Hepatitis C virus-induced prion protein expression facilitates hepatitis C virus replication

Hepatitis C virus (HCV) infects approximately 180 million people worldwide. Significant progress has been made since the establishment of in vitro HCV infection models in cells. However, the replication of HCV is complex and not completely understood. Here, we found that the expression of host prion protein (PrP) was induced in an HCV replication cell model. We then showed that increased PrP expression facilitated HCV genomic replication. Finally, we demonstrated that the KKRPK motif on the N-terminus of PrP bound nucleic acids and facilitated HCV genomic replication. Our results provided important insights into how viruses may harness cellular protein to achieve propagation.

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Physical and functional interaction between hepatitis C virus NS5A protein and ovarian tumor protein deubiquitinase 7B

Hepatitis C virus (HCV) NS5A protein plays crucial roles in viral RNA replication, virus assembly, and viral pathogenesis. Although NS5A has no known enzymatic activity, it modulates various cellular pathways through interaction with cellular proteins. HCV NS5A (and other HCV proteins) are reportedly degraded through the ubiquitin–proteasome pathway; however, the physiological roles of ubiquitylation and deubiquitylation in HCV infection are largely unknown. To elucidate the role of deubiquitylation in HCV infection, an attempt was made to identify a deubiquitinase (DUB) that can interact with NS5A protein. An ovarian tumor protein (OTU), deubiquitinase 7B (OTUD7B), was identified as a novel NS5A‐binding protein. Co‐immunoprecipitation analyses showed that NS5A interacts with OTUD7B in both Huh‐7 and HCV RNA replicon cells. Immunofluorescence staining revealed that HCV NS5A protein colocalizes with OTUD7B in the cytoplasm. Moreover, HCV infection was found to enhance the nuclear localization of OTUD7B. The OTUD7B‐binding domain on NS5A was mapped using a series of NS5A deletion mutants. The present findings suggest that the domain I of NS5A is important and the region from amino acid 121 to 126 of NS5A essential for the interaction. Either V121A or V124A mutation in NS5A disrupts the NS5A‐OTUD7B interaction. The results of this in vivo ubiquitylation assay suggest that HCV NS5A enhances OTUD7B DUB activity. Taken together, these results suggest that HCV NS5A protein interacts with OTUD7B, thereby modulating its DUB activity.     

Establishment of interferon alpha-resistant hepatitis C virus using cell culture system

To investigate the molecular mechanisms underlying interferon alpha (IFNα) treatment failure in hepatitis C virus (HCV) patients with chronic hepatitis, we aimed to develop an IFNα-resistant clone of HCV. By treating JFH-1-infected Huh7.5 cells with a prolonged low-dose treatment of IFNα, we selected a clone of HCV that survived against 100 U/ml of IFNα. By genetic analysis of this clone, we found four substitution mutations in the C-terminal coding sequence of non-structural 5A (NS5A). By introducing these four mutations into wild-type JFH-1, we established a new HCV clone that acquired IFNα resistant phenotype. These data suggest that four amino acid substitutions in NS5A are involved in IFNα resistance and thus this newly established HCV may be a useful tool for elucidating the molecular mechanisms of IFNα resistance in HCV patients.     

Overexpression of hepatitis C virus NS5A protein induces chromosome instability via mitotic cell cycle dysregulation

Hepatocellular carcinoma (HCC) is a common primary cancer associated with high incidences of genetic variations including chromosome instability. Moreover, it has been demonstrated that hepatitis C virus (HCV) is one of the major causes of HCC. However, no previous work has assessed whether HCV proteins are associated with the induction of chromosome instability. Here, we found that liver cell lines constitutively expressing full-length or truncated versions of the HCV genome show a high incidence of chromosome instability. In particular, the overexpression of HCV NS5A protein in cultured liver cells was found to promote chromosome instability and aneuploidy. Further experiments showed that NS5A-induced chromosome instability is associated with aberrant mitotic regulations, such as, an unscheduled delay in mitotic exit and other mitotic impairments (e.g. multi-polar spindles). Thus, our results indicate that HCV NS5A protein may be directly involved in the induction of chromosome instability via mitotic cell cycle dysregulation, and provide novel insights into the molecular mechanisms of HCV-associated hepatocarcinogenesis.     

Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins

Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines—HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A—D2303H, S2362G, and E2414K—enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC₅₀ values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation.     

Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is involved both in the viral replication and particle production. Its third domain (NS5A-D3), although not absolutely required for replication, is a key determinant for the production and assembly of novel HCV particles. As a prerequisite to elucidate the precise functions of this domain, we report here the first molecular characterization of purified recombinant HCV NS5A-D3. Sequence analysis indicates that NS5A-D3 is mostly unstructured but that short structural elements may exist at its N-terminus. Gel filtration chromatography, circular dichroism and finally NMR spectroscopy all point out the natively unfolded nature of purified recombinant NS5A-D3. This lack of stable folding is thought to be essential for primary interactions of NS5A-D3 domain with other viral or host proteins, which could stabilize some specific conformations conferring new functional features.     

A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 (a.a. 3-631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.     

Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain‐containing 3 and induces activator protein 1 activation

Hepatitis C virus (HCV) non‐structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A‐interacting protein, SET and MYND domain‐containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon‐harboring and HCV‐infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N‐SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A‐SMYD3 interaction. NS5A co‐localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP‐1) activity, this being potentiated by co‐expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP‐1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP‐1 activation in HCV‐infected cells.     

The protein phosphatase 2A represents a novel cellular target for hepatitis C virus NS5A protein

It is well established that HCV NS5A protein when expressed in mammalian cells perturbs the extracellular signal regulated kinase (ERK) pathway. The protein serine/threonine phosphatase 2A controls the phosphorylation of numerous proteins involved in cell signaling and one characterized function is the regulation of Ras-Raf mitogen activated protein (MAP) kinase signaling pathways. Our results showed that expression of HCV NS5A protein stimulates phosphatase 2A (PP2A) activity in cells, indicating the relevance of NS5A as a regulator of PP2A in vivo. We found that transient expression of the full length NS5A protein in different cell lines leads to a significant increase of the PP2A activity and this activity is specifically inhibited by the addition of okadaic acid, a PP2A inhibitor, in living cells. Further investigation showed that NS5A protein interacts in vivo and in vitro with the scaffolding A and the catalytic C subunits of PP2A. We propose that HCV NS5A represents a viral PP2A regulatory protein. This is a novel function for the NS5A protein which may have a key role in the ability of the virus to deregulate cell growth and survival.     

Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells

The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Delta4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Delta4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Delta4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Delta4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-alpha (IFN-alpha). Consequently, this EG(Delta4AB)SEAP reporter can be used to screen for NS3/4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells.     

丙型肝炎病毒NS5A蛋白对Huh7细胞周期的影响

应用PCR技术从含有丙型肝炎病毒（HCV）全长开放阅读框的质粒pBRTM／HCV1～3011中获得NS5A全长基因片段,利用基因重组技术将其克隆至真核表达载体pcDNA3．1（-）中。通过酶切、PCR及测序鉴定证实,NS5A基因已正确插入到pcDNA3．1（-）中。再利用脂质体介导转染Huh7细胞,30h后收获细胞,经Western blot验证,证实HCV的NS5A基因在Huh7细胞中已经获得表达。在培养条件完全一致的条件下,表达NS5A基因的Huh7细胞与pcDNA3．1（-）转染的细胞在转染30h后被收集起来,乙醇固定,PI染色后利用流式细胞仪检测细胞周期变化。G0／G1期由60．6％下降到49．7％,S期由23．9％上升到32．7％,而转染pcDNA3．1（-）细胞的细胞周期与正常的Huh7细胞则差别不大。从而证明HCV NS5A蛋白对Huh7细胞周期具有调节作用。     

Unconjugated interferon‐stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

Interferon‐stimulated gene 15 (ISG15), a ubiquitin‐like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti‐ or pro‐viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation‐independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C‐terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein–protein interaction.

     

RNA template-mediated inhibition of hepatitis C virus RNA-dependent RNA polymerase activity

The enzymatic activity of hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is modulated by the molar ratio of NS5B enzyme and RNA template. Depending on the ratio, either template or enzyme can inhibit activity. Inhibition of NS5B activity by RNA template exhibited characteristics of substrate inhibition, suggesting the template binds to a secondary site on the enzyme forming an inactive complex. Template inhibition was modulated by primer. Increasing concentrations of primer restored NS5B activity and decreased the affinity of template for the secondary site. Conversely, increasing template concentration reduced the affinity of primer binding. The kinetic profiles suggest template inhibition results from the binding of template to a site that interferes with primer binding and the formation of productive replication complexes.     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.