scholarly journals Identification of Alpha Interferon-Induced Envelope Mutations of Hepatitis C VirusIn VitroAssociated with Increased Viral Fitness and Interferon Resistance

2013 ◽  
Vol 87 (23) ◽  
pp. 12776-12793 ◽  
Author(s):  
Stéphanie B. N. Serre ◽  
Henrik B. Krarup ◽  
Jens Bukh ◽  
Judith M. Gottwein
Keyword(s):  
Hepatitis C ◽  
Viral Entry ◽  
Experimental Studies ◽  
Viral Fitness ◽  
Alpha Interferon ◽  
Close Link ◽  
Treatment Regimens ◽  
Viral Spread ◽  
Infection Resistance

Alpha interferon (IFN-α) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-α resistancein vitro. Long-term culture with IFN-α2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-α resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-α therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-α resistance. In conclusion,in vitroescape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-α resistance. While we established a close link between viral fitness and IFN-α resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance.

scholarly journals Factors Influencing the Prevalence of Resistance-Associated Substitutions in NS5A Protein in Treatment-Naive Patients with Chronic Hepatitis C

Biomedicines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 80
Author(s):  
Karen K. Kyuregyan ◽  
Vera S. Kichatova ◽  
Anastasiya A. Karlsen ◽  
Olga V. Isaeva ◽  
Sergei A. Solonin ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Immune Escape ◽  
Viral Fitness ◽  
Treatment Regimens ◽  
Immune Pressure ◽  
Treatment Naïve ◽  
Infection Resistance ◽  
Total Proportion ◽  
N 93 ◽  
Direct Acting

Direct-acting antivirals (DAAs) revolutionized treatment of hepatitis C virus (HCV) infection. Resistance-associated substitutions (RASs) present at the baseline impair response to DAA due to rapid selection of resistant HCV strains. NS5A is indispensable target of the current DAA treatment regimens. We evaluated prevalence of RASs in NS5A in DAA-naïve patients infected with HCV 1a (n = 19), 1b (n = 93), and 3a (n = 90) before systematic DAA application in the territory of the Russian Federation. Total proportion of strains carrying at least one RAS constituted 35.1% (71/202). In HCV 1a we detected only M28V (57.9%) attributed to a founder effect. Common RASs in HCV 1b were R30Q (7.5%), L31M (5.4%), P58S (4.4%), and Y93H (5.4%); in HCV 3a, A30S (31.0%), A30K (5.7%), S62L (8.9%), and Y93H (2.2%). Prevalence of RASs in NS5A of HCV 1b and 3a was similar to that worldwide, including countries practicing massive DAA application, i.e., it was not related to treatment. NS5A with and without RASs exhibited different co-variance networks, which could be attributed to the necessity to preserve viral fitness. Majority of RASs were localized in polymorphic regions subjected to immune pressure, with selected substitutions allowing immune escape. Altogether, this explains high prevalence of RAS in NS5A and low barrier for their appearance in DAA-inexperienced population.

scholarly journals Alpha Interferon Inhibits Hepatitis C Virus Replication in Primary Human Hepatocytes Infected In Vitro

2002 ◽  
Vol 76 (16) ◽  
pp. 8189-8199 ◽  
Author(s):  
Valérie Castet ◽  
Chantal Fournier ◽  
Alexandre Soulier ◽  
Rozenn Brillet ◽  
Joliette Coste ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Primary Cultures ◽  
Indirect Evidence ◽  
Human Hepatocytes ◽  
Alpha Interferon ◽  
Healthy Human

ABSTRACT Chronic hepatitis C is a common cause of liver disease, the complications of which include cirrhosis and hepatocellular carcinoma. Treatment of chronic hepatitis C is based on the use of alpha interferon (IFN-α). Recently, indirect evidence based on mathematical modeling of hepatitis C virus (HCV) dynamics during human IFN-α therapy suggested that the major initial effect of IFN-α is to block HCV virion production or release. Here, we used primary cultures of healthy, uninfected human hepatocytes to show that: (i) healthy human hepatocytes can be infected in vitro and support HCV genome replication, (ii) hepatocyte treatment with IFN-α results in expression of IFN-α-induced genes, and (iii) IFN-α inhibits HCV replication in infected human hepatocytes. These results show that IFN-α acts primarily through its nonspecific antiviral effects and suggest that primary cultures of human hepatocytes may provide a good model to study intrinsic HCV resistance to IFN-α.

Detection of hepatitis C virus RNA in alpha interferon derived from in vitro culture of leukocytes of human origin

Biologicals ◽  
2001 ◽  
Vol 29 (1) ◽  
pp. 45-53 ◽  
Author(s):  
D. Forčić ◽  
R. Zgorelec ◽  
M. Šantak ◽  
T. Košutić ◽  
R. Mažuran
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
In Vitro Culture ◽  
Alpha Interferon ◽  
Human Origin ◽  
Hepatitis C Virus Rna ◽  
Virus Rna

scholarly journals Modulation of alpha interferon anti-hepatitis C virus activity by ISG15

2009 ◽  
Vol 90 (12) ◽  
pp. 2929-2939 ◽  
Author(s):  
Pong Kian Chua ◽  
Matthew F. McCown ◽  
Sonal Rajyaguru ◽  
Simran Kular ◽  
Ram Varma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Effect ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Moderate Increase ◽  
Knock Down ◽  
Inducible Genes

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-α) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-α, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-α-specific effect, which was not observed with IFN-γ, correlated with an increase in expression of the IFN-α-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-α-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-α after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-α-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-α treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-α pathway and its antiviral effect.

scholarly journals Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus

2015 ◽  
Vol 89 (19) ◽  
pp. 9962-9973 ◽  
Author(s):  
Virgínia Gondar ◽  
Francisca Molina-Jiménez ◽  
Takayuki Hishiki ◽  
Luisa García-Buey ◽  
George Koutsoudakis ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Low Density Lipoprotein ◽  
Free Particle ◽  
Density Lipoprotein ◽  
Host Cells ◽  
Viral Spread ◽  
Cell Transmission ◽  
Hcv Transmission

ABSTRACTHepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmissionin vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis.IMPORTANCEThis work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention.

scholarly journals Regulated and Liver-Specific Tamarin Alpha Interferon Gene Delivery by a Helper-Dependent Adenoviral Vector

2005 ◽  
Vol 79 (11) ◽  
pp. 6772-6780 ◽  
Author(s):  
Luigi Aurisicchio ◽  
Amedeo De Tomassi ◽  
Nicola La Monica ◽  
Gennaro Ciliberto ◽  
Cinzia Traboni ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Transcriptional Control ◽  
Hepatoma Cell ◽  
Adenovirus Vector ◽  
Alpha Interferon ◽  
Hepatoma Cell Line ◽  
Virus Challenge ◽  
Regulated Gene Expression

ABSTRACT Gene therapy approaches based on liver-restricted and regulated alpha interferon (IFN-α) expression, recently shown to be effective in different murine hepatitis models, appear promising alternatives to inhibit hepatitis C virus (HCV) replication in patients and minimize side effects. Tamarins (Saguinus species) infected by GB virus B (GBV-B) are considered a valid surrogate model for hepatitis C to study the biology of HCV infection and the development of new antiviral drugs. To test the efficacy of local delivery and expression of IFN-α in this model, we have developed HD-TET-tIFN, a helper-dependent adenovirus vector expressing tamarin IFN-α (tIFN) under the control of the tetracycline-inducible transactivator rtTA2s-S2. Expression of tIFN was successfully induced both in vitro and in vivo in rodents by doxycycline administration with consequent activation of IFN-responsive genes. More importantly, tIFN efficiently inhibited GBV-B replicon in a Huh-7 hepatoma cell line at low HD-TET-tIFN doses. A certain degree of transcriptional control of tIFN was achieved in tamarins injected with HD-TET-tIFN, but under the conditions used in this study, infection and replication of GBV-B were only delayed and not totally abrogated upon virus challenge. Hepatic delivery and regulated expression of IFN-α appear to be a possible approach for the cure of hepatitis, but this approach requires more studies to increase its efficacy. To our knowledge, this is the first report showing a regulated gene expression in a nonhuman primate hepatitis model.

scholarly journals Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells

2004 ◽  
Vol 48 (12) ◽  
pp. 4784-4792 ◽  
Author(s):  
Kai Lin ◽  
Ann D. Kwong ◽  
Chao Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adverse Effects ◽  
Protease Inhibitor ◽  
Standard Of Care ◽  
Rna Replication ◽  
Viral Rna ◽  
Alpha Interferon ◽  
Hcv Rna

ABSTRACT The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

scholarly journals Screening and Rational Design of Hepatitis C Virus Entry Inhibitory Peptides Derived from GB Virus A NS5A

2012 ◽  
Vol 87 (3) ◽  
pp. 1649-1657 ◽  
Author(s):  
Xiuying Liu ◽  
Yibing Huang ◽  
Min Cheng ◽  
Ling Pan ◽  
Youhui Si ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Entry ◽  
Rational Design ◽  
Antiviral Drug ◽  
Inhibitory Effect ◽  
Content Type ◽  
Peptide Helicity

ABSTRACTChronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selectedFlaviviridaeviruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCVin vitro, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.

scholarly journals VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

2006 ◽  
Vol 50 (5) ◽  
pp. 1813-1822 ◽  
Author(s):  
Kai Lin ◽  
Robert B. Perni ◽  
Ann D. Kwong ◽  
Chao Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Agents ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Resistance Mutations ◽  
In Vitro Characterization ◽  
Antiviral Activities

ABSTRACT The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log10 was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log10 reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

scholarly journals NP-1, a Rabbit α-Defensin, Prevents the Entry and Intercellular Spread of Herpes Simplex Virus Type 2

2003 ◽  
Vol 47 (2) ◽  
pp. 494-500 ◽  
Author(s):  
Sara Sinha ◽  
Natalia Cheshenko ◽  
Robert I. Lehrer ◽  
Betsy C. Herold
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Viral Entry ◽  
Herpes Simplex Virus Type ◽  
Viral Envelope ◽  
Viral Spread ◽  
Simplex Virus

ABSTRACT Rabbit neutrophil peptide-1 (NP-1), a prototypic α-defensin, protects cells in vitro from infection by clinical and laboratory isolates of herpes simplex virus type 2 (HSV-2). Incubation of concentrated virus stocks for 1 h with noncytotoxic concentrations of NP-1 reduces subsequent infection by >98%. Pretreating cells with NP-1 for 1 h prior to inoculation with untreated virus also prevents infection. NP-1, a cationic peptide, does not compete with viral envelope glycoproteins for binding to cellular heparan sulfate receptors, but it prevents viral entry. No VP16, a major viral tegument protein, is transported to the cell nucleus in the presence of NP-1. Infectious center assays demonstrate that NP-1 also inhibits cell-to-cell viral spread. Thus, NP-1 prevents virally mediated fusion events, entry, and cell-to-cell spread. This unique mechanism of anti-HSV activity, coupled with established antibacterial and possible anti-human immunodeficiency virus type 1 activities of defensins, render this family of compounds excellent candidates for further development as topical microbicides.

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