Investigation of the mechanism of translation and contribution to pathogenesis of Kaposi's sarcoma associated herpesvirus FLICE Inhibitory Protein (vFLIP)

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the most recently identified human herpesvirus and is the causative agent of not only Kaposi's sarcoma but also B-cell primary effusion lymphomas and some forms of multicentric Castleman's disease. During latency, translation of viral open reading frames is tightly regulated. Only two transcripts, encoding ORFs 71-73 (LANA, vCyclin and vFLIP), are truly type 1; that is, there is no increase in the rate of their transcription after the induction of lytic replication. Both transcripts are transcribed from a common start point and are subsequently spliced to produce a tricistronic transcript, encoding ORFs 71-73, and a bicistronic transcript encoding ORFs 71 and 72 (vFLIP and vCyclin). Translation of ORF 71, vFLIP, has been shown to be mediated via an internal ribosome entry site (IRES) contained within the upstream vCyclin coding sequence. This is the first example of cap-independent translation in a DNA virus. vFLIP has been demonstrated to have anti-apoptotic activity and is also capable of activating the cellular transcription factor NF-kB.In this thesis the contribution of vFLIP toward viral pathogenesis has been investigated through the construction and use of recombinant murine gammaheipesviruses (MHV-76). KSHV vFLIP under the control of either the Cytomegalovirus immediate early (CMV-IE) or the murine phosphoglycerol kinase (PGK) promoter, together with enhanced green fluorescent protein (EGFP) and a hygromycin resistance marker, has been inserted into the left-hand end of the MHV76 genome to produce recombinant viruses that have been used for in vivo and in vitro studies. All recombinant viruses display similar in vitro growth kinetics to wild-type MHV-76 during infection of BHK cells. Intranasal infection of Balb/c mice with the CMV vFLIP viruses resulted in a 2 fold greater infectious virus titre in the lungs, at day 5, compared with the control and wild-type MHV-76 viruses. However, this enhanced viral replication was not observed following infection with the PGK vFLIP viruses. In all cases the vFLIP expressing and control viruses displayed a decreased establishment of splenic latency. The influence of vFLIP on viral replication in an in vitro system has been investigated through the infection of NSO cells, a murine myeloma cell line. These data indicate that the expression of vFLIP, in the context of a herpesvirus infection, increases the initial establishment of a latently infected pool of cells, by up to 3-fold at day 8 post infection. The correlation of this process with the activation of NF-kB has been investigated.A number of techniques have been applied to investigate the nature of any IRES interacting factors (ITAFs) necessary for translation of vFLIP from the KSHV IRES. Through the use of an in vitro pull-down assay it has been possible to demonstrate that a known ITAF, poly-pyrimidine tract binding protein (PTB), can associate with the KSHV IRES. Electrophoretic mobility shift assays indicate that additional cellular proteins interact with the IRES sequence and investigations indicate that one of these may be the translation initiation factor eIF4G.