vectors,
followed by DNA sequence analysis using 377 ABI automatic sequencers. Results of
DNA sequence comparison indicated that no nucleotide substitution was found between
these two inbred lines (data not shown).

Discussion

In an attempt to reveal the potential possibility of cGH to be involved in MD
susceptibility, a genetic approach using 272 F2 chickens selected from inbred line 6 x
line 7 for the association studies was performed. One of the major approaches of MD
resistance research in chicken has been the "top-down" approach; that is, mapping and
identification of QTLs through the use of linked molecular markers. Recent studies have
successfully identified 14 QTLs involved in MD susceptibility using genetic markers
(Yonash et al. , 1999).

Kuhnlein et al. (1997) reported that the cGH gene was found to be significantly
associated with MD resistance in non-inbred selected White Leghorn strains. Moreover,
previous studies demonstrated that cGH is capable of binding to MDV SORF2 protein.
These results suggested that cGH might play a role in MD oncogenesis. cGH has been
proven to have a wide variety of physiological effects on traits such as appetite control,
growth, body composition, aging and reproduction (Byatt et al. , 1993; Copras et al. ,
1993; Apa et al., 1994; Vasilatos-Younken, 1995) as well as immune responsiveness
(Marsh, 1992; Blulock, 1994; Kelley and Felton, 1995). In this study, the importance of

the cGH gene as a candidate QTL was examined by investigating the possible ca-

87

segregation of the cGH gene and variation in MD susceptibility among intercross F2
chickens.

First cGH was mapped to E59 in the East Lansing genetic map. Previous work on
QTL identification found no QTL on E59 (Yonash et al., 1999). However, due to the
large intervals between each marker, single marker analysis was carried out using another
population derived from the 6 x 7 intercross F2. Several components of MD traits based
an MD susceptibility were tested for association with cGH alleles. A direct association
of MD incidence with the cGH gene was not found in this particular population. Overall,
preliminary analyses from this study showed no significant cGH allelic co-segregation
with the genetic susceptibility to MD in 6x7 F2 White Leghorn chickens. However, we
cannot exclude the possibility that other cGH alleles exist outside the lines 6 and 7 which
could significantly affect MD resistance.

Digestion of the cGH PCR fragment reveals one polymorphic recognition site for
SacI at Intron 4. However, this polymorphic region is located in an intron. DNA
sequence from each exon indicated that there is no nucleotide substitution detected
between chicken lines 6 and 7. Results suggest that cGH is quite conserved among
strains at the amino acids level.

Although there is no amino acid difference of cGH between lines 6 and 7, it is
noticed that immune response organs, such as spleen, thymus, and bursa, have a greater
weight (percent relative to body weights) in chickens from line 7. It has been reported
that cGH is involved in cell proliferation of immune response organs (reviewed by
Harvey and Hull, 1997). Thus, it is reasonable to suspect that the expression of cGH may

be varied between these two lines in terms of amount or timing. Therefore, genes

88

involved in regulation of cGH, e.g., somatostatin, growth hormone releasing factor,
growth hormone receptor, and cGH specific transcription factors, would be potential
candidates to test for QTLs. Once polymorphisms at these genes locus are identified,
combined genotypic analysis can be further used to define the basis of cGH effects in

ADOL lines 6 and 7.

89

CHAPTER IV
Conclusion and Future Directions
Summary of Results

In an attempt to study the biological characteristics of MDV genes, a unique gene,
SORF 2, came to our attention. An interesting phenomenon was discovered in the MDV .
recombinant strain RMl which was found to have a REV LTR insertion upstream of the
SORF2 gene. The unregulated overexpression of SORF2 due to the LTR promoter
resulted in the loss of MDV oncogenicity. This finding prompted further investigation of
SORF 2.

In this study we have shown that the unique MDV gene SORF2 is expressed in
naturally infected culture cells and in viva. In a screen to identify host proteins that
interact with SORF2, a genetic approach using the yeast two-hybrid system was
employed. The chicken growth hormone structural polypeptide was found to interact
with SORF2. To test this interaction directly, in vitro protein binding assays and co-
irnmunoprecipitations were used to demonstrate that these two proteins associated with
one another. In addition, SORF2 and a truncated form of cGH lacking its signal
sequence were shown to co-localize in cultured CEFs. Sections of neoplastic tissues
induced by oncogenic MDV were examined using antibodies that specifically recognize
either cGH or SORF 2. Positive staining was observed for both proteins. Electron
microscopy of neoplastic spleen sections using colloidal gold markers confirmed a
common cytoplasmic localization for the two proteins. These results provided biological

evidence that these two protein are capable of interacting with each other.

90

To further investigate if the cGH gene was associated with genetic disease
resistance, analysis was carried out in an intercross 6x7 F2 population. We did not detect
any significant association between the polymorphic alleles of cGH and the variation of
MD incidence among F2 animals. To provide a precise location of the cGH gene on the
chicken linkage map, segregation analysis was done using the East Lansing BC resource
family. The cGH locus was tested for linkage against 1130 genetic markers previously
scored in this pedigree. cGH was firmly assigned to the E59 linkage group.

In summary, we successfully identified and confirmed that cGH and SORF 2 are
capable of binding with each other. Further studies will be required to elucidate the
biological meaning of this specific interaction.

Future Directions

It has been well studied that MDV infection in genetically susceptible chickens
results in an extensive lymphoproliferation leading to the formation of gross lymphomas,
while in resistant birds there is only limited lymphoproliferation and tumor formation is
very rare. The cGH polypeptide is found to interact with viral SORF 2 protein, moreover,
it is involved in cell proliferation of immune response organs. It is important to reveal
the meaning of this interaction. There are several immediate questions that need to be
addressed.

Does MDV infection induce cGH alternative splicing?

The cDNA library used for yeast two-hybrid screen was constructed from
mitogen activated splenic cells. As mentioned in chapter 11 an altered form of cGH was
identified from this library by its interaction with SORF 2 protein. It will be necessary to

determine if this altered clone resulted fi'om alternative splicing or initiation of

91

transcription or is just an artifact form generated during the synthesis of cDNA. To
answer this, mRNA directly isolated from MDV infected splenic cells will be examined.
' There are two strategies that can be employed in this study: the primer extension assay
and S1 nuclease protection assay. In the primer extension assay, a primer designed
downstream of the suspected splice acceptor or transcription start site will be used to
prime cDNA synthesis from the total mRNA pool. If two products of correct size are
generated by extension of the primer, it might suggest an alternative splicing event
occured. Similarly, the 3’ isotope-labeled single strand DNA which complementary to
the full length mRNA of GH will be hybridized to total mRNA. Following S1 nuclease
digestion, if two different labeled DNA products are detected, this might also suggest that
the altered form of cGH is a product of alternative splicing. If MDV are capable of
inducing the production of the altered cGH, which will be retained inside the cell instead
of secretion, it can be suggested that SORF2 —GH interaction may preserve a particular
function, and it will be fiuther evaluated.

Does this interaction have any efl'ect on cell proliferation?

Studies showed that the administration of antiserum against bovine GH to mice
results in decreased growth and large reductions in the weights of thymus and of spleen
(Pierpauli and Sorkin, 1968; Pandian and Talwar, 1971). This effect can be reversed by
the concomitant administration of GH (Pierpauli and Sorkin, 1968). Our studies indicate
that both SORF 2 and cGH proteins can be detected in immune cells. An important
question is whether this interaction has any meaning in terms of cell proliferation.

Amino acid sequence alignment indicates that there is no similarity between

SORF2 and the growth hormone receptor. Will SORF2-GB interaction interfere GH-

92

GHR interaction? To answer this, the chicken adenohypophyseal cells will be used for
this study. Cultured adenohypophyseal cells were originated from pituitary gland that
express cGH and have been used broadly for the study of interaction among the cGH and
other factors. The SORF 2 gene will be cloned into an eukaryotic expression vector then
transfected into CEF cells. Protein SORF 2 purified from cultured cells will be added to
cultured adenohypophyseal cells. In vitro cell proliferation assays will be performed to
examine the effect of interaction in cell proliferation.

Once these specific questions are answered, one can propose a possible test for
the role of the SORF2-cGH interaction, if any, with respect to the biological role of
SORF 2 in MD pathogenesis. Then, further studies will evaluate whether the SORF2 acts
as an antagonist to block the normal cGH signaling pathway. Other functions during

viral oncogenesis via interaction with cGH are also possible.

93

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