Hepatitis c virus rna polymerase and ns5a complex with a. Baseline amino acid substitutions in the ns5a isdr and pkr binding domain of hepatitis c and different fibrosis levels and levels of development of hepatocellular carcinoma. Ns5a ras testing is recommended for genotype 3infected, treatmentnaive patients with cirrhosis and treatmentexperienced patients without cirrhosis being considered for 12 weeks of sofosbuvirvelpatasvir. The point mutation in ns5a, a protein that has been implicated in inhibiting pkr function,36 could modify pkrdependent repression of innate immunity. While no known enzymatic function has been ascribed to ns5a, it is an essential component of the hcv. There are multiple ways by which various proteins of the hepatitis c virus interact with host cells, undermine cellular protective mechanisms, and modulate cellular growth in advantage of. Ns5a may therefore influence irf1 function by regulating pkr activity during hcv rna replication.
Finally the inhibition of the pkr protein kinase, by ns5a is the first described function for this hcv protein. It appears to be a dimeric form without trans membrane helices. Pkr binds the hcv ires rna, cooperates with some functions of the hcv core protein and may represent a target for ns5a or e2. Cypi, by disrupting ns5a cypa complexes, block hcv replication both in vitro and in patients. The role of such interaction in modulating the antiviral effect of ifn is still controversial. Pdf baseline amino acid substitutions in the ns5a isdr and. Implication of protein kinase r gene quantification in. Ns5a is derived from a large polyprotein that is translated from the hcv genome, and undergoes post. The clinical correlation between amino acid mutations within the hcv ns5a region and response to antiviral treatment is controversial. Elbasvir is an inhibitor of hcv ns5a, which is essential for viral rna replication and virion assembly. Pdf baseline amino acid substitutions in the ns5a isdr. Activation of the interferoninducible protein kinase pkr by. Chronic infection with hepatitis c virus hcv is associated with progressive liver damage, including the development of cirrhosis and. The aim of the study was to investigate a relationship between isdrpkr substitutions and their association with liver fibrosis or hcc development.
Overall, comparing isdr and pkr bd substitutions between svr and failingtreatment patients, no signi. Nonstructural protein 5a ns5a is a zincbinding and prolinerich hydrophilic phosphoprotein that plays a key role in hepatitis c virus rna replication. The ns5a and the e2 proteins of hepatitis c virus hcv1b can bind and inhibit in vitro the interferon ifninduced cellular kinase pkr. The hcv ns5a drug resistance assay is a genotypic sequencing resistance assay that analyzes the nonstructural ns 5a region of hepatitis c virus hcv genotypes 1a or 1b using nextgeneration sequencing ngs techniques.
Protein kinase rna pkr regulated is a doublestranded rna activated protein kinase whose expression is induced by interferon. We have analyzed the e2 and the ns5a sequences in hcv1binfected patients treated with ifn to assess whether and how different combinations. Hepatitis c virus hcv ns5a is a phosphoprotein that possesses a cryptic transactivation activity. 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. Baseline amino acid substitutions in the ns5a isdr and pkr. Two pkr inhibitor hcv proteins correlate with early but not. An overview of hcv molecular biology, replication and. The aasld and idsa guidelines do not currently recommend routine use of ns5a resistance testing in this circumstance. Daclatasvir, asunaprevir plus ribavirin for hcv genotype. Most isolates of hepatitis c virus hcv infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined. Listing a study does not mean it has been evaluated by the u. In addition, we expressed recombinant gbvc ns5a protein to determine if it interferes with rnaactivated protein kinase pkr function in vitro. Hcv often causes a prolonged and persistent infection and plays an important role in the pathogenesis of virusassociated hepatocellular carcinoma hcc 35.
The protein is phosphorylated on multiple sites by host cell kinases and interacts with host cell membranes. Ns5a inhibition of pkr correlated with a block in irf1 activation and subsequently the dsrnainduced stimulation of irf1dependent gene expression. The ns5a and e2 proteins of hcv genotype 1 were reported to inhibit the doublestranded ds rnadependent protein kinase pkr, which is involved in the cellular antiviral response induced by interferon ifn. The hepatitis c virus hcv nonstructural 5a ns5a protein has generated wide interest in hcv research because of its ability to modulate the host cell interferon ifn response. 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.
Hepatitis c virus nonstructural 5a protein membrane anchor provide feedback. Relationship of hepatitis c genotype 1 ns5a sequence. Extending the analysis to the pkrbinding domain codons 22092274 revealed no additional differences between the hcv1a prototype sequence hcv1 25 and the consensus sequence. Naturally occurring resistance mutations to inhibitors of hcv. Activation of pkr in hcv infected cells is antagonized by interaction with ns5a, an hcv nonstructural protein. Protein kinaseinteracting domain in the hepatitis c virus. If y93h is present, weightbased ribavirin should be added or another recommended regimen should be used. Hepatitis c virus controls interferon production through. Daclatasvir, asunaprevir plus ribavirin for hcv genotype 1b without ns5a rav the safety and scientific validity of this study is the responsibility of the study sponsor and investigators. The ns5a protein has a substantial role in viral replication, packaging, assembly and complex interactions with cellular functions.
It is thought to interact with the ds rna dependent interferon inducible kinase pkr, p19525 1,2. Our studies demonstrate that the ns5a protein can inhibit pkr during hcv rna replication. Bms790052 is a specific hcv inhibitor since it has been shown to be inactive against a panel of rna and dna viruses. However, selection of daaresistant viral variants may hamper treatment. The current treatment of care, pegylated interferon. Hepatitis c virus hcv 3 is the main causative agent for transfusionassociated and sporadic nona, nonb hepatitis throughout the world. The ns5a protein is phosphorylated when expressed in mammalian cells.
Dashes indicate that amino acid is identical to prototype sequence. Ns5a inhibitors most likely act on the nterminus of ns5a. Directacting antiviral daa agents target hcv proteins. Repression of the pkr protein kinase by the hepatitis c virus ns5a. Nonstructural protein 5a ns5a inhibitors are a directacting antiviral agent used to treat hepatitis c virus hcv. Apr 02, 2002 our studies demonstrate that the ns5a protein can inhibit pkr during hcv rna replication. Although hepatitis c virus hcv infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of hcv pathogenesis remain elusive. Another hcv mechanism to overcome cellular antiviral strategies has been described for the ns5a protein of hcv. Mutations in the protein kinasebinding domain of the ns5a. We now show that hcv infection triggers phosphorylation and activation. Activation of pkr in hcvinfected cells is antagonized by interaction with ns5a, an hcv nonstructural protein. Hepatitis c viral rna genotype 1 ns5a drugresistance. Prevalence of wildtype in ns5apkr protein kinase binding.
The placebocontrolled, phase 3 polaris1 trial evaluated a 12week course of the daily fixeddose combination of sofosbuvir 400 mgvelpatasvir 100 mgvoxilaprevir 100mg in patients with a prior ns5a inhibitorcontaining daa regimen. The role of pkr in cell growth regulation is controversial, with some studies supporting a tumour suppressor function and others suggesting a growthpromoting role. Sequencing of e2 and ns5a regions of hcv genotype 3a in. Recently the structure of domain 1 has been determined, revealing a structural scaffold with a novel zinc. Evidence that hepatitis c virus resistance to interferon is. Regulation of pkr and irf1 during hepatitis c virus rna. Hepatitis c virus hcv is a major cause of liver disease throughout the world. Hepatitis c virus hcv is resistant to the antiviral cytokine type i interferon, representing a major clinical problem. Hepatitis c virus ns5a and subgenomic replicon activate nf. The ns5a nonstructural protein of hcv contains several prolinerich sequences consistent with src homology sh 3binding sites found in cellular signaling molecules. Nonstructural protein 5a protein ns5a of hepatitis c virus hcv plays an important role in the regulation of viral replication, interferon resistance, and apoptosis. Activation of the interferoninducible protein kinase pkr. A compound, bms790052 daclatasvir with the structure as 36, developed by bristolmyerssquibb bms, was found to produce dramatic reductions in viral load and few side effects in a phase i clinical trial gao et al.
Correlation between mutations in the interferon sensitivity. This compound was found to target ns5a protein of hcv. Inhibition of the interferon inducible protein kinase pkr. Garaigorta and chisari 2009 reveal that hcv uses the activation of the dsrnadependent protein kinase r, which phosphorylates and inhibits the translation initiation factor eif2. Hepatitis c virus rna polymerase and ns5a complex with a snarelike protein. Gbvc ns5a amplified from a person whose virus was cleared by ifn therapy ifn sensitive demonstrated unique amino acid changes occurring in the region that aligns with the hepatitis c virus hcv ifn.
Hcv ns5a cooperates with pkr in modulating hcv ires. Despite no inherent enzymatic activity being attributed to ns5a, its function is mediated through interaction with other nonstructural ns viral and cellular proteins. Naturally occurring resistance mutations to inhibitors of. Hepatitis c virus blocks interferon effector function by inducing. Despite improvements in the treatment of chronic hepatitis c virus hcv infection, only 7%12% of hcv genotype 1infected patients demonstrate sustained virologic response svr to interferon ifn. Here, we demonstrate that ns5a specifically bound to growth.
Hepatitis c viral rna genotype 1 ns5a drug resistance. Cypi, by disrupting ns5acypa complexes, block hcv replication both in vitro and in patients. Hepatitis c virus is a poor inducer of interferon ifn, although its structured viral rna can bind the rna helicase rigi, and activate the ifninduction pathway. Naturally occurring, resistanceassociated hepatitis c virus ns5a variants are linked to interleukin28b genotype and are sensitive to interferonbased therapy. The hepatitis c virus hcv envelope glycoprotein2 inhibits the interferon ifninduced. Hepatitis c virus hcv causes acute and chronic hepatitis which can eventually lead to permanent liver damage, hepatocellular carcinoma and death. Thus, inactivation of pkr may be one mechanism by which hcv avoids the antiviral effects of ifn. Experimental studies have demonstrated that the wildtype pkr ns5a strain of hepatitis c virus hcv may have oncogenic potential through the binding and functional repression of pkr protein kinase. Elbasvir mk8742 is a hepatitis c virus nonstructural protein 5a hcv ns5a inhibitor with ec 50 s of 4, 3 and 3 nm against genotype 1a, 1b, and 2a, respectively. Effects of resistanceassociated ns5a mutations in hepatitis. The hepatitis c virus hcv ns5a drug resistance for genotype 2 assay detects ns5 nonstructural protein 5a mutations and polymorphisms in hcv genotype 2 that are associated with resistance to directacting antivirals. When compared with each of the reference strainshcv1bj for hcv1b, hcg9 for hcv1c, and hcj6 for hcv2athe hcv isolates tested possessed 15 to 30 amino acid substitutions in ns5a excluding the pkrbinding region. An interaction of the hepatitis c virus hcv ns5a protein with the interferon ifn. Moreover, expression of ns5a alone was sufficient to block induction of an irf1dependent promoter by dsrna.
The specificity of bms790052 has been further demonstrated by isolation and mapping of primary resistance mutations to ns5a. Hepatitis c virus controls interferon production through pkr. Currently, there is no vaccine available for prevention of hcv infection due to high degree of strain variation. Here it is shown that the hcv envelope protein e2 contains a sequence identical with phosphorylation sites of the interferoninducible protein kinase pkr and the translation initiation factor eif2. Test code 92447, hepatitis c viral rna genotype 1 ns5a drugresistance test code 93871, hepatitis c viral rna genotype, lipa with reflex to hcv ns5a drugresistance if the hcv genotype is 1a, ns5a drugresistance test will be performed at additional charge. The researchers at bms have shown that small molecule ns5a inhibitors are potent and specific. Here, we have investigated the early events of ifn induction upon hcv infection, using the cellcultured hcv jfh1. The current hypothesis is that hcvassociated hcc may be resulted from immune. Finally, the inhibition of the pkr protein kinase by ns5a is. Since ns5a also binds to pkr, a central component of the ifn response, we investigated the possibility of a relationship between cypa, ns5a and pkr in the ifn response to hcv. Grazoprevir is an inhibitor of hcv ns34a protease, which is necessary for the proteolytic cleavage of the hcvencoded polyprotein into mature forms of the ns3, ns4a, ns4b, ns5a, and ns5b proteins and is essential for viral replication.
The current hypothesis is that hcv associated hcc may be resulted from immunemediated. Citeseerx domain 2 of nonstructural protein 5a ns5a of. Control of pkr protein kinase by hepatitis c virus. The assay is intended to be used for patients with hcv. Dec 17, 20 directacting antiviral daa agents target hcv proteins. Both cyclophilin inhibitors and directacting antivirals. The majority 61% experienced a failure with a combination regimen of an ns5b inhibitor plus an ns5a.
Two pkr inhibitor hcv proteins correlate with early but. In this study, we investigate the molecular mechanisms of nf. Hepatitis c virus blocks interferon effector function by. Hepatitis c virus ns5a protein downregulates the expression.
We and others demonstrated that the contact between ns5a and the host factor cypa is critical for hcv replication. Inhibition of the interferon inducible protein kinase pkr by. Efficient acute and chronic infection of stem cellderived. The ability of the hepatitis c virus to cause lifelong infection and liver damage together with limited therapeutic success in hcv elimination represent ongoing challenges to both clinicians and scientists. Hcv infection strongly induces antiviral interferonstimulated gene isg expression in the liver, yet it persists, suggesting that hcv can block isg effector function. Apr 01, 2002 amino acid sequence alignment of ns5ainterferon ifn sensitivitydetermining region isdr protein sequence aa 22092248 for all 22 patients numbered as in table 2, compared with ifnresistant prototype sequences of hepatitis c virus hcvj and hcvh. However, it is possible that the function of pkr varies with the type of cancer in question. Citeseerx document details isaac councill, lee giles, pradeep teregowda. Isoform specific products your search returned no results. Hcv infected cells treated with cypi, daas or ifn were analyzed for the expression and activation of various components of the innate response.
Variations in the interferon sensitivitydetermining region isdr within the ns5a region were related to the development of hepatocellular carcinoma hcc in patients infected with hepatitis c virus hcv. Protein kinase rna pkrregulated is a doublestranded rna activated protein kinase whose expression is induced by interferon. Therefore ns5a inhibitors treat viral infections by reducing the ability of the virus to replicate. We demonstrate herewith that hcv core proteins encoded by sequences isolated from. To investigate its to investigate its potential role in viral replication, we searched for the cellular proteins interacting with ns5a protein by yeast twohybrid. Twentyfour to thirtysix hours later, cotransfection with polyi.
An overview of hcv molecular biology, replication and immune. Effect of hcv ns5a on pkr mediated hcv ires activation cotransfection in hepg2 a and huh7 cells b of the plasmid carrying the cat ires hcv luc transcriptional unit and the hcv ns5a expression plasmid 0. Ns5a is derived from a large polyprotein that is translated from the hcv genome, and undergoes posttranslation processing by nonstructural protein 3 ns3 viral protease. Ns5a, a nonstructural protein of hepatitis c virus, binds. Mutations in different parts of ns5a 21882318 region in patients infected with hepatitis c virus hcv1a. Hepatitis c virus nonstructural protein 5a ns5a has been implicated in the hcv antiviral resistance, replication, and transactivation of cellular gene expression. The aim of the study was to investigate a relationship between isdr pkr substitutions and their association with liver fibrosis or hcc development. Low ifn induction has been attributed to hcv ns34a proteasemediated cleavage of the mitochondriaadapter mavs. Evidence that hepatitis c virus resistance to interferon. Hepatitis c virus hcv is a major etiological agent of chronic liver disease and hepatocellular carcinoma hcc. To assess whether the ns5a pkr binding domain may be involved in hcv related liver carcinogenesis, its sequence was analyzed in the sera of 85 patients with hepatocellular carcinoma.
These results suggest that the ability of hcv to activate pkr may, paradoxically. Hepatitis c virus hcv is a singlestranded rna virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site ires. Svr has been enhanced by the addition of ribavirin, although only 25% of genotype 1infected patients develop svr 3, 4. Jul 16, 2010 activation of pkr in hcvinfected cells is antagonized by interaction with ns5a, an hcv nonstructural protein. Dec 17, 2009 hepatitis c virus hcv is resistant to the antiviral cytokine type i interferon, representing a major clinical problem. The researchers at bms have shown that small molecule ns5a inhibitors are potent and specific inhibitors of hcv replication. Ns5a inhibits pkr activity, which is postulated to allow hcv replication to continue in the presence of an. The aim of this study was to illustrate potential natural daaresistance mutations in the hcv ns5a and ns5b regions of hcv genotypes 1a and 1b from daa. The molecular function of the nonstructural 5a protein is uncertain.
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