An entropic safety catch controls hepatitis C virus entry and antibody resistance
- Institute of Immunity and Transplantation, Division of Infection and Immunity, University College London, United Kingdom
- Institute of Structural and Molecular Biology, Birkbeck College, United Kingdom
- MRC-University of Glasgow Centre for Virus Research, United Kingdom
- Shared Research Facilities, The Institute of Cancer Research, United Kingdom
- Division of Infection and Immunity, University College London, United Kingdom
- International Agency for Research on Cancer, World Health Organization, France
- The Pirbright Institute, United Kingdom
- Division of Medicine, Institute for Liver and Digestive Health, University College London, United Kingdom
- Department of Genetics, University of Cambridge, United Kingdom
- Institut für Biologische Physik, Universität zu Köln, Germany
- MRC Biostatistics Unit, University of Cambridge, United Kingdom
Figures
Figure 1 with 2 supplements
HCV evolves to optimise entry through altered receptor dependency.
J6/JFH HCVcc mutants were isolated following continuous propagation in Huh-7.5 cells. (A) Infectivity of WT, I438V and I438V A524T HCVcc, expressed as foci forming units per ml. Values were …
Figure 1—figure supplement 1
In vitro evolution of HCV.
J6/JFH HCVcc was continuously propagated in Huh-7.5 cells for 20 weeks with viral evolution monitored by NGS. (A) The proportion of synonymous (white) and non-synonymous (black) substitutions …
Figure 1—figure supplement 2
Inhibition of HCVcc infection by BLT-4.
Huh-7.5 cells, treated with a serial dilution of a small molecule inhibitor of SR-B1 (BLT-4), were infected with WT and mutant HCVcc. Infection is expressed as % inhibition relative to untreated …
Figure 2 with 1 supplement
HCV I438V A524T is hyper-reactive.
(A) WT and I438V A524T HCVcc were incubated for 0–8 hr at 37 °C before infection of Huh-7.5 cells. Remaining infectivity is expressed relative to t=0 time point. Data points represent the mean of …
Figure 2—figure supplement 1
Mathematical modelling of HCV entry.
We previously developed a mathematical model to explore HCV receptor engagement and entry (Kalemera et al., 2019). Receptor dependency data from WT and I43V A524T HCVcc (Figures 1 and 2) was …
Figure 3 with 3 supplements
Hyper-reactive HCV is acutely sensitive to all neutralising antibodies.
(A) Neutralisation of WT and I438V A524T HCVcc by a serial dilution of HCV patient IgG. (B) Molecular cartoon of major antigenic sites targeted by nAbs. (C) Neutralisation curves for three …
Figure 3—figure supplement 1
Antibody sensitivity and antigenicity of WT and I438V A524T HCV.
(A) 50% inhibitory concentrations of mAbs (as provided in Figure 3D) separated according to antigenic targets. mAb names are provided for each target. FL and BL refer to Front Layer and Back Layer. …
Figure 3—figure supplement 2
Hyper-reactive phenotype is recapitulated in HCV pseudoparticles.
We characterised WT and I438V A524T E1E2 in the context of HCV pseudoparticles. (A) HCVpp infection of parental or receptor knockout Huh-7, data is expressed relative to parental cells. (B) …
Figure 3—figure supplement 3
pH sensitivity of HCV or Chikungunya (CHIKV) E1E2 pseudovirus .
Virus particles were immobilised and incubated in PBS at pH 7, 6 or 5 prior to addition of Huh-7.5 target cells, infections were read after 72hr. (A) Example raw data demonstrating luciferase …
Figure 4 with 5 supplements
Hyper-reactive HCV exhibits stabilisation of HVR-1.
The conformational dynamics of WT and I438V A524T E2 ectodomain were explored by five independent 1µs MD simulations. (A) Images summarising two representative simulations; superimposed snapshots of …
Figure 4—figure supplement 1
Limited proteolysis of soluble E2.
Soluble E2 (WT or I438V A524T) was incubated at 37 °C with endproteinase GluC for up to 4 hr. (A) Representative western blot images of digestion over time (minutes and hours as indicated); …
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Figure 4—figure supplement 1—source data 1
Raw unedited blot images (A & C) and annotated blots indicating band and sample identities (B & D) for WT (A & B) and I438V A524T E2 (C & D).
- https://cdn.elifesciences.org/articles/71854/elife-71854-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
Five independent 1µs MD simulations of WT E2.
The conformational dynamics of WT E2 ectodomain were explored by MD simulations. E2 RMSF (left), images illustrating HVR-1 mobility (middle), and HVR-1 RMSD (right) are provided for each simulation.
Figure 4—figure supplement 3
Five independent 1µs MD simulations of I438V A524T E2.
The conformational dynamics of I438V A524T E2 ectodomain were explored by MD. E2 RMSF (left), images illustrating HVR-1 mobility (middle), and HVR-1 RMSD (right) are provided for each simulation.
Figure 4—figure supplement 4
Biophysical analysis of E2 by circular dichroism spectroscopy.
(A) Circular dichroism spectra for WT, I438V A524T, and ΔHVR-1 soluble E2. (B) Estimates of the secondary structure content found in E2, data is expressed as a proportion of total. Asterisks …
Figure 4—figure supplement 5
Biophysical analysis of E2 by nano differential scanning fluorimetry.
(A) Intrinsic fluorescence ratio (330 nm over 350 nm) for WT, I438V A524T, and ΔHVR-1 soluble E2 at increasing temperatures. (B) First derivative transformation of fluorescence ratio; peak indicates …
Figure 5
HVR-1 is an entropic safety catch.
(A) The entropic safety catch model. On free virions HVR-1 is dynamic and it acts as an autoinhibitory safety catch on E1E2 function. Interactions with SR-B1 at the cell surface stabilise HVR-1, …
Figure 6 with 1 supplement
Consistent emergence of hyper-reactive mutants in the absence of antibody selection.
(A) Mean infectivities of WT, I438V A524T and S449P HCVcc; foci forming units are corrected for input particle numbers (B) HCVcc infection of parental Huh-7 cells or those CRISPR/Cas9 edited to …
Figure 6—figure supplement 1
Five independent 1µs MD simulations of S449P E2.
The conformational dynamics of S449P E2 ectodomain were explored by MD. E2 RMSF (left), images illustrating HVR-1 mobility (middle), and HVR-1 RMSD (right) are provided for each simulation.
Author response image 1
HCV E1E2 fusion assay.
Two populations of Huh-7 cells expressing matched components of the split-GFP/luciferase system were co-seeded and transfected with the stated E1E2 expression plasmid (or pD603 empty plasmid). 48 …
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71854/elife-71854-transrepform1-v2.pdf
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Source data 1
All underlying data from each experimental measurement is included.
Large data sets from molecular dynamic experiments are excluded, however, full MD trajectories are available for download.
- https://cdn.elifesciences.org/articles/71854/elife-71854-data1-v2.xlsx
