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.