A polypyrimidine tract facilitates the expression of Kaposi's sarcoma-associated herpesvirus vFLIP through an internal ribosome entry site

Abstract

We have identified a novel internal ribosome entry site (IRES) within a latently expressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene (vCyclin) that controls the expression of a downstream open reading frame encoding an inhibitor of apoptosis (vFLIP). This IRES is the first such element to be identified in a DNA virus and may represent a novel mechanism through which this virus controls gene expression. We have used a dual luciferase reporter assay to identify important sequence elements essential for the activity of the IRES. A sequence of 32 nucleotides incorporating a polypyrimidine tract (PPT) was found to be required for the proper functioning of the IRES. We also show, using an electrophoretic mobility shift assay (EMSA), that proteins specific to a KSHV-infected cell line (BCP-1) but not a KSHV-negative cell line (HEK293) were able to form complexes with the IRES. By using an in vitro RNA binding assay, the cellular polypyrimidine tract binding protein (PTB, hnRNP-I) was found to bind to the IRES RNA. These results suggest that the interaction of PTB with the PPT may contribute to the correct functioning of the KSHV IRES in infected cells. INTRODUCTIONKaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is the most recently identified member of the herpesvirus family to infect humans (Chang et al., 1994). KSHV, a gamma-2-herpesvirus, has been proposed as the aetiological agent for Kaposi's sarcoma as well as other malignancies such as primary effusion lymphoma (PEL) (Cesarman et al., 1995) and multicentric Castleman's disease (MCD) (Soulier et al., 1995).KSHV is closely related to three other herpesviruses with oncogenic potential; herpesvirus saimiri (HVS), murine gammaherpesvirus (MHV-68) and, more distantly, to Epstein-Barr virus (EBV). The complete nucleotide sequence of KSHV DNA has revealed several genes which have probably been captured from the host cell during virus evolution, and whose products could also play a role in cellular transformation and tumour induction (Neipel et al., 1997;Russo et al., 1996) The observation that a bicistronic transcript (Talbot et al., 1999) encodes vCyclin and vFLIP led to the investigation of the mechanism of translation of the vFLIP ORF. We (Bieleski & Talbot, 2001) and others (Grundhoff & Ganem, 2001;Low et al., 2001) were able to identify a novel internal ribosome entry site (IRES) within the latently expressed vCyclin gene that controls the expression of the downstream vFLIP ORF. This IRES is the first such element to be identified in a DNA virus. Recently, an IRES element has been described in the untranslated region of the Epstein-Barr nuclear antigen-1 (EBNA1) gene, which may contribute to the regulation of latent gene expression (Isaksson et al., 2003).IRES elements were first identified in the 59 untranslated regions (UTR) of picornaviruses and are essential for the cap-independent translation of the viral polyprotein (Jang et al., 1988;Pelletier & Sonenberg, 1988). More recently IRES elements have been chara...

Searching for IRES

“…As an example, PTB has been connected with several functions such as splicing repression, pre-mRNA 39-end processing, mRNA localization, and mRNA stability. It has also been shown to be involved in IRES activation with both viral (Sanderbrand et al 2000;Wollerton et al 2001;Bieleski et al 2004) and cellular (Giraud et al 2001;Mitchell et al 2003Mitchell et al , 2005Pickering et al 2003) IRES but has been also shown to be inhibitory to IRES activity in Unr or Bip . Several IRESes have a polypyrimidine tract at the 39-end that has been shown to be important for activity (Kaminski et al 1994) and a recognition site for PTB (Kolupaeva et al 1996).…”

Transcripts Encoding K12, v-FLIP, v-Cyclin, and the MicroRNA Cluster of Kaposi's Sarcoma-Associated Herpesvirus Originate from a Common Promoter

“…The LT cluster is transcribed from a constitutively active promoter (LT c ) and through alternative splicing gives rise to a 5.4-kb mRNA containing ORFs 71, 72, and 73 and a 1.7-kb transcript containing ORFs 71 and 72. It is likely that LANA (ORF 73) is the principal translation product of the longer mRNA, whereas both v-Cyclin and v-FLIP are synthesized from the shorter transcript, the latter by way of an internal ribosome entry site upstream of ORF 71 (5,6,22,34). Although not shown in the figure, a 1.1-kb monocistronic (ORF 71) transcript can also be detected in PELs at low abundance compared to the 1.7-kb mRNA and is derived from LT c by additional splicing (22).…”

Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus

“…Previous studies by Adair et al (1) found that HCMV infection can induce a response from cellular splicing factors, including PTB, and implied that PTB might be involved in the interaction of HCMV and cells. Other studies of PTB showed that it has positive effects on viral infection by activating the internal ribosome entry site (4,42). Therefore, PTBs are a group of multifunctional proteins and are important in maintaining the equilibrium of gene regulation.…”