Interaction of poly(rC)-binding protein 2 with the 5′-terminal stem loop of the hepatitis C virus genome
ABSTRACT The 5' noncoding region (NCR) of hepatitis C virus (HCV) contains an internal ribosome entry site for translation initiation. Cellular proteins (e.g. La, polypyrimidine tract-binding protein, and p25) that interact with HCV 5' NCR have been implicated in facilitating efficient internal initiation. The 5' NCR may also contain RNA structures and specific RNA sequences that interact with cellular proteins to promote RNA replication. UV crosslinking experiments revealed a 43-kDa cellular protein (p43) also interacts with the HCV 5' NCR. Further UV crosslinking experiments with deletion mutants of HCV 5' NCR demonstrated that p43 bound specifically to the 5'-terminal stem-loop of the HCV 5' NCR. Achromobactor proteinase I digests, competition experiments, and immunoprecipitation confirmed that p43 was identical to human poly(rC)-binding protein 2 (PCBP2). We prepared a PCBP2-immunodepleted rabbit reticulocyte lysate with an anti-PCBP2 antibody. Translation activity promoted by the HCV internal ribosome-entry site was the same in PCBP2-depleted lysates as in mock-depleted lysates. In conclusion, PCBP2 specifically interacted with the 5' terminus of HCV genome but had no effect on HCV translation. We speculate that PCBP2's interaction with HCV 5' NCR may be involved in the replication-initiation complex of HCV.
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- "On the other hand, a number of transacting cellular factors have been shown to interact with the HCV IRES. These include the polypyrimidine-tract-binding protein (Ali & Siddiqui, 1995), the human La antigen (Pudi et al., 2003; Izumi et al., 2004), the poly(rC)-binding protein 2 (Fukushi et al., 2001a), the heterogeneous nuclear ribonucleoprotein L (Hahm et al., 1998) and ribosomal protein factors S9 (Fukushi et al., 1999) and S5 (Fukushi et al., 2001b). Furthermore, viral sequences located at distal regions from the HCV IRES (Ito et al., 1998; Ito & Lai, 1999; Wang et al., 2000; Murakami et al., 2001; Imbert et al., 2003; Kim et al., 2003) as well as selected viral proteins (Shimoike et al., 1999; Kato et al., 2002; Zhang et al., 2002; He et al., 2003; Li et al., 2003) appear to modulate the efficiency of the HCV IRES activity. "
ABSTRACT: Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located mainly within the 5' non-translated region of the viral genome. In this study, the effect of the HCV non-structural 5A (NS5A) protein on the HCV IRES-dependent translation was investigated by using a transient transfection system. Three different cell lines (HepG2, WRL-68 and BHK-21) were co-transfected with a plasmid vector containing a bicistronic transcript carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase genes separated by the HCV IRES sequences, and an expression vector producing the NS5A protein. Here, it was shown that the HCV NS5A protein inhibited HCV IRES-dependent translation in a dose-dependent manner. In contrast, NS5A had no detectable effect on cap-dependent translation of the upstream gene (CAT) nor on translation from another viral IRES. Further analysis using deleted forms of the NS5A protein revealed that a region of about 120 aa located just upstream of the nuclear localization signal of the protein is critical for this suppression. Overall, these results suggest that HCV NS5A protein negatively modulates the HCV IRES activity in a specific manner.Journal of General Virology 05/2005; 86(Pt 4):1015-25. DOI:10.1099/vir.0.80728-0 · 3.53 Impact Factor
- Developments in Food Science 01/2000; 41:39-75. DOI:10.1016/S0167-4501(00)80005-9