Synthesis of New Indanyl Nucleoside Analogues and their Biological Evaluation on Hepatitis C Virus (HCV) Replicon

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.

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Comparison of Daclatasvir Resistance Barriers on NS5A from Hepatitis C Virus Genotypes 1 to 6: Implications for Cross-Genotype Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02788-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5155-5163 ◽

Cited By ~ 50

Author(s):

Chunfu Wang ◽

Lingling Jia ◽

Donald R. O'Boyle ◽

Jin-Hua Sun ◽

Karen Rigat ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Rank Order ◽

Replicon Cell ◽

Hcv Genotypes ◽

Resistance Selection ◽

Virus Genotypes ◽

Hepatitis C Virus Genotypes

ABSTRACTA comparison of the daclatasvir (DCV [BMS-790052]) resistance barrier on authentic or hybrid replicons containing NS5A from hepatitis C virus (HCV) genotypes 1 to 6 (GT-1 to -6) was completed using a replicon elimination assay. The data indicated that genotype 1b (GT-1b) has the highest relative resistance barrier and genotype 2a (GT-2a M31) has the lowest. The rank order of resistance barriers to DCV was 1b > 4a ≥ 5a > 6a ≅ 1a > 2a JFH > 3a > 2a M31. Importantly, DCV in combination with a protease inhibitor (PI) eliminated GT-2a M31 replicon RNA at a clinically relevant concentration. Previously, we reported the antiviral activity and resistance profiles of DCV on HCV genotypes 1 to 4 evaluated in the replicon system. Here, we report the antiviral activity and resistance profiles of DCV against hybrid replicons with NS5A sequences derived from HCV GT-5a and GT-6a clinical isolates. DCV was effective against both GT-5a and -6a hybrid replicon cell lines (50% effective concentrations [EC50s] ranging from 3 to 7 pM for GT-5a, and 74 pM for GT-6a). Resistance selection identified amino acid substitutions in the N-terminal domain of NS5A. For GT-5a, L31F and L31V, alone or in combination with K56R, were the major resistance variants (EC50s ranging from 2 to 40 nM). In GT-6a, Q24H, L31M, P32L/S, and T58A/S were identified as resistance variants (EC50s ranging from 2 to 250 nM). Thein vitrodata suggest that DCV has the potential to be an effective agent for HCV genotypes 1 to 6 when used in combination therapy.

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Hepatitis C Virus-Like Particle Morphogenesis

Journal of Virology ◽

10.1128/jvi.76.8.4073-4079.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4073-4079 ◽

Cited By ~ 77

Author(s):

Emmanuelle Blanchard ◽

Denys Brand ◽

Sylvie Trassard ◽

Alain Goudeau ◽

Philippe Roingeard

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Semliki Forest Virus ◽

Virus Like Particle ◽

Genes Encoding ◽

Host Cell Interactions ◽

Viral Morphogenesis ◽

First Time

ABSTRACT Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

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Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽

10.1006/viro.2002.1461 ◽

2002 ◽

Vol 297 (2) ◽

pp. 298-306 ◽

Cited By ~ 35

Author(s):

I.Wayne Cheney ◽

Suhaila Naim ◽

Vicky C.H. Lai ◽

Shannon Dempsey ◽

Daniel Bellows ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Enzymatic Activity ◽

Rna Replication ◽

Viral Rna ◽

Subgenomic Replicon ◽

Replicon Cell ◽

Ns5b Polymerase

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In Vitro Resistance Study of AG-021541, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00834-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 675-683 ◽

Cited By ~ 34

Author(s):

Stephanie T. Shi ◽

Koleen J. Herlihy ◽

Joanne P. Graham ◽

Shella A. Fuhrman ◽

Chau Doan ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

High Throughput Screening ◽

Subgenomic Replicon ◽

Replicon Cell ◽

Resistance Selection ◽

Polymerase Inhibitors ◽

Virus Rna ◽

Allosteric Sites

ABSTRACT A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 Å away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 μM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

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Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

Journal of Virology ◽

10.1128/jvi.02674-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6922-6928 ◽

Cited By ~ 27

Author(s):

Kazuo Sugiyama ◽

Kenji Suzuki ◽

Takahide Nakazawa ◽

Kenji Funami ◽

Takayuki Hishiki ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Genetic Analysis ◽

Phylogenetic Analyses ◽

Structural Proteins ◽

Infectious Particle ◽

First Time ◽

Self Replication

ABSTRACT Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5′ untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.

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