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
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.