Browsing by Subject "KSHV"
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Item Prevalent and differential herpesviral gene regulation mediated by 3'-untranslated regions(2012-08) McClure, Lydia Virginia; Sullivan, Christopher S.Herpesviral infections are currently incurable and are associated with severe human diseases, such as cancer. Kaposi’s Sarcoma-associated Herpesvirus (KSHV), like all herpesviruses, undergoes a long-term, latent infection where few viral products are made as a mechanism to evade the host immune system. Recently, the KSHV latent genome was shown to have bivalent histone marks thought to keep the virus poised for replication. However, it is unclear how the virus prevents spurious leaky transcription from this primed state. The 3' untranslated region (3'-UTR) of transcripts is a common site of gene expression regulation, however less than half of the KSHV 3'-UTRs have been mapped and few studies have interrogated their role during infection. The work presented here is the first large-scale map and analysis of the KSHV 3'-UTRs. Four methods were used to identify the 3'-UTRs expressed by the ~85 KSHV genes, including prediction algorithms, 3'-RACE, DNA tiling array, and next generation deep sequencing analysis. The role of each KSHV 3'-UTR in gene expression was then examined using luciferase reporter assays and showed a surprising prevalence of negative regulation conveyed during latent infection. Sequential deletions across numerous 3'-UTRs indicated RNA structure is likely involved in this regulation. In addition, several KSHV 3'-UTRs conveyed an increase in translation during lytic infection through enhanced recognition by the cap-dependent translation initiation machinery activated via the MNK1 kinase. A second mechanism of KSHV gene regulation was identified through motifs encoded in the K7 3'-UTR. This work indicated that a previously characterized RNA element and a novel putative hairpin are both partially responsible for negative regulation conveyed by the K7 3'-UTR. We hypothesize that these structural motifs control expression of the K7 transcript by altering its sub-cellular location and/or via RNA stability. This work represents a broad 3'-UTR study that mapped the KSHV 3'-UTRs and is the first large-scale functional analysis of 3'-UTRs from a large genome virus. We have implicated post-transcriptional mechanisms, along with known transcriptional regulation, in viral evasion of the immune response during latency and the escape of viral-mediated host shutoff. These results identify new potential targets for therapeutic intervention of KSHV-associated disease.Item Study of a tumor virus unveils a novel function for the miRNA biogenesis machinery(2012-12) Lin, Yao-Tang; Sullivan, Christopher S.Kaposi’s Sarcoma-associated Herpes Virus (KSHV) is a human herpesvirus associated with cancers. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. This work presented here is a novel approach to profile small RNAs from populations of cells undergoing predominantly lytic infection. Using two different next generation sequencing platforms, I cloned and sequenced both pre-microRNAs and derivative microRNAs (miRNAs). This analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5 prime end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. I report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miRs-K8 and K12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. I identify several novel small RNAs of low abundance, including viral microRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. This work also shows that much of the KSHV genome is transcribed in both the top and bottom strand orientations during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, I observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs). From the small RNA deep-sequencing, I also detected a low abundant small RNA fragment (23 nt) that maps to a putative hairpin structure (named hairpin K) within the KSHV PAN transcript. I demonstrate that hairpin K is a cis-negative regulatory element in PAN. It is well-appreciated that viruses utilize host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often utilize host-encoded factors to optimize replication. This work shows that Drosha, an endoribonuclease best known for its role in the biogenesis of miRNAs, can also function to directly regulate viral gene expression. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. I demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses.