The biological role of viral tRNA-like molecules in a murine gammaherpesvirus infection

Members of the subfamily Gammaherpesvirinae commonly establish latency within lymphoid cells and are associated with lymphoproliferative disease. Gammaherpesviruses include the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpes virus. Due to the narrow host range of infection exhibited by these viruses and their limited productive growth in vitro, the events occurring during lytic replication and the establishment of latency are not well characterised. Murine gammaherpesvirus 68 (MHV-68) is able to undergo productive replication in a number of cell types in vitro and infects laboratory mice; consequently it provides an excellent model for study of gammaherpesvirus infection. MHV-68 encodes eight viral tRNA-like molecules (vtRNAl-8), which resemble cellular tRNAs in that they have a predicted cloverleaf-like secondary structure and are transcribed by RNA polymerase III. However unlike cellular tRNAs they are not amino-acylated and therefore do not function directly during protein synthesis. They are known to be expressed to high levels during both latency and lytic replication. However their role within infection is not known.

The aim of this project was to characterise the vtRNAs. The presence of the vtRNAs within purified, RNase treated viral stocks indicated their packaging within the MHV-68 virion. Although both viral and cellular mRNAs were also present, it appeared that the major RNA species packaged by MHV-68 were small RNA molecules, such as the vtRNAs. Incorporation of RNA molecules into the virion is not unique to MHV-68 as other herpesviruses have been found to package RNA, although the vtRNAs represent the only packaged small viral non-coding RNA molecules discovered so far. In addition, this is the first study to demonstrate the preferential incorporation of small RNA molecules by a herpesvirus. The mechanism by which the vtRNAs assemble into the virion is not clear. In situ hybridization demonstrated that within infected cells the vtRNAs localized to globular areas within the nucleus and were also found at high levels within the cytoplasm. Electrophoretic mobility shift assays performed using vtRNAl and vtRNA4 indicated binding to protein complexes present within both the nucleus and cytoplasm of infected cells. Inhibition of vtRNA-protein binding by an anti-MHV-68 antibody indicated direct interaction of the vtRNAs with viral protein(s). Hence it is likely that their incorporation is mediated through binding to viral protein(s) during virion assembly in either the nucleus or cytoplasm.

MHV-76 is a deletion mutant of MHV-68, which lacks all eight vtRNAs along with four other genes (M1-M4). The contribution of the vtRNAs to viral pathogenesis has been investigated by construction of recombinant MHV-76, which expressed vtRNAsl-5 under their natural promoters. The insertion of the vtRNAs into MHV-76 had no effect on the ability of the virus to replicate in vitro. In addition, the recombinant viruses displayed identical characteristics to MHV-76 following intranasal infection of BALB/c mice, demonstrated by the levels of lytic virus present within the lung and the levels of latent virus within the spleen. Therefore the role of the vtRNAs within infection remains to be determined and the recombinant viruses produced in this project will provide excellent tools to investigate their function further through both in vitro and in vivo analysis.