A Functional Global Gene Profiling of Kaposi’s Sarcoma-Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with several human malignancies including the cancers Kaposi’s sarcoma and primary effusion lymphoma. As with other herpesviruses, KSHV can enter either latency or undergo lytic replication within its host. Several latency-associated open reading frames (ORF) and a few lytic cycle-associated ORFs are relatively well-studied. However, the majority of the 140.5kb long unique region of the genome, which contains about 90 ORFs, is largely uncharacterized outside of in silico studies. We took advantage of the BAC16, a bacterial artificial chromosome clone of a KSHV genome, as well as the iSLK cell line, a permissive cell line with tight latency when infected and efficient reactivation upon chemical induction, in order to generate targeted KSHV mutants to better understand the functionality of KSHV ORFs./nIn chapter 2, I explain how we generated 90 mutants using the BAC16, each characterized by the disruption of a single ORF. Mutant BACs were introduced into iSLK cells by transfection and classified as essential or nonessential for the maintenance of latency in this cell model. Then transfected cells were chemically induced into lytic replication and the amount of infectious virus generated and released into the supernatant was quantified. This allowed us to classify ORFs as essential or nonessential for reactivation and infectious virus generation. Notably, we identified several core herpesvirus ORFs as non-essential which had previously been classified as essential in other herpesviruses, including ORF 17, 42, 57, 64, 66, 67, 69, glycoprotein L, glycoprotein H, and glycoprotein B./nI then further characterized the nonessential mutants by using growth curve assays under conditions favoring latency, reactivation, and lytic replication in chapter 3. This allowed me to isolate the roles of KSHV ORFs to specific viral life cycle stages, and to take a high-level approach of understanding which ORFs disrupt or promote infectious virus generation under these conditions. We found that ORFs 16, 46, 58, 60, 61, and 67 are deficient in virus generation under lytic conditions and likely play an important role in the early stages of viral infection to prime the virus for successful reactivation. We also identified several KSHV-specific ORFs (K3, K4, and K5), which function to inhibit reactivation. Finally, we discovered that ORFs 49, 50, 56, and K11 promote spontaneous reactivation during latency while ORFs 11AA, 72, K6, and K7 repress it, underscoring the biological importance of viral control of this phenomenon, which plays a large role in pathogenesis and is not yet fully understood. In chapter 4, I further dissected the effects of ORF disruption on the viral life cycle. Here we monitored lytic antigen expression of BAC16 or mutant infected cells by measuring the percentage of infected cells expressing immediate-early (ORF45), early (K8α), or late (K8.1A/B) lytic antigens under conditions favoring latency or reactivation. We found that ORF20 tempers lytic gene expression upon reactivation. Additionally, ORF 10.1, 20, 23, 27, 58, and 67 seem to facilitate the burst of lytic gene expression shortly after infection, while ORF 11, 28, K10, K10.5, and K9 promote the establishment of latency after this.

Advisor: Liu, Fenyong