The reduction and induction of Kaposi's sarcoma-associated herpesvirus lytic replication
In humans, Kaposi's Sarcoma-associated Herpesvirus (KSHV) is capable of establishing latent infection in B-cells. During latency no infectious virions are produced, the viral episome is maintained and few viral genes are expressed. Latently infected cells, however, retain the capacity to enter the lytic cycle. KSHV ORF50 encodes the transcription factor RTA whose expression is sufficient to initiate the full lytic cycle. RNA interference (RNAi) involves the sequence specific silencing of gene expression. Although extensively validated and widely available at present, lentiviral vector-mediated RNAi was unavailable in 2002. This thesis describes the generation and characterisation of shRNA expression cassettes that can be delivered using self- inactivating lentiviral vectors. Using shRNAs designed to interfere with KSHV ORF50 expression, the number of cells expressing RTA following chemical induction of the lytic cycle was greatly reduced. Analysis of viral lytic antigens and virus production suggests that reducing RTA expression prevents latent KSHV from initiating the lytic cycle. Although KSHV infects B-cells in vivo, most B-cell lines are recalcitrant to KSHV infection in vitro. This thesis describes the in vitro susceptibility of a panel of B-cell lines, representing different stages of B-cell differentiation, and other common cell-lines to recombinant KSHV infection. Interestingly, all the adherent cell-lines examined were susceptible to recombinant KSHV infection whereas we were unable to identify any B-cell lines which were efficient targets for recombinant KSHV infection KSHV positive primary effusion lymphoma (PEL) cells are phenotypically similar to plasmablasts, which represent a late stage in B-cell differentiation, immediately preceding terminal differentiation into plasma cells. This thesis examines the stage at which B-cell development is arrested in PEL, focussing on the transcription factor X- box binding protein-1 (XBP-1) and the unfolded protein response (UPR). Using exogenously expressed XBP-1, the role of XBP-1 as the possible molecular switch linking terminal differentiation and KSHV lytic reactivation is considered.