Abstract
Murine gammaherpesvirus 68 (MHV-68) is a lymphotropic virus which infects wild
murid rodents and can readily infect experimental mice. Thus, MHV-68 is an
invaluable small animal model for elucidating gammaherpesvirus pathogenesis and
immune evasion strategies. The MHV-68 genome has been fully sequenced (Virgin et
al., 1997) and comparative sequence analysis has shown that it is more closely related to
Kaposi's sarcoma-associated herpesvirus (KSHV) and herpesvirus saimiri (HVS) than
Epstein-Barr virus (EBV). There are at least 80 open reading frames (ORFs) within the
genome, many of which are present in other gammaherpesviruses. There are, however,
4 ORFs, termed M1-M4, which are unique to MHV-68. Gammaherpesviruses have the
propensity to abduct immunomodulatory elements, therefore it is likely that such
elements will be encoded within the MHV-68 genome. One such candidate is the M4
gene. The aim of the project was to functionally characterise the M4 gene and elucidate
its role in the pathogenesis of MHV-68.
Half of the M4 protein was expressed in a bacterial expression system and the purified
protein was used to generate anti-sera. Immunoprecipitation studies demonstrated the
M4 sera bound to a protein of ~44kDa, the predicted size of the M4 product. Analysis
of M4 transcription in vitro showed that the gene was transcribed both early, prior to
DNA synthesis, and late in the virus lifecycle.
The M4 gene was cloned into a mammalian expression construct and transfected into
COS-7 cells. Cells containing the construct were selected under G418 (neomycin
derivative) antibiotic selection. However, we were unable to demonstrate that the COS7 cells expressed the M4 protein.
The M4 gene was inserted into MHV-76, a virus which lacks part of the left hand end of
the genome including the M4 gene, generating a M4 knock-in (M4KI) recombinant
virus. Apart from this deletion, MHV-76 harbours the full contigency of MHV-68 genes. Polymerase chain reaction (PCR) and Southern analysis were used to
demonstrate the presence of the M4 gene in the M4KI virus. Reverse trancription-PCR
(RT-PCR) and northern analysis were used to show that the M4KI virus was expressing
M4 RNA in vitro. The M4KI virus was used for comparative studies with MHV-76,
which lacks the M4 gene, and MHV-68, which contains the M4 gene. The growth
kinetics of MHV-76 are similar to MHV-68 in vitro but in contrast the virus appears to
be cleared more efficiently in vivo in the lungs of mice. The establishment of latency is
also impaired in the spleens of MHV-76-infected mice (Macrae et al., 2001). In vitro
studies revealed the M4KI virus growth kinetics were similar to MHV-68 and MHV-76.
Infection of BALB/c mice with the M4KI virus revealed that it replicates in the lung
with the same kinetics as MHV-76. Thus, compared to MHV-68, MHV-76 and M4KI
virus titres rise slightly faster, and levels of virus are reduced or cleared quicker. M4KI
virus infective centres were much reduced in the mediastinal lymph nodes in comparison
to MHV-76 and MHV-68, and were not detectable in the spleen. Thus, the M4 gene
product, in the context of the MHV-76 genome prevented the establishment of
detectable latent infection in the spleen.
