Differential Sensitivity of 5′UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1−6 to Protease and NS5A Inhibitors

Li, Yiping; Ramírez, Santseharay; Humes, Daryl; Jensen, Sanne B.; Gottwein, Judith M.; Bukh, Jens

doi:10.1053/j.gastro.2013.11.009

Cited by 56 publications

(96 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…in the adaptation of JFH1-based 5-5A recombinants of genotypes 1 to 6 (46). In this study, we further demonstrated that LSG combined with NS5B mutation Y2981F (49) (interestingly, most genotype 2b isolates naturally have F2981 [11]) has the potential to initiate adaptation of additional genotypes, such as the genotype 1a strain TN.…”

Section: Discussionmentioning

confidence: 60%

“…We previously identified the LSG mutations (F1464L, A1672S, and D2979G), which permitted development of full-length HCV infectious culture systems for genotypes 1a (TNcc) (9), 2a (J6cc) (10), and 2b (J8cc, DH8cc, and DH10cc) (10,11), as well as 5-5A recombinants with JFH1 NS5B-3=UTR for genotypes 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) (46). In this study, we initially attempted to use the LSG mutations and a similar approach previously applied to J6cc and TNcc cultures to generate an HCV-1 infectious culture system.…”

Section: Resultsmentioning

confidence: 99%

“…After our success in adapting genotype 1a strains TN and HCV-1 for efficient growth in vitro, we wanted to determine whether the key adaptive mutations that we had uncovered could be used to adapt H77C, another important HCV prototype strain. We recently demonstrated that the LSGF and LSGF/TNm mutations were not sufficient to adapt the H77C genome after transfection in Huh7.5 cells (46) into HCV entry-deficient S29 cells (43). All clones contained LSGF/S399F/TNm plus specific mutations as indicated under each bar graph.…”

Section: Resultsmentioning

confidence: 99%

“…Q1773H" in reference 46). After passage, the virus acquired a partial change in the NS3 helicase, S1368P (46), which became dominant after the second passage. Interestingly, this mutation was also identified here in the two cell culture-adapted HCV-1 5-5A viruses ( Table 2).…”

Section: Resultsmentioning

confidence: 99%

“…Likewise, amino acid 2919 in NS5B is a variable position, and A, V, and T can be found in various genotypes. Among the remaining cell culture-adaptive mutations present in (46) and HCV-1 (Table 2) 5-5A recombinant viruses; the first-and second-passage H77C_LSGF/TNmr/ S1368P viruses both acquired noncoding nucleotide changes A2558G, A3089G, G3860A, C4403T, T4904C, A6437G, A6713G, A7727G, A8804G, and T9227C. b Nucleotides at positions 9321 and 9322 are in the same codon for the F2994R change.…”

Section: Discussionmentioning

confidence: 99%

See 4 more Smart Citations

Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

Ramírez

Mikkelsen

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5=UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3=UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/ D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10 4.0 focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10 3.5 and 10 4.4 FFU/ml, respectively. IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes. Hepatitis C virus (HCV) has chronically infected over 130 million people worldwide and is a leading cause of liver fibrosis, cirrhosis, a...

show abstract

Section: Discussionmentioning

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

Ramírez

Mikkelsen

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a

et al. 2014

Self Cite

View full text Add to dashboard Cite

Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization investigations across genotypes, subtypes, and isolates. We investigated the breadth of neutralization of 10 HMAbs with therapeutic potential against a panel of 16 JFH1-based HCVcc expressing patient-derived Core-NS2 from genotypes 1a (strains H77, TN, and DH6), 1b (J4, DH1, and DH5), 2a (J6, JFH1, and T9), 2b (J8, DH8, and DH10), 2c (S83), and 3a (S52, DBN, and DH11). Virus stocks used for in vitro neutralization analysis contained authentic E1/E2, with the exception of full-length JFH1 that acquired the N417S substitution in E2. The 50% inhibition concentration (IC50) for each HMAb against the HCVcc panel was determined by dose-response neutralization assays in Huh7.5 cells with antibody concentrations ranging from 0.0012 to 100 μg/ml. Interestingly, IC50-values against the different HCVcc’s exhibited large variations among the HMAbs, and only three HMAbs (HC-1AM, HC84.24, and AR4A) neutralized all 16 HCVcc recombinants. Furthermore, the IC50-values for a given HMAb varied greatly with the HCVcc strain, which supports the use of a diverse virus panel. In cooperation analyses, HMAbs HC84.24, AR3A, and, especially HC84.26, demonstrated synergistic effects towards the majority of the HCVcc’s when combined individually with AR4A. Conclusion: Through a neutralization analysis of 10 clinically relevant HMAbs against 16 JFH1-based Core-NS2 recombinants from genotypes 1a, 1b, 2a, 2b, 2c, and 3a, we identified at least 3 HMAbs with potent and broad neutralization potential. The neutralization synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV.

show abstract

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

Jensen¹,

Fahnøe²,

Pham³

et al. 2019

Hepatology

Self Cite

View full text Add to dashboard Cite

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.

show abstract

Differential Sensitivity of 5′UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1−6 to Protease and NS5A Inhibitors

Cited by 56 publications

References 38 publications

Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a

Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

Contact Info

Product

Resources

About