Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti SARS-CoV-2 activity against fully infectious virus of dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as the concentrations that inhibited viral replication by 50%; CC50s were also calculated and defined as the concentrations that kill 50% of cells. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet its hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.

Artemisia annua L. extracts inhibit the in vitro replication of SARS-CoV-2 and two of its variants.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. has been used for millennia in Southeast Asia to treat "fever". Many infectious microbial and viral diseases have been shown to respond to A. annua and communities around the world use the plant as a medicinal tea, especially for treating malaria. AIM OF THE STUDY: SARS-CoV-2 (the cause of Covid-19) globally has infected and killed millions of people. Because of the broad-spectrum antiviral activity of artemisinin that includes blockade of SARS-CoV-1, we queried whether A. annua suppressed SARS-CoV-2. MATERIALS AND METHODS: Using Vero E6 and Calu-3 cells, we measured anti viral activity SARS-CoV-2 activity against fully infectious virusof dried leaf extracts of seven cultivars of A. annua sourced from four continents. IC50s were calculated and defined as (the concentrations that inhibited viral replication by 50%.) and CC50s (the concentrations that kill 50% of cells) were calculated. RESULTS: Hot-water leaf extracts based on artemisinin, total flavonoids, or dry leaf mass showed antiviral activity with IC50 values of 0.1-8.7 µM, 0.01-0.14 µg, and 23.4-57.4 µg, respectively. Antiviral efficacy did not correlate with artemisinin or total flavonoid contents of the extracts. One dried leaf sample was >12 years old, yet the hot-water extract was still found to be active. The UK and South African variants, B1.1.7 and B1.351, were similarly inhibited. While all hot water extracts were effective, concentrations of artemisinin and total flavonoids varied by nearly 100-fold in the extracts. Artemisinin alone showed an estimated IC50 of about 70 µM, and the clinically used artemisinin derivatives artesunate, artemether, and dihydroartemisinin were ineffective or cytotoxic at elevated micromolar concentrations. In contrast, the antimalarial drug amodiaquine had an IC50 = 5.8 µM. Extracts had minimal effects on infection of Vero E6 or Calu-3 cells by a reporter virus pseudotyped by the SARS-CoV-2 spike protein. There was no cytotoxicity within an order of magnitude above the antiviral IC90 values. CONCLUSIONS: A. annua extracts inhibit SARS-CoV-2 infection, and the active component(s) in the extracts is likely something besides artemisinin or a combination of components that block virus infection at a step downstream of virus entry. Further studies will determine in vivo efficacy to assess whether A. annua might provide a cost-effective therapeutic to treat SARS-CoV-2 infections.

Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses.

Antiviral therapies that impede virus entry are attractive because they act on the first phase of the infectious cycle. Drugs that target pathways common to multiple viruses are particularly desirable when laboratory-based viral identification may be challenging, e.g., in an outbreak setting. We are interested in identifying drugs that block both Ebola virus (EBOV) and Lassa virus (LASV), two unrelated but highly pathogenic hemorrhagic fever viruses that have caused outbreaks in similar regions in Africa and share features of virus entry: use of cell surface attachment factors, macropinocytosis, endosomal receptors, and low pH to trigger fusion in late endosomes. Toward this goal, we directly compared the potency of eight drugs known to block EBOV entry with their potency as inhibitors of LASV entry. Five drugs (amodiaquine, apilimod, arbidol, niclosamide, and zoniporide) showed roughly equivalent degrees of inhibition of LASV and EBOV glycoprotein (GP)-bearing pseudoviruses; three (clomiphene, sertraline, and toremifene) were more potent against EBOV. We then focused on arbidol, which is licensed abroad as an anti-influenza drug and exhibits activity against a diverse array of clinically relevant viruses. We found that arbidol inhibits infection by authentic LASV, inhibits LASV GP-mediated cell-cell fusion and virus-cell fusion, and, reminiscent of its activity on influenza virus hemagglutinin, stabilizes LASV GP to low-pH exposure. Our findings suggest that arbidol inhibits LASV fusion, which may partly involve blocking conformational changes in LASV GP. We discuss our findings in terms of the potential to develop a drug cocktail that could inhibit both LASV and EBOV.IMPORTANCE Lassa and Ebola viruses continue to cause severe outbreaks in humans, yet there are only limited therapeutic options to treat the deadly hemorrhagic fever diseases they cause. Because of overlapping geographic occurrences and similarities in mode of entry into cells, we seek a practical drug or drug cocktail that could be used to treat infections by both viruses. Toward this goal, we directly compared eight drugs, approved or in clinical testing, for the ability to block entry mediated by the glycoproteins of both viruses. We identified five drugs with approximately equal potencies against both. Among these, we investigated the modes of action of arbidol, a drug licensed abroad to treat influenza infections. We found, as shown for influenza virus, that arbidol blocks fusion mediated by the Lassa virus glycoprotein. Our findings encourage the development of a combination of approved drugs to treat both Lassa and Ebola virus diseases.

Arbidol: a broad-spectrum antiviral compound that blocks viral fusion.

Arbidol (ARB; ethyl-6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate), is a Russian-made potent broad-spectrum antiviral with demonstrated activity against a number of enveloped and non-enveloped viruses. ARB is well known in Russia and China, although to a lesser extent in western countries. Unlike other broad-spectrum antivirals, ARB has an established molecular mechanism of action against influenza A and B viruses, which is different from that of available influenza antivirals, and a more recently established mechanism of inhibition of hepatitis C virus (HCV). For both viral infections the anti-viral mechanism involves ARB inhibition of virus-mediated fusion with target membrane and a resulting block of virus entry into target cells. However, ARB inhibition of fusion exploits different ARB modalities in case of influenza viruses or HCV. This review aims to summarize the available evidence of ARB effects against different groups of viruses, also, to compare various aspects of ARB anti-fusion mechanisms against influenza virus and HCV (with reference to different stringency of pH-dependence of these two viral fusogens) and to discuss further prospects for ARB and its improved derivatives of the parent compounds.

Prospective multicenter clinical evaluation of AMPLICOR and COBAS AMPLICOR hepatitis C virus tests.

We conducted a multicenter clinical evaluation of the second versions of the manual AMPLICOR and the semiautomated COBAS AMPLICOR tests for hepatitis C virus (HCV) RNA (Roche Molecular Systems, Inc., Pleasanton, Calif.). The performance characteristics of these HCV RNA tests for diagnosis of active viral infection were determined by comparison to anti-HCV serological test results, alanine aminotransferase levels, and liver biopsy histology results. A total of 878 patients with clinical or biochemical evidence of liver disease were enrolled at four hepatology clinics. A total of 1,089 specimens (901 serum and 188 plasma) were tested with the AMPLICOR test. Sensitivity compared to serology was 93.1% for serum and 90.6% for plasma. The specificity was 97% for serum and 93.1% for plasma. A total of 1,084 specimens (896 serum and 188 plasma) were tested with the COBAS test. Sensitivities for serum and plasma were the same as with the AMPLICOR test. The specificity was 97.8% for serum and 96.6% for plasma. Of the 69 specimens with false-positive and false-negative AMPLICOR test results relative to those of serology, alternative primer set (APS) reverse transcription (RT)-PCR analysis showed that the AMPLICOR test provided the correct result relative to the specimens containing HCV RNA in 64 (92.7%) specimens. Similarly, 66 of 67 (98.5%) false-positive and false-negative COBAS test results were determined to be correct by APS RT-PCR analysis. There were no substantive differences in clinical performances between study sites, patient groups, specimen types, storage conditions (-20 to -80 degrees C versus 2 to 8 degrees C), or anticoagulants (EDTA versus acid citrate dextrose) for either test. Both tests showed >99% reproducibility within runs, within sites, and overall. We conclude that these tests can reliably detect the presence of HCV RNA, as evidence of active infection, in patients with clinical or biochemical evidence of liver disease.

Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-DeltaN110), 222 (NS5A-DeltaN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-DeltaN110 and NS5A-DeltaN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-kappaB and AP-1 were important in NS5A-DeltaN222 transactivation in the presence of tumor necrosis factor alpha and that NF-IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

Elevated levels of interleukin-8 in serum are associated with hepatitis C virus infection and resistance to interferon therapy.

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.

Analyzing the mechanisms of interferon-induced apoptosis using CrmA and hepatitis C virus NS5A.

The dsRNA-dependent protein kinase, PKR, is a key component of interferon (IFN)-mediated anti-viral action and is frequently inhibited by many viruses following infection of the cell. Recently, we have demonstrated that IFN and PKR can sensitize cells to apoptosis predominantly through the FADD/caspase-8 pathway (S. Balachandran, P. C. Roberts, T. Kipperman, K. N. Bhalla, R. W. Compans, D. R. Archer, and G. N. Barber. (2000b) J. Virol. 74, 1513-1523). Given these findings, it is thus plausible that rather than specifically target IFN-inducible genes such as PKR, viruses could also subvert the mechanisms of IFN action, in part, at locations that could block the apoptotic cascade. To explore this possibility, we analyzed whether the poxvirus caspase-8 inhibitor, CrmA, was able to inhibit IFN or PKR/dsRNA-mediated apoptosis. Our findings indicated that CrmA could indeed inhibit apoptosis induced by both viral infection and dsRNA without blocking PKR activity or inhibiting IFN signaling. In contrast HCV-encoded NS5A, a putative inhibitor of PKR, did not appear to inhibit cell death mediated by a number of apoptotic stimuli, including IFN, TRAIL, and etoposide. Our data imply that viral-encoded inhibitors of apoptosis, such as CrmA, can block the innate arms of the immune response, including IFN-mediated apoptosis, and therefore potentially constitute an alternative family of inhibitors of IFN action in the cell.

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.

The protein kinase-interacting domain in the hepatitis C virus envelope glycoprotein-2 gene is highly conserved in genotype 1-infected patients treated with interferon.

The hepatitis C virus (HCV) envelope glycoprotein-2 inhibits the interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) via the PKR eukaryotic initiation factor-2alpha phosphorylation homology domain (PePHD). The present study examined the genetic variability of the PePHD in patients receiving IFN therapy. The PePHD from 12 HCV genotype 1 (HCV-1)-infected patients receiving daily IFN therapy was amplified by reverse-transcriptase polymerase chain reaction and analyzed by direct and clonal sequencing. The PePHD was highly conserved in 38 HCV GenBank isolates. There was no difference in pretreatment PePHD sequences isolated from IFN responders versus nonresponders. The major PePHD quasi-species variant did not change after 6 weeks of daily IFN therapy, and in 1 patient the major quasi-species variant did not change during 9 months of observation. Sequencing of 25 pretreatment PePHD clones from 3 patients confirmed that there was extremely low sequence variability surrounding the PePHD. The PePHD is highly conserved in HCV-1-infected IFN responders and nonresponders and does not appear to evolve in response to IFN therapy.

The molecular basis for responsiveness to anti-viral therapy in hepatitis C.

Hepatitis C virus (HCV) infection is an important clinical problem, with a world-wide prevalence of approximately 1-2%. HCV infection is associated with an increased risk for the development of severe liver disease. HCV is inherently resistant to anti-viral therapy with interferon (IFN). The virus circulates in infected individuals as a mixture of related, yet genetically distinct variants, or quasispecies. Many studies have implicated HCV quasispecies in IFN responsiveness. Effective containment of HCV quasispecies mutation and selection through more aggressive therapy (e.g. daily induction), combination therapy (e.g. IFN plus ribavirin), or longer lasting therapy (e.g. pegylated IFN) is required for IFN responsiveness. Recently, several HCV proteins including the non-structural 5A and envelope gene 2-glycoprotein have been implicated in HCV anti-viral resistance. It is likely that multiple HCV genes disrupt IFN-induced anti-viral responses at many levels and that these virus-host cell interactions are associated with IFN resistance. Characterisation of HCV-encoded mechanisms of anti-viral resistance has important implications for the development of new anti-virals.

Evidence for sequence selection within the non-structural 5A gene of hepatitis C virus type 1b during unsuccessful treatment with interferon-alpha.

Resistance of the hepatitis C virus (HCV) to interferon-alpha (IFN-alpha) therapy in patients with hepatitis C may be genetically controlled by an IFN sensitivity-determining region (ISDR) within the non-structural 5A (NS5A) gene. To assess whether HCV 1b strains carrying a 'resistant' type of ISDR are selected during unsuccessful IFN therapy, we analysed the evolution of the NS5A quasispecies, as detected by the clonal frequency analysis technique, and of the ISDR sequence by nucleotide sequence determination, in 11 patients showing no virological response during two consecutive cycles of IFN-alpha therapy. IFN-resistant patients had a homogeneous ISDR quasispecies with sequences identical to those described as 'resistant-' or 'intermediate-' type ISDR. After retreatment with IFN, further selection towards a homogeneous viral population was observed and 10 out of 11 patients had only one variant of HCV with no or just one single amino acid mutation within the ISDR sequence. Treatment and retreatment with IFN was associated in our non-responder patients with evolution of the ISDR quasispecies towards a rather homogeneous viral population carrying a conserved or minimally mutated ISDR motif, supporting the idea that this motif may be relevant for IFN resistance in HCV 1b-infected individuals.

Effect of retreatment with interferon alone or interferon plus ribavirin on hepatitis C virus quasispecies diversification in nonresponder patients with chronic hepatitis C.

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.

Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN-induced, RNA-dependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A-expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2-OS) cell lines were generated that express NS5A under tight regulation by the tetracycline-dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half-life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high-level NS5A expression. Full-length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A-1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40-fold, whereas a full-length NS5A-1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A-1a-triangle upISDR) isolated from a genotype 1a IFN-nonresponsive patient showed no rescue activity. The NS5A-1b and NS5A-1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two- to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN-sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection.

Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.

The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Evolution of hepatitis C virus quasispecies in hypervariable region 1 and the putative interferon sensitivity-determining region during interferon therapy and natural infection.

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.

Mutations in the NS5A gene of hepatitis C virus in North American patients infected with HCV genotype 1a or 1b.

Previous studies from Japan have described an association between a conserved sequence within the hepatitis C virus (HCV) genome and resistance to interferon (IFN) therapy for patients infected with HCV genotype 1b [Enomoto et al. (1995): Journal of Clinical Investigation 96: 224-230; Enomoto et al. (1996): New England Journal of Medicine 334:77-81]. The present study examines amino acid sequences surrounding the putative Interferon Sensitivity Determining Region (ISDR) of the NS5A gene of HCV in 21 North American patients with genotype 1a or 1b infection receiving recombinant IFN therapy. The ISDR consensus or intermediate pattern was observed in 13 of 14 NS5A clones from North American patients infected with genotype 1b. However, we found no evidence of the consensus ISDR sequence in any NS5A clones isolated from 15 patients with genotype 1a infection. In select cases, gel shift analysis showed no significant changes in the clonal frequency of the putative ISDR domain of HCV genotype 1a or 1b infected patients who were either nonresponsive to IFN therapy, or relapsed following withdrawal of IFN therapy. These results suggest that a conserved ISDR domain is neither associated with, nor responsible for, IFN resistance in North American patients infected with HCV genotype 1a, and demonstrate a need for further investigation into the reported association between ISDR consensus sequences and IFN resistance in genotype 1b clones.

Assessment of hepatitis C virus quasispecies heterogeneity by gel shift analysis: correlation with response to interferon therapy.

Hepatitis C virus (HCV) quasispecies heterogeneity was assessed in pretreatment sera of 22 patients treated with interferon (IFN). Quasispecies heterogeneity was quantitated by analysis of 490 hypervariable region 1 (HVR1) clones using gel shift analysis (GSA), which allowed determination of two components of HCV quasispecies heterogeneity: genetic complexity (number of variants) and genetic diversity (mean genetic distance between variants). HCV genotype and pretreatment RNA titer were similar between responders (n = 12) and nonresponders (n = 10). GSA correlated well with nucleotide sequencing for estimating HCV genetic diversity (R = .952; P < .001). A positive correlation of .666 (P < .001) was observed between genetic diversity and complexity, suggesting that these components of quasispecies heterogeneity were related. However, while there were no significant differences in pretreatment complexity or genetic diversity among response groups, higher pretreatment genetic diversity was predictive of response failure (P = .041). Assessment of HVR1 heterogeneity by GSA revealed important relationships between quasispecies genetic diversity, complexity, and response to IFN therapy and suggested a need for analysis of other HCV genes.

Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein.

Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally the inhibition of the PKR protein kinase, by NS5A is the first described function for this HCV protein.

Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes.

Hepatitis C virus (HCV), a positive-strand RNA virus, is the major infectious agent responsible for causing chronic hepatitis. Currently, there is no vaccine for HCV infection, and the only therapy for chronic hepatitis C is largely ineffective. To investigate new genetic approaches to the management of HCV infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library, characterized, and expressed from recombinant adenovirus vectors. The expressed ribozymes individually or in combination were efficient at reducing or eliminating the respective plus- or minus-strand HCV RNAs expressed in cultured cells and from primary human hepatocytes obtained from chronic HCV-infected patients. This study demonstrates the potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection.