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.     

Relation between Reactivity to the NS-4 Region Peptides of Hepatitis C Virus (HCV) and Clinical Features among Patients Infected with HCV Genotype 1b

Nearly all patients infected with hepatitis C virus (HCV) genotype 1b have reactivity to the core (c22-3) or non-structural (NS)-3 region (c33c) protein in a second-generation recombinant immunoblot assay (RIBA-2). However, reactivities to the NS-4 region antigens (5-1-1, c100-3) vary among patients. To clarify whether differences in serological reactivities to the NS-4 antigens are associated with the clinical features or response to interferon (IFN) therapy of patients infected with hepatitis C virus (HCV) genotype 1b, we clinically investigated 115 such patients. Positive reactions to 5-1-1 and c100-3 were seen in 75.7 and 79.1%, respectively, of the patients. There were no differences between the patients with and those without antibodies to NS-4 region antigens (5-1-1, c100-3) with regard to age, duration of HCV infection, severity of liver disease and virus load. Fifty-one of the patients were treated with recombinant IFN-α, and 17 of the 51 patients showed sustained response to the therapy. The sustained response was more frequently seen in the patients positive for antibodies to both 5-1-1 and c100-3 as compared with those negative for either or both antibodies (41.0% vs. 8.3%, P < 0.05).     

Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells

The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.     

Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A–NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.     

双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制

α干扰素为治疗丙型肝炎病毒(HCV)感染的主要药物,但部分患者呈干扰素耐受而不能获得持久的病毒阴转,其可能的原因之一是病毒通过其编码的蛋白(NS5A及E2)抑制干扰素诱导的抗病毒效应分子——双链RNA激活的蛋白激酶(PKR)的活性.而关于PKR是否在IFN-α抗HCV的机理中起抑制作用目前仍有争议.为研究PKR对HCV蛋白合成环节是否有抑制作用,通过构建野生型 PKR真核表达载体（pPKRwt）及主要起负性调节作用的缺失突变PKR真核表达载体（pPKRΔ6）,并将pPKRwt /pPKRΔ6 与HCV复制子RNA同时转染Huh7细胞进行共表达, 用Western印迹检测 HCV IRES 下游的NPTⅡ蛋白表达水平,与转染空载体的对照细胞及单用IFN-α处理的细胞相比较.结果显示：表达PKRwt的细胞中NPTⅡ蛋白水平低于转染空载体的对照细胞,但高于经IFN-α单独处理的细胞;表达PKRΔ6的细胞中NPTⅡ蛋白水平与对照细胞无明显差别,但PKRΔ能部分抵消IFN-α的抑制作用,说明在IFN-α抑制HCV IRES指导的蛋白合成中,PKR有一定的抑制作用,但可能还有其它的PKR非依赖机制参与.     

Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2alpha (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208-5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262-1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2alpha after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2alpha phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.     

Determination of Sustained Virological Response in Hepatitis C Virus Genotypes by the Number of Mutations in the E2 and NS5A-ISDR Regions: A Meta-Analysis

Since last few decades hepatitis C virus (HCV) has been a major cause of death due to the involvement of acute and chronic type of liver diseases throughout the world. Genotype variability and mutations occurring at different regions of HCV genome provides a critical parameter for the study of sustained virological response (SVR) against mono and combinational therapies. Most of these mutations occurring in E2 and NS5A-ISDR regions in HCV genotypes play a significant role in SVR against Interferon-monotherapy and combination therapy. Therefore, the aim of the present study is to evaluate the SVR in various genotypes and the role of mutations in specific regions. In line with this, the NS5A and E2 proteins of HCV genotype 1 were found to suppress the double-stranded (ds) RNA-dependent protein kinase (PKR), which in turn is entailed in the cellular antiviral response stimulated by interferon (IFN). The response to IFN therapy varies between genotypes, with response rates among patients infected with types 2 and 3 nearly two-three-fold greater than in patients infected with type 1. Surprisingly, a considerable percentage of HCV genotype 3a infected patients do not react to treatment at all. In Japan, a link was observed between the numbers of mutations in an “interferon sensitivity determining region” (ISDR) and the result of interferon treatment in genotype 1b infected patients. Therefore, we published data on E2 (PePHD) and NS5A-ISDR regions including our data on SVR of different HCV genotypes, the relationship between the number and patterns of mutations in the E2 (PePHD) and NS5A-ISDR regions and responsiveness to IFN therapy.     

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture-produced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.     

Anti-interferon-α neutralizing antibody induced telaprevir resistance under the interferon-α plus telaprevir treatment in vitro

Although the development of anti-interferon (IFN)-α neutralizing antibodies (NAbs) is likely to be a common clinical problem for patients with various diseases treated with IFN, anti-IFN-α NAb has been exceptionally considered to have no clinical significance in the treatment of chronic hepatitis C with pegylated IFN-α (Peg-IFN-α). However, we recently clarified that the presence of NAb was associated with a non-response to the Peg-IFN plus ribavirin (RBV) therapy. In this study, we used the HCV-replicon system with genotype 1b, and investigated the role of anti-IFN-α NAb in the response to telaprevir (TVR)-containing new antiviral therapy for hepatitis C virus (HCV). Anti-IFN-α NAb-positive sera specifically inhibited the anti-HCV effects of IFN-α, without any effect on the activity of IFN-β in vitro. The NAb-positive sera also inhibited the IFN-α-dependent induction of interferon-stimulated genes, MxA and OAS-1, in a dose-dependent manner. Although TVR monotherapy decreased the HCV-RNA in vitro, the HCV-RNA was increased again with the development of TVR-resistant mutations. When IFN-α was administrated with TVR, the replication of HCV was continuously suppressed for more than a month. However, in the presence of anti-IFN-α NAb-positive sera, even when IFN-α was combined with TVR, the levels of HCV-RNA exhibited a time-course similar to that with TVR monotherapy, and HCV with TVR-resistant mutations emerged. In conclusion, our findings suggest that the presence of IFN-α NAb decreases the antiviral effects of IFN-α and may be related to the development of TVR-resistant mutated viruses.     

Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.     

Analysis of Functional Differences between Hepatitis C Virus NS5A of Genotypes 1–7 in Infectious Cell Culture Systems

Hepatitis C virus (HCV) is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN)-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1–7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a) and QC69(7a) recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a). Reduced virus production was linked to attenuated replication in all cases, but ED43(4a) and SA13(5a) also displayed impaired particle assembly. Compared to the original H77C(1a) NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a) and TN(1a) NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important for understanding of HCV NS5A function and therapeutic targeting.     

Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.     

Contribution of Genome-Wide HCV Genetic Differences to Outcome of Interferon-Based Therapy in Caucasian American and African American Patients

Background

Hepatitis C virus (HCV) has six major genotypes, and patients infected with genotype 1 respond less well to interferon-based therapy than other genotypes. African American patients respond to interferon α-based therapy at about half the rate of Caucasian Americans. The effect of HCV''s genetic variation on treatment outcome in both racial groups is poorly understood.

Methodology

We determined the near full-length pre-therapy consensus sequences from 94 patients infected with HCV genotype 1a or 1b undergoing treatment with peginterferon α-2a and ribavirin through the Virahep-C study. The sequences were stratified by genotype, race and treatment outcome to identify HCV genetic differences associated with treatment efficacy.

Principal Findings

HCV sequences from patients who achieved sustained viral response were more diverse than sequences from non-responders. These inter-patient diversity differences were found primarily in the NS5A gene in genotype 1a and in core and NS2 in genotype 1b. These differences could not be explained by host selection pressures. Genotype 1b but not 1a African American patients had viral genetic differences that correlated with treatment outcome.

Conclusions & Significance

Higher inter-patient viral genetic diversity correlated with successful treatment, implying that there are HCV genotype 1 strains with intrinsic differences in sensitivity to therapy. Core, NS3 and NS5A have interferon-suppressive activities detectable through in vitro assays, and hence these activities also appear to function in human patients. Both preferential infection with relatively resistant HCV variants and host-specific factors appear to contribute to the unusually poor response to therapy in African American patients.     

An objective assessment of conformational variability in complexes of hepatitis C virus polymerase with non-nucleoside inhibitors

A major target for antiviral therapy against hepatitis C virus (HCV) is the HCV polymerase nonstructural protein 5B (NS5B). Huge efforts have been devoted to the development of nucleoside and non-nucleoside inhibitors (NNIs) of NS5B. An offshoot of these efforts has been the structural characterization of the interaction of NS5B with NNIs by X-ray crystallography. These works have shown that the conformation of recombinant NS5B is very similar across strains, constructs and complexes, making evaluation of the long-range conformational effects of NNIs nontrivial.Using procedures appropriate to the evaluation of such minor but potentially important differences, we objectively assessed the conformational diversity in the 78 available genotype 1b NS5B structures in the Protein Data Bank. We find that there are 20 significantly different NS5B conformations available, but all are geometrically close to a closed, RNA synthesis initiation-competent one. Within this fairly restricted range, differences can be mapped to movements of NS5B domains and subregions. Most of this information is actually defined by small but significant changes in complexes with NNIs. We thus establish rigorously the moving parts of the NS5B molecular machine and the previously unrecognized hinge points that come into play upon NNI binding.We propose that NNIs binding at three of the four distinct sites specifically inhibit the initiation step by the same mechanism: they prevent NS5B's “thumb” from quite reaching the proper initiation-competent position. Furthermore, we suggest that a small number of critical hinges in the NS5B structure may emerge as sites of resistance mutations during future antiviral treatment.     

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.     

Both nonstructural proteins NS1 and NS2 of pneumonia virus of mice are inhibitors of the interferon type I and type III responses in vivo

Infection of mice with pneumonia virus of mice (PVM) provides a convenient experimental pathogenesis model in a natural host for a human respiratory syncytial virus-related virus. Extending our previous work showing that the PVM nonstructural (NS) proteins were pathogenicity factors in mice, we identify both the NS1 and NS2 proteins as antagonists of alpha/beta interferon (IFN-α/β) and IFN-λ by use of recombinant PVM (rPVM) with single and combined deletions of the NS proteins (ΔNS1, ΔNS2, and ΔNS1 ΔNS2). Wild-type and NS deletion PVMs were evaluated for growth and pathogenesis by infecting knockout mice that lack functional receptors to IFN-α/β, IFN-λ, or both. The absence of the receptor to IFN-α/β (IFNAR) or IFN-λ (interleukin-28 receptor α chain [IL-28Rα]) individually did not reverse the attenuated virulence of the NS deletion viruses although loss of IFNAR partially restored replication efficiency. When both receptors were deleted, replication and virulence were largely rescued for rPVM ΔNS1 and were significantly but not completely rescued for rPVM ΔNS2. As for rPVM ΔNS1 ΔNS2, the effect was mostly limited to partial enhancement of replication. This indicates that both IFN-α/β and IFN-λ contributed to restricting the NS deletion viruses, with the former playing the greater role. Interestingly, the replication and virulence of wild-type PVM were completely unaffected by the presence or absence of functional receptors to IFN-α/β and IFN-λ, indicating that both systems are strongly suppressed during infection. However, pretreatment of mice with IFN-α/β was protective against lethal rPVM challenge, whereas pretreatment with IFN-λ delayed but did not prevent disease and, in some cases, reduced mortality. The fact that virulence of rPVM lacking NS2 was not recovered completely when both interferon receptors were deleted suggests that NS2 may have further functions outside the IFN system.     

Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections.     

Ferrocene-based inhibitors of hepatitis C virus replication that target NS5A with low picomolar in vitro antiviral activity

An unprecedented series of organometallic HCV (hepatitis C virus) NS5A (nonstructural 5A protein) replication complex inhibitors that incorporates a 1,1′-ferrocenediyl scaffold was explored. This scaffold introduces the elements of linear flexibility and non-planar topology that are unconventional for this class of inhibitors. Data from 2-D NMR spectroscopic analyses of these complexes in solution support an anti (unstacked) arrangement of the pharmacophoric groups. Several complexes demonstrate single-digit picomolar in vitro activity in an HCV genotype-1b replicon system. One complex to arise from this investigation (10a) exhibits exceptional picomolar activity against HCV genotype 1a and 1b replicons, low hepatocellular cytotoxicity, and good pharmacokinetic properties in rat.