Hepatitis C virus NS5A protein inhibits interferon antiviral activity, but the effects do not correlate with clinical response

Paterson, Morris; Laxton, Carl; Thomas, Howard; Ackrill, Andrew M.; Foster, Graham R.

doi:10.1016/s0016-5085(99)70405-1

Cited by 83 publications

(62 citation statements)

References 30 publications

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confidence: 76%

“…Both the HCV envelope (E) 2 protein and the nonstructural (NS)-5A protein of HCV-1a/b isolates were shown to inhibit IFN-␣-induced double-stranded RNA-activated protein kinase (PKR) in vitro (14, 51). Moreover, cellular expression of the NS5A protein from HCV-1a/b isolates confirmed inhibitory effects on IFN-␣ activity (13,33,34,36).In 1995 and 1996 Enomoto et al demonstrated clinically that an increasing number of mutations within a restricted region of the HCV NS5A protein named the IFN sensitivity determining region (ISDR; amino acids [aa] 2209 to 2248 according to ) was correlated with treatment response in isolates of HCV-1b-infected patients (10, 11). The importance of mutations within the NS5A ISDR of HCV-1b-infected patients was confirmed by other investigations from Japan (7,20).…”

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confidence: 98%

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Hepatitis C Virus Nonstructural 5A Protein and Interferon Resistance: a New Model for Testing the Reliability of Mutational Analyses

Sarrazin

Herrmann

Bruch

et al. 2002

J Virol

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Presumably due to the capability of the hepatitis C virus (HCV) to evade the antiviral effects of alpha interferon, treatment is ineffective in more than half of chronically genotype HCV type 1 (HCV-1)-infected patients. Previous approaches to correlate the number of amino acid mutations within regions of HCV nonstructural (NS)-5A protein with virologic treatment response provided conflicting results. In the present study, we developed a new mathematical model to investigate NS5A sequences of HCV-1-infected patients. The mean number of all mutations within the complete NS5A protein was significantly higher in virologic responders compared to nonresponders (P ‫؍‬ 0.008 and P ‫؍‬ 0.0001 for amino acid residues predicted on the surface of the NS5A protein). Differences did not achieve statistical significance for NS5A regions that are currently assumed to be functionally relevant (e.g., the interferon sensitivity-determining region, the RNA-activated protein kinase-binding domain, etc.). Analyses of smoothed mutational frequencies showed that the number of mutations in other NS5A regions correlated with virologic response. Such a correlation was observed for both genuine and randomly generated NS5A sequences. The existence of local accumulations of mutations within genuine NS5A isolates that truly correlated with treatment response was defined by a refined test procedure. Upon considering the predicted residue accessibility, we identified the main focus of mutations correlating with treatment response to be the sequence from amino acids 2350 to 2370. Thus, evaluation of NS5A mutations in correlation with treatment response is improved by consideration of functional and predicted conformational amino acid properties. As shown by simulations with randomly generated sequences, multiple analyses of simple counts of local NS5A amino acid mutations and correlation with treatment response are insufficient. For improvement of mutational analysis, a refined specific functional data test procedure is proposed.Chronic hepatitis C virus (HCV) infection frequently progresses to liver cirrhosis and is associated with an elevated risk for development of hepatocellular carcinoma (1,30,42). The only effective treatment is based on alpha interferon (IFN-␣). IFN-␣ alone or in combination with ribavirin leads to sustained virologic response in 20 to 56% of patients with chronic hepatitis C (24,26,37,53). The strongest predictive parameter for a sustained virologic response to treatment is the HCV genotype, with HCV genotype 1 (HCV-1) being the least sensitive to IFN-␣-based therapy (24, 37).For HCV as for other eukaryotic viruses, including the influenza, vaccinia, and human immunodeficiency viruses (6,22,27), the ability to evade the antiviral effects of IFN-␣ has been reported. Both the HCV envelope (E) 2 protein and the nonstructural (NS)-5A protein of HCV-1a/b isolates were shown to inhibit IFN-␣-induced double-stranded RNA-activated protein kinase (PKR) in vitro (14, 51). Moreover, cellular expression of the NS5A protein from HCV...

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confidence: 76%

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confidence: 98%

Hepatitis C Virus Nonstructural 5A Protein and Interferon Resistance: a New Model for Testing the Reliability of Mutational Analyses

Sarrazin

Herrmann

Bruch

et al. 2002

J Virol

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“…A few Japanese studies have confirmed the fact that HCV virus with multiple amino-acid substitutions in this region is sensitive to IFN, [16][17][18][19] but studies from Europe and the United States have reported that such an association was not observed in their patients. [20][21][22][23][24][25][26][27][28] One recent study from Europe did however show the partial predictive value of amino-acid analysis in the ISDR. 29,30 More recently, Taylor et al 31 reported that HCV E2 protein contains a 12 amino-acid-sequence domain that is highly homologous to the autophosphorylation site of PKR and initiation factor eIF2 ␣, a target of PKR (the PKR-eIF2 ␣ phosphorylation homology domain [PePHD]).…”

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confidence: 97%

Association of Amino Acid Sequence in the Pkr–Eif2 Phosphorylation Homology Domain and Response to Interferon Therapy

et al. 2000

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Hepatitis C virus (HCV) usually causes chronic infection, which can result in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. [1][2][3][4] The only drug that effectively reduces the virus load is interferon (IFN), but complete eradication of the virus occurs in only some treated patients. [5][6][7][8] Currently, genotype 1b and a high virus titer are well-established predictive factors of a poor response to IFN. 9-11 Enomoto et al., 12,13 recently reported that amino-acid-sequence substitutions in a 40 amino acid stretch (codons 2209-2248) in the NS5A region of HCV genotype 1b are tightly associated with the outcome of IFN therapy, and designated this region as the IFN-sensitivity-determining-region (ISDR). A recent in vitro study by Gale et al., 14,15 suggested that the ISDR (or the amino-acid stretch adjacent to it) binds to IFN-inducible protein kinase (PKR), and inhibits its action as a blocker of viral protein synthesis. However, whether this region is really predictive of the outcome of IFN therapy is still controversial. A few Japanese studies have confirmed the fact that HCV virus with multiple amino-acid substitutions in this region is sensitive to IFN, [16][17][18][19] but studies from Europe and the United States have reported that such an association was not observed in their patients. [20][21][22][23][24][25][26][27][28] One recent study from Europe did however show the partial predictive value of amino-acid analysis in the ISDR. 29,30 More recently, Taylor et al. 31 reported that HCV E2 protein contains a 12 amino-acid-sequence domain that is highly homologous to the autophosphorylation site of PKR and initiation factor eIF2 ␣, a target of PKR (the PKR-eIF2 ␣ phosphorylation homology domain [PePHD]). They showed that the E2 protein inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth, suggesting that the interaction of E2 and PKR may be one of the mechanisms by which HCV circumvents the antiviral effect of IFN. They also reported that E2 proteins with a PePHD sequence identical to genotypes 2 and 3 HCV, which are known IFN-sensitive genotypes, showed only a weak inhibitory effect against PKR activity.The above results prompted us to investigate whether aminoacid substitutions in the PePHD domain correlate with outcome of IFN therapy in patients with genotype 1b HCV infection. Furthermore, we sequenced multiple clones of PePHD in patients before and during IFN to investigate the behavior of HCV quasispecies in this region and to determine whether an IFN-resistant strain was selected during IFN treatment. We also studied amino-acid sequences of the ISDR and virus load in the same population using 3 different quantitative measurements of HCV, namely, branched (b)DNA assay and real time Abbreviations: ALT, alanine transaminase; HCV, hepatitis C virus; ISDR, interferon sensitivity determining region; PCR, polymerase chain reaction, PePHD, PKR-eIF2 ␣ phosphorylation homology domain; RT, reverse transcription; PKR, IFN-inducible prot...

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“…It is noteworthy that the E2 envelope protein also interacts with and inhibits PKR (54), indicating that HCV may employ multiple strategies to perturb a major host cell antiviral function. NS5A can also confer IFN resistance on encephalomyocarditis virus and vesicular stomatitis virus, viruses normally sensitive to the antiviral actions of IFN (1,19,38,40,49), and it reverses the IFN-sensitive phenotype of a vaccinia virus (VV) lacking the E3L gene (25). NS5A provides resistance to apoptosis induced by PKR agonists, such as dsRNA, and can cause cell transformation and solid-tumor growth in vivo through both PKR-dependent and -independent mechanisms (19).…”

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confidence: 99%

Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase

Nakao²,

Tan³

et al. 2002

J Virol

162

128

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Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.Hepatitis C virus (HCV), a Flaviviridae family member, contains a positive-sense, single-stranded RNA genome that encodes about 10 mature viral structural and nonstructural (NS) proteins (41). Infecting approximately 2% of the world population, HCV is the global leading cause of chronic liver disease and has become a major public health problem in the United States (10, 11). In the majority of cases, acute infection with HCV results in persistent viral replication and establishment of a chronic infection. Chronic hepatitis C frequently leads to progressive liver disease, including liver fibrosis and cirrhosis, and is strongly associated with the onset of hepatocellular carcinoma. HCV research has been hampered by the lack of an efficient tissue culture system or an adequate animal model of HCV infection (18). As a result, the mechanisms of HCV replication, persistence, and pathogenesis remain poorly understood. Consequently, our general understanding of the impact of HCV infection on cellular signaling is far from complete or clear.HCV-host interactions have been intensely investigated despite the lack...

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Hepatitis C virus NS5A protein inhibits interferon antiviral activity, but the effects do not correlate with clinical response

Cited by 83 publications

References 30 publications

Hepatitis C Virus Nonstructural 5A Protein and Interferon Resistance: a New Model for Testing the Reliability of Mutational Analyses

Hepatitis C Virus Nonstructural 5A Protein and Interferon Resistance: a New Model for Testing the Reliability of Mutational Analyses

Association of Amino Acid Sequence in the Pkr–Eif2 Phosphorylation Homology Domain and Response to Interferon Therapy

Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase

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