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 <i>in vitro</i> 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 COS-7 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 contingency 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 transcription-PCR (RT-PCR) and northern analysis were used to show that the M4KI virus was expressing M4 RNA <i>in vitro. </i>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 <i>in vitro</i> but in contrast the virus appears to be cleared more efficiently <i>in vivo</i> in the lungs of mice. The establishment of latency is also impaired in the spleens of MHV-76-infected mice (Macrae <i>et al.,</i> 2001). <i>In vitro</i> studies revealed that 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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663443 |
| Date | January 2002 |
| Creators | Wan, Flora |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/30039 |
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