Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant

Mathiesen, Christian K.; Prentoe, Jannick; Meredith, Luke W.; Jensen, Tanja B.; Krarup, Henrik; McKeating, Jane A.; Gottwein, Judith M.; Bukh, Jens

doi:10.1128/jvi.00039-15

Cited by 27 publications

(54 citation statements)

References 83 publications

(186 reference statements)

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“…In general, our data do not support the concept of virion assembly-independent HCV transmission. In agreement with our findings, adaptive assembly-enhancing mutations conferred significantly increased cell-to-cell transmission (41).…”

Section: Discussionsupporting

confidence: 82%

Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System

Zhao

Deng

et al. 2017

J Virol

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Hepatitis C virus (HCV) infects 2 to 3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology and antiviral drug development to drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, the HCV-dependent fluorescence relocalization (HDFR) system allowed live-cell visualization of infection without modifying the viral genome, but this strategy required careful recognition of the fluorescence relocalization pattern for its high fluorescence background in the cytoplasm. In this study, to achieve background-free visualization of HCV infection, a viral infection-activated split-inteinmediated reporter system (VISI) was devised. Uninfected Huh7.5.1-VISI cells show no background signal, while HCV infection specifically illuminates the nuclei of infected Huh7.5.1-VISI cells with either green fluorescent protein (GFP) or mCherry. Combining VISI-GFP and VISI-mCherry systems, we revisited HCV cell-to-cell transmission with clearcut distinction of donor and recipient cells in a live-cell manner. Independently of virion assembly, exosomes have been reported to transfer HCV subgenomic RNA to initiate replication in uninfected cells, which suggested an assembly-free pathway. However, our data demonstrated that HCV structural genes and the p7 gene were essential for not only cell-free infectivity but also cell-to-cell transmission. Additionally, depletion of apolipoprotein E (ApoE) from donor cells but not from recipient cells significantly reduced HCV cell-to-cell transmission efficiency. In summary, we developed a background-free cell-based reporter system for convenient live-cell visualization of HCV infection, and our data indicate that complete HCV virion assembly machinery is essential for both cell-free and cell-to-cell transmission. IMPORTANCE Hepatitis C virus (HCV) infects hepatocytes via two pathways: cell-free infection and cell-to-cell transmission. Structural modules of the HCV genome are required for production of infectious cell-free virions; however, the role of specific genes within the structural module in cell-to-cell transmission is not clearly defined. Our data demonstrate that deletion of core, E1E2, and p7 genes individually results in no HCV cell-to-cell transmission and that ApoE knockdown from donor cells causes less-efficient cell-to-cell transmission. Thus, this work indicates that the complete HCV assembly machinery is required for HCV cell-to-cell transmission. At last, this work presents an optimized viral infection-activated split-intein-mediated reporter system for easy live-cell monitoring of HCV infection.

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Section: Discussionsupporting

confidence: 82%

Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System

Zhao

Deng

et al. 2017

J Virol

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“…However, viral escape mutants are generally difficult to generate with HCVcc because of the inherently high antibody resistance of most HCV isolates. In addition, we have shown that the high fitness of certain viruses, like core-NS2 recombinants J6/JFH1 and SA13/JFH1, permits the viruses to spread in culture, even at high concentrations of antibody, without developing resistance substitutions (24,31), possibly aided by high levels of cell-to-cell spread (32,33). Previously, we studied AR5A resistance by using novel HCVcc with a deletion of hypervariable region 1 (HVR1) (24).…”

mentioning

confidence: 99%

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms

Velázquez-Moctezuma

Galli

Law

et al. 2019

J Virol

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Yearly, ϳ2 million people become hepatitis C virus (HCV) infected, resulting in an elevated lifetime risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although viable, conferred only low-level AR3A resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1 ΔHVR1 and J6(core-NS2)/JFH1 ΔHVR1 , we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1 ΔHVR1 , or J6/ JFH1 ΔHVR1 with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1 ΔHVR1 , and D431G in J6/JFH1 ΔHVR1 . M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1 ΔHVR1 . L438S and F442Y conferred high-level AR3A resistance to H77/ JFH1 ΔHVR1 but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1 ΔHVR1 but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1 D431G but not J6/JFH1 ΔHVR1/D431G while decreasing scavenger receptor class B type I coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 2a recombinants [JFH1 or T9(core-NS2)/JFH1]. Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects on AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage, with important implications for HCV vaccine development. IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver-related mortality, and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype-reactive antibody AR3A has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we succeeded in inducing only low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B type I HCV coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. Citation Velázquez-Moctezuma R, Galli A, La...

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“…These HCVcc have been used to further define viral entry pathways (Brimacombe et al, 2011;Carlsen et al, 2013;Mathiesen et al, 2015;Timpe et al, 2008) and to show that broadly neutralizing monoclonal antibodies (bNAbs) can prevent HCV infection in animal models (Forns et al, 2000;Morin et al, 2012;Youn et al, 2005). Panels of HCVcc expressing E1E2 from multiple genotypes have also been used to measure neutralizing breadth of bNAbs (Carlsen et al, 2014;Keck et al, 2008Keck et al, , 2013Law et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles

Wasilewski

Ray

Bailey

2016

Journal of General Virology

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A better understanding of natural variation in neutralization resistance and fitness of diverse hepatitis C virus (HCV) envelope (E1E2) variants will be critical to guide rational development of an HCV vaccine. This work has been hindered by inadequate genetic diversity in viral panels and by a lack of standardization of HCV entry assays. Neutralization assays generally use lentiviral pseudoparticles expressing HCV envelope proteins (HCVpp) or chimeric full-length viruses that are replication competent in cell culture (HCVcc). There have been few systematic comparisons of specific infectivities of E1E2-matched HCVcc and HCVpp, and to our knowledge, neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of human broadly neutralizing monoclonal antibodies (bNAbs) targeting distinct epitopes. Here, we describe an efficient method for introduction of naturally occurring E1E2 genes into a full-length HCV genome, producing replication-competent chimeric HCVcc. We generated diverse panels of E1E2-matched HCVcc and HCVpp and measured the entry-mediating fitness of E1E2 variants using the two systems. We also compared neutralization of E1E2-matched HCVcc and HCVpp by a diverse panel of human bNAbs targeting epitopes across E1E2. We found no correlation between specific infectivities of E1E2-matched HCVcc versus HCVpp, but found a very strong positive correlation between relative neutralization resistance of these same E1E2-matched HCVcc and HCVpp variants. These results suggest that quantitative comparisons of neutralization resistance of E1E2 variants can be made with confidence using either HCVcc or HCVpp, allowing the use of either or both systems to maximize diversity of neutralization panels.

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Adaptive Mutations Enhance Assembly and Cell-to-Cell Transmission of a High-Titer Hepatitis C Virus Genotype 5a Core-NS2 JFH1-Based Recombinant

Cited by 27 publications

References 83 publications

Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System

Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms

Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles

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