Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

ABSTRACT 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-ΔN110), 222 (NS5A-ΔN222), 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-ΔN110 and NS5A-ΔN222 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-κB and AP-1 were important in NS5A-ΔN222 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.

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Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

Journal of Virology ◽

10.1128/jvi.74.19.9028-9038.2000 ◽

2000 ◽

Vol 74 (19) ◽

pp. 9028-9038 ◽

Cited By ~ 86

Author(s):

J.-B. Nousbaum ◽

S. J. Polyak ◽

S. C. Ray ◽

D. G. Sullivan ◽

A. M. Larson ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Antiviral Therapy ◽

Variable Region ◽

Response To Therapy ◽

Full Length ◽

Variable Regions ◽

C Terminus

ABSTRACT 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 (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 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.

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The N-terminal amino acids are essential for the antigenic structure of the hepatitis C virus core protein

Hepatology ◽

10.1016/0270-9139(93)91843-h ◽

1993 ◽

Vol 18 (4) ◽

pp. A79

Author(s):

T GOESER

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Antigenic Structure ◽

Virus Core ◽

Terminal Amino

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A 2a/1b full-length p7 inter-genotypic chimeric genome of hepatitis C virus is infectious in vitro

Virology ◽

10.1016/j.virol.2006.10.014 ◽

2007 ◽

Vol 360 (1) ◽

pp. 17-26 ◽

Cited By ~ 15

Author(s):

G. Haqshenas ◽

X. Dong ◽

G. Ewart ◽

S. Bowden ◽

E.J. Gowans

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Full Length

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Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6663 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6663-6669 ◽

Cited By ~ 50

Author(s):

L Trieschmann ◽

Y V Postnikov ◽

A Rickers ◽

M Bustin

Keyword(s):

Amino Acids ◽

Structural Features ◽

Binding Domain ◽

Full Length ◽

Modular Structure ◽

Chromosomal Proteins ◽

Nucleosome Core ◽

Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

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Hepatitis C Virus NS5A Protein In Vitro Modulates Template Selection by the RNA-dependent RNA Polymerase

Antiviral Research ◽

10.1016/j.antiviral.2009.02.048 ◽

2009 ◽

Vol 82 (2) ◽

pp. A28-A29

Author(s):

Olga Ivanova ◽

Vera Tunitskaya ◽

Alexander Ivanov ◽

Vladimir Mitkevich ◽

Vladimir Prassolov ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Dependent Rna Polymerase ◽

Ns5a Protein ◽

Template Selection

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Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

Journal of Virology ◽

10.1128/jvi.80.7.3332-3340.2006 ◽

2006 ◽

Vol 80 (7) ◽

pp. 3332-3340 ◽

Cited By ~ 28

Author(s):

Tetsuro Shimakami ◽

Masao Honda ◽

Takashi Kusakawa ◽

Takayuki Murata ◽

Kunitada Shimotohno ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Polymerase Activity ◽

Subgenomic Replicon ◽

Huh7 Cells ◽

Hcv Ns5b

ABSTRACT We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

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Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase.

Journal of Virology ◽

10.1128/jvi.71.10.7187-7197.1997 ◽

1997 ◽

Vol 71 (10) ◽

pp. 7187-7197 ◽

Cited By ~ 121

Author(s):

K E Reed ◽

J Xu ◽

C M Rice

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Ns5a Protein

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Interfering with hepatitis C virus assembly in vitro using affinity peptides directed towards core protein

Canadian Journal of Microbiology ◽

10.1139/w2012-009 ◽

2012 ◽

Vol 58 (4) ◽

pp. 475-482

Author(s):

Jean-Baptiste Duvignaud ◽

Nathalie Majeau ◽

Priscilla Delisle ◽

Normand Voyer ◽

Stéphane M. Gagné ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Self Assembly ◽

Core Protein ◽

Virus Assembly ◽

Viral Assembly ◽

Structural Protein ◽

Ns5a Protein ◽

The Core

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.

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Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

Journal of Virology ◽

10.1128/jvi.00684-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1385-1399 ◽

Cited By ~ 20

Author(s):

Thomas H. R. Carlsen ◽

Troels K. H. Scheel ◽

Santseharay Ramirez ◽

Steven K. H. Foung ◽

Jens Bukh

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Core Protein ◽

Particle Density ◽

Virus Production ◽

Envelope Proteins ◽

Late Step

ABSTRACTThe hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexesin vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

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