|dc.description.abstract||All mammalian cells encode microRNAs (miRNAs), which are small non-coding RNAs
(~ 22 nucleotides) that control numerous physiological processes via regulation of gene
expression. A number of viruses, in particular herpesviruses, also encode miRNAs.
Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma
associated herpesvirus (KSHV) are associated with lymphoproliferative disorders and
some types of cancer in humans. Gammaherpesvirus-encoded miRNAs are predicted to
contribute to pathogenesis and virus life cycle by suppressing host and viral target genes.
However, the exact functions of these miRNAs during virus infection in the natural host
are largely unknown. Strict species specificity has limited research on the human
gammaherpesviruses mainly to in vitro studies.
Murine gammaherpesvirus 68 (MHV-68) encodes at least 15 miRNAs and provides a
unique tractable small animal model to investigate in vivo gammaherpesvirus
pathogenic features that are difficult to assess in humans. Following intranasal infection
of lab mice, the virus undergoes primary lytic infection in the lung epithelial cells and
then spreads to the spleen establishing latent infection in splenic B lymphocytes,
macrophages, and dendritic cells. The peak of the latent viral load occurs in the spleen at
14 dpi and then it decreases over time, but the virus is not completely eliminated and the
latent viral genomes remain in the host cells for lifetime and can reactivate to produce
infectious virus under certain conditions.
The aims of my project were to: (1) establish and develop quantitative reverse
transcription polymerase chain reaction (qRT-PCR) assays for quantification of the
MHV-68 miRNAs, (2) determine the miRNAs expression profiles during the two stages
of virus infection (lytic and latent infection), (3) investigate the kinetics of the miRNAs
expression during latency in vivo, (4) construct an MHV-68 miRNA mutant virus
lacking 9 miRNAs (designated MHV-68.ΔmiRNAs), and (5) carry out thorough
phenotypic characterisation of this mutant virus in order to determine the possible
functions MHV-68 miRNAs in the context of natural host infection.
It was found that the MHV-68 miRNAs expression pattern varied during different stages
of infection, suggesting a differential regulation of the expression of these miRNAs
depending on the phase of infection. In order to investigate the kinetics of miRNAs
expression during latency in vivo, BALB/c mice were infected intranasally with MHV-
68 virus and spleens were harvested at days 10, 14, 21, and 32 post infection. The levels
of miRNAs expression were determined by qRT-PCR in the splenocytes from infected
mice. Interestingly, in contrast to the lytic MHV-68 protein coding genes, the expression
of the miRNAs increased over time after 21 dpi, suggesting that the MHV-68-encoded
miRNAs may play more fundamental roles during later stages of latent infection.
In order to determine the potential roles of the MHV-68 miRNAs in virus pathogenesis,
a miRNA mutant virus lacking the expression of 9 miRNAs, named MHV-
68.ΔmiRNAs, was constructed. The miRNA mutant virus replicated with the same
kinetics as wild type virus in vitro and in vivo demonstrating that the deleted MHV-68
miRNAs are dispensable for virus lytic replication. To examine the roles of the miRNAs
during virus latency, the MHV-68.ΔmiRNAs virus was characterised throughout a 49-
day course of infection.
Although the level of ex vivo reactivation of the MHV-68.ΔmiRNAs virus was
comparable to that of the WT virus during the establishment of latency and as late as 28
dpi, the reactivation of the MHV-68.ΔmiRNAs virus was approximately 18-times higher
than that of the WT virus at 49 dpi despite the similar levels of the genomic viral DNA
loads at the same time-point. This suggests that the MHV-68 miRNAs suppress virus
reactivation and promote maintenance of long-term latency. Moreover, the lytic viral
gene expression levels were higher in splenocytes from the MHV-68.ΔmiRNAs-infected
mice than the basal expression levels in the splenocytes from WT MHV-68-infected
mice, suggesting that the MHV-68 miRNAs may suppress viral lytic gene expression
during long-term latency in vivo and thus help the virus lay low.||en