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Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. There is no effective therapy or vaccine available to HCV-infected patients other than interferon–ribavarin combination, which is effective in a relatively small percentage of infected patients. Our previous results have shown that a synthetic peptide (LAP) corresponding to the N-terminal 18 amino acids of the Lupus autoantigen (La) was a potent inhibitor of HCV IRES-mediated translation. We demonstrate here that LAP efficiently blocks HCV replication of infectious JFH1 virus in cell culture. Our data suggest that LAP forms complexes with IRES-transacting factors (ITAFs) PTB and PCBP2. LAP-mediated inhibition of HCV IRES-mediated translation in vitro could be fully rescued by recombinant PCB and PCBP2. Also transient expression of PTB / PCBP2 combination significantly restores HCV replication in LAP-inhibited cultures. These results suggest that ITAFs could be potential targets to block HCV replication.

We have previously identified HSC70 in complex with viral NS5A along with other heat shock proteins. HSC70 has been reported to modulate HCV infectivity. Here, we further analyze the NS5A/HSC70 interaction biochemically and investigate the role of HSC70 in viral life cycle. We demonstrate that HSC70 knockdown significantly affects virus production with no cytotoxicity. We observed that HSC70 knockdown did not affect viral RNA replication and protein production. In fact, we found a slight, but statistically significant increase in viral RNA and protein levels likely caused by compensatory upregulation of HSP70, which we have previously shown to be required for viral protein production. NS5A-augmented IRES-mediated translation was also not inhibited by HSC70 knockdown and, rather, slightly increased. In contrast, we discovered that intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains direct...

Poliovirus (PV) is the prototype virus of a large group of medically important viruses (picornaviruses) that include those inducing poliomyelitis (polioviruses), infectious hepatitis (hepatitis A virus), the common cold (rhinoviruses), and encephalitis and myocarditis (coxsackie viruses) (31). The single-stranded, plus-polarity RNA genome (∼7,500 nucleotides) of PV (18, 30) is translated into one large polyprotein, which is cotranslationally processed by virally encoded proteases 2Apro, 3Cpro, and 3CDpro into mature viral structural and nonstructural proteins (22). The viral proteases have been studied extensively and found to be very specific in polyprotein cleavage; 3Cpro and 3CDpro cleave the polyprotein at glutamine-glycine (Q-G) bonds while the 2Apro cleaves only at tyrosine-glycine (Y-G) bonds (20). The proteases do not cleave every potential cleavage site within the polyprotein; other determinants such as accessibility and context of the cleavage site are also important. Accurate initiation of transcription by RNA polymerase (Pol) II requires the assembly of a multiprotein complex on the core promoter around the mRNA start site (13). The multiprotein complex, consisting of at least seven to eight general transcription factors (GTFs) and RNA Pol II form a preinitiation complex at specific cis-acting elements (promoters) on the DNA template. The most common cis-acting element, the TATA box, is situated approximately 25 nucleotides upstream of the transcription start site. Among the GTFs in the complex, the TATA-binding protein (TBP) has been studied extensively (29). The TBP was first identified through its role in Pol II transcription, where it associates with 13 or 14 TBP-associated factors to form the TFIID complex (34). The unique aspect of TBP is its additional presence in complexes required for RNA Pol I (SL1) and RNA Pol III (TFIIIB) transcription. In both SL1 and TFIIIB complexes, TBP associates with a distinct set of TBP-associated factors (7, 33). Thus, TBP appears to be involved in all three RNA polymerase-mediated transcriptions. TBP has a bipartite structure with a highly conserved C-terminal core domain (amino acids 159 to 339), which folds as a molecular saddle, and is responsible for DNA binding via the concave underside. Additionally, the core also interacts with GTFs and other regulatory proteins via the solvent-exposed convex side. In contrast, the N-terminal region (amino acids 1 to 141) is variable in both length and sequence but is well conserved among vertebrates. It modulates DNA binding by interacting with the core domain and is characterized by a glutamine repeat region (Fig. ​(Fig.1).1). Recent studies have underscored the importance of the TBP N-terminal domain in transcriptional regulation both in yeast and mammalian systems (15, 21). Moreover, deletion of 55 and 96 amino acids from the N-terminal domain of TBP leads to inactivation of TATA-mediated transcription from the U6 small nuclear RNA promoter (24). FIG. 1. Shutoff of RNA polymerase II-mediated transcription does not correlate with 3Cpro-induced cleavage at the 18th glutamine-glycine site in TBP. (A) The domain structure of TBP consisting of the core, N-terminal with the glutamine stretch (Q), and acidic ... Infection of HeLa cells with PV causes a severe decrease in cellular transcription catalyzed by all three cellular RNA polymerases (10). Transcription mediated by RNA polymerase I (Pol I) is inhibited first, at 1 to 2 h postinfection, followed by inhibition of Pol II and Pol III transcription at approximately 3 and 4 h postinfection, respectively. Crawford et al. first showed that PV-induced inhibition of transcription observed in vivo could be recapitulated in vitro (8). Further studies showed that the viral protease 3Cpro alone was responsible for the shutoff of transcription by all three cellular RNA polymerases (36). Recent studies have shown that 3Cpro enters the nucleus in the form of its precursor 3CD (32), which presumably undergoes autocatalysis to generate 3Cpro in the nucleus of infected cells. A primary target of 3Cpro in PV-infected cells was previously identified to be the TATA-binding protein (6). Consistent with this observation, the TFIID complex isolated from PV-infected HeLa cells was transcriptionally inactive in an in vitro reconstituted transcription assay compared to the TFIID isolated from uninfected cells (19). Moreover, purified TBP could be directly cleaved by the purified 3Cpro in vitro, and the addition of purified TBP could completely restore both basal and activated transcription from the TATA and initiator promoters in HeLa cell extracts from PV-infected cells (37). Examination of the human TBP sequence revealed three Q-G sites at positions 12, 18, and 112 (Fig. ​(Fig.1).1). Subsequent in vitro studies using purified components showed that only the 18th Q-G site in TBP could be efficiently cleaved by 3Cpro both in vitro and in vivo (9). To determine if cleavage of TBP at the 18th Q-G bond is responsible for the shutoff of transcription in PV-infected cells, we expressed and purified a recombinant TBP that lacks the N-terminal 18 amino acids (called ΔN18 TBP). If cleavage at the 18th Q-G bond is the primary cause of transcriptional inactivation of TBP, then ΔN18 TBP should not be able to restore transcription in PV-infected cell extracts. We found that ΔN18 TBP was as active as the wild-type (wt) TBP in fully restoring Pol II transcription in PV-infected cell extracts from the adenovirus major late promoter (Ad MLP). We also found that the transcriptional activity of ΔN18 TBP was comparable to that of wt TBP in an in vitro transcription reconstitution assay. Since both genetic and biochemical evidence suggested that 3Cpro was involved in the shutoff of transcription and that TBP was the primary target of 3Cpro, we set out to determine if TBP was being cleaved by 3Cpro at sites other than the 18th Q-G bond. We report here identification of a Q-S site at position 104 to 105 of TBP, which appears to be cleaved by the viral protease 3Cpro. We also demonstrate that an alanine residue at P4 is critical for 3C-mediated cleavage of the 104th Q-S bond. A truncated form of TBP lacking the first 100 amino acids is significantly less active transcriptionally than the wt TBP in vitro. Finally, we demonstrate that a stable HeLa cell line expressing a recombinant TBP (rTBP) resistant to cleavage by the viral proteases is significantly refractive to the shutoff of transcription by poliovirus compared to the control cell line expressing wt TBP. Infection with poliovirus of the HeLa cell line expressing the noncleavable form of TBP shows small plaques and significantly reduced viral RNA synthesis compared to the control TBP cell line, suggesting that a defect in the shutoff of transcription can lead to inefficient replication of the virus.

Mammalian cells infected with poliovirus, the prototype member of the picornaviridae family, undergo rapid macromolecular and metabolic changes resulting in efficient replication and release of virus from infected cells. Although this virus is predominantly cytoplasmic, it does shut-off transcription of all three cellular transcription systems. Both biochemical and genetic studies have shown that a virally encoded protease, 3C(pro), is responsible for host cell transcription shut-off. The 3C protease cleaves a number of RNA polymerase II transcription factors including the TATA-binding protein (TBP), the cyclic AMP-responsive element binding protein (CREB), the Octamer binding protein (Oct-1), p53, and RNA polymerase III transcription factor IIICalpha, and Polymerase I factor SL-1. Most of these cleavages occur at glutamine-glycine bonds. Additionally, a second viral protease, 2A(pro), also cleaves TBP at a tyrosine-glycine bond. The latter cleavage could be responsible for shut-off of small nuclear RNA transcription. Recent studies indicate that the viral protease-polymerase precursor 3CD can enter nucleus in poliovirus-infected cells. The nuclear localization signal (NLS) present within the 3D sequence appears to play a role in the nuclear entry of 3CD. Thus, 3C may be delivered to the infected cell nucleus in the form the precursor 3CD or other 3C-containing precursors. Auto-proteolytic cleavage of these precursors could then generate 3C. Thus, for a small RNA virus that strictly replicates in the cytoplasm, a portion of its life cycle does include interaction with the host cell nucleus.

Host cell transcription mediated by all three RNA polymerases is rapidly inhibited after infection of mammalian cells with poliovirus (PV). Both genetic and biochemical studies have shown that the virus-encoded protease 3C cleaves the TATA-binding protein and other transcription factors at glutamine–glycine sites and is directly responsible for host cell transcription shut-off. PV replicates in the cytoplasm of infected cells. To shut-off host cell transcription, 3C or a precursor of 3C must enter the nucleus of infected cells. Although the 3C protease itself lacks a nuclear localization signal (NLS), amino acid sequence examination of 3D identified a potential single basic type NLS, KKKRD, spanning amino acids 125–129 within this polypeptide. Thus, a plausible scenario is that 3C enters the nucleus in the form of its precursor, 3CD, which then generates 3C by auto-proteolysis ultimately leading to cleavage of transcription factors in the nucleus. Using transient transfection of enhanced green fluorescent protein (EGFP) fusion polypeptides, we demonstrate here that both 3CD and 3D are capable of entering the nucleus in PV-infected cells. However, both polypeptides remain in the cytoplasm in uninfected HeLa cells. Mutagenesis of the NLS sequence in 3D prevents nuclear entry of 3D and 3CD in PV-infected cells. We also demonstrate that 3CD can be detected in the nuclear fraction from PV-infected HeLa cells as early as 2 h postinfection. Significant amount of 3CD is found associated with the nuclear fraction by 3–4 h of infection. Taken together, these results suggest that both the 3D NLS and PV infection are required for the entry of 3CD into the nucleus and that this may constitute a means by which viral protease 3C is delivered into the nucleus leading to host cell transcription shut-off.

We have previously demonstrated that quercetin, a bioflavonoid, blocks hepatitis C virus (HCV) proliferation by inhibiting NS5A-driven internal ribosomal entry site (IRES)-mediated translation of the viral genome. Here, we investigate the mechanisms of antiviral activity of quercetin and six additional bioflavonoids. We demonstrate that catechin, naringenin, and quercetin possess significant antiviral activity, with no associated cytotoxicity. Infectious virion secretion was not significantly altered by these bioflavonoids. Catechin and naringenin demonstrated stronger inhibition of infectious virion assembly compared to quercetin. Quercetin markedly blocked viral translation whereas catechin and naringenin demonstrated mild activity. Similarly quercetin completely blocked NS5A-augmented IRES-mediated translation in an IRES reporter assay, whereas catechin and naringenin had only a mild effect. Moreover, quercetin differentially inhibited HSP70 induction compared to catechin and naringenin. Thus, the antiviral activity of these bioflavonoids is mediated through different mechanisms. Therefore combination of these bioflavonoids may act synergistically against HCV.

We previously identified HSP70 and HSC70 in complex with NS5A in a proteomic screen. Here, coimmunoprecipitation studies confirmed NS5A/HSC70 complex formation during infection, and immunofluorescence studies showed NS5A and HSC70 to colocalize. Unlike HSP70, HSC70 knockdown did not decrease viral protein levels. Rather, intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains directly bind NS5A whereas only the HSP70 nucleotide binding domain does. Knockdown of both HSC70 and HSP70 demonstrated an additive reduction in virus production. This data suggests that HSC70 and HSP70 play discrete roles in the viral life cycle. Investigation of these different functions may facilitate developing of novel strategies that target host proteins to treat HCV infection.