Interaction of poly(rC)-binding protein 2 with the 5′-terminal stem loop of the hepatitis C virus genome
R&D Center, BioMedical Laboratories, 1361-1, Matoba, Kawagoe-shi, 350-1101, Saitama, Japan.Virus Research (Impact Factor: 2.32). 02/2001; 73(1):67-79. DOI: 10.1016/S0168-1702(00)00228-8
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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- "In addition to miR-122/AGO2, several cellular proteins, including the heterogeneous nuclear ribonuclear proteins PCBP2 (hnRNP E2) and hnRNP L, bind to the 5 0 end of the HCV genome and facilitate its replication (Fukushi et al., 2001; Li et al., 2014; Wang et al., 2011). PCBP2 is of particular interest, as it regulates the IRES-initiated translation of poliovirus, another positive-strand RNA virus, and facilitates both circularization and translation of the HCV genome (Perera et al., 2007; Wang et al., 2011). "
ABSTRACT: The liver-specific microRNA, miR-122, stabilizes hepatitis C virus (HCV) RNA genomes by recruiting host argonaute 2 (AGO2) to the 5' end and preventing decay mediated by exonuclease Xrn1. However, HCV replication requires miR-122 in Xrn1-depleted cells, indicating additional functions. We show that miR-122 enhances HCV RNA levels by altering the fraction of HCV genomes available for RNA synthesis. Exogenous miR-122 increases viral RNA and protein levels in Xrn1-depleted cells, with enhanced RNA synthesis occurring before heightened protein synthesis. Inhibiting protein translation with puromycin blocks miR-122-mediated increases in RNA synthesis, but independently enhances RNA synthesis by releasing ribosomes from viral genomes. Additionally, miR-122 reduces the fraction of viral genomes engaged in protein translation. Depleting AGO2 or PCBP2, which binds HCV RNA in competition with miR-122 and promotes translation, eliminates miR-122 stimulation of RNA synthesis. Thus, by displacing PCBP2, miR-122 reduces HCV genomes engaged in translation while increasing the fraction available for RNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.
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- "Some researchers have reported that PCBP2 is induced in Japanese Fulminating Hepatitis-1 (JFH-1) infected cells and required for JFH-1 replication . Other studies report that PCBP2 and PTB can rescue the inhibition of HCV IRES-mediated translation in vitro , while other findings indicate that PCBP2 has no direct effect on HCV replication or IRES-mediated translation , , . "
ABSTRACT: Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.
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- "Earlier, PCBP2 (an IRES trans acting factor, ITAF) and 3CD (viral protein) have been shown to play role in the switch for poliovirus RNA 4. The study provided evidence of a complex interplay between the viral and cellular protein in binding to viral RNA and influencing the switch from translation to replication. Some canonical initiation factors (eIF2 and eIF3) and non-canonical ITAFs, have been shown to interact with the HCV-IRES and influence its function 5678. Earlier, results from our laboratory and others have shown, that human La autoantigen binds to HCV IRES RNA both in vitro and ex vivo and help in ribosome assembly during internal initiation of translation of HCV RNA 5910. "
ABSTRACT: HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3(pro)) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3(pro) binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3(pro) as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3(pro) binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.
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