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  1. Evaluation Summary:

    HCV is unique in its glycoprotein structure, complex receptor usage and its unusual persistence for a (+)RNA virus. This is a well done study that explains a number of observations regarding receptor usage and how HCV may evade antibody control via HVR1 due to its disordered nature, enable mutation to continually evade antibody responses. This manuscript should be of substantial interest to those in the fields of virus entry, vaccination against human viruses, and the study of how intrinsically disordered regions can play regulatory roles.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript, the authors isolate a cell culture adapted HCV with 2 point mutation in E2. This is associated with increased infection, rate of entry, and less reliance on the entry factor SR-B1. Tradeoffs are that the virion is less stable in culture and is much more sensitive to antibody neutralization. Biophysical analysis suggests that the mutations stabilize HVR1 with properties that resemble a deletion of HVR1. Indeed deletion of HVR1 has enhanced properties of the double point mutant: higher infection, but less thermal stability and resistance to neutralization. This produces a model wherein HVR1 is a disordered peptide tail that blocks antibody neutralization and inhibits CD81 interaction. Binding of E2 to SR-B1would alter HVR1 conformation, exposing the CD81 binding site and leading to productive entry.

    I found the experiments to be well done and interpreted. The model explains a number of observations regarding receptor usage and how HCV evades antibody control via HVR1, whose disordered nature enables mutation to continually evade antibody responses.

    I thought the manuscript was complete with only minor corrections necessary.

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  3. Reviewer #2 (Public Review):

    The molecular mechanism of how HCV enters a host cell remain undefined. Envelope glycoprotein 1 and 2 (E1 and E2) are responsible for facilitating entry. E2 demonstrates conformational flexibility with three hypervariable regions (HVR) and several disordered regions. Using a series of diverse virology and computational methods, the authors claim that HVR1 acts as a "safety catch" that regulates entry. The employment of MD simulations to look at conformational mobility is compelling.

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  4. Reviewer #3 (Public Review):

    The complex mechanism of HCV entry has long been an area of focus for both fundamental virologists as well as groups interested in generating vaccines. The virus employs multiple surface receptors to mediate cell entry, which occurs via clathrin-mediated endocytosis and is linked to a complex cascade of both host signalling and the induction of additional responses.

    The study utilises cell culture adaptation to decipher how HCV might improve its ability to enter and infect cells. This is done twice, once in the absence of antibodies, once with them present. Resultant mutations in the absence of antibody selection lead to lesser dependence on SR-B1, increased affinity for CD81, and enhanced susceptibility to neutralising antibodies. In addition, the thermal stability of virions is reduced. This phenotype is recapitulated by genetic deletion of the hypervariable region 1 (HVR1). The enhanced ability for these mutants to enter cells is supported by mathematical models and MD simulations support that the disordered HVR1 region becomes stabilised as a result of these changes.

    The authors therefore propose that a loss of entropy in the E2 protein is responsible for this "hyper-reactive" phenotype. In nature, this would be achieved through SR-B1 interactions, hence the loss of dependence upon this receptor in vitro.

    Strengths:

    This paper provides a new concept for how the multi-step entry process in HCV takes place, based upon the two known physical interactions with cellular receptors. The enhanced affinity for CD81 appears directly related to the loss of entropy in the system, and according to the mathematical model, ensuing events post-CD81 binding might also be accelerated. The authors propose that by understanding the nature of the switch that the development of vaccines might be enhanced. The combination of MD, virus culture, biophysical methods and mathematical models provides a strong argument in support of the hypothesis. The work has been carried out to a very high standard and presented very nicely.

    Weaknesses:

    Whilst the mathematical models support accelerated entry at multiple stages, steps following the interaction with CD81 are not explored beyond determination of ensuing viral titres.

    It is also unclear whether there might be a region of E2 in which the changes in overall structure can be rationalised in terms of mechanism. For example, do regions interacting with CD81 and/or E1 show any alterations that might explain altered behaviour? It is difficult to make out from the RMSF plots whether this might be the case.

    Given that HCV enters cells via clathrin-mediated endocytosis, it would be of interest to determine whether the enhanced entry phenotype was also related to pH. HCV is unusual compared to e.g. influenza in that the loss of infectivity following acid pH treatment can be restored by re-buffering virions to neutral conditions - could this be related to the entropic catch and associated structural changes?

    Whilst this may be difficult to address, would antibody binding to HVR1 also be expected to reduce entropy? Could non-neutralising, yet HVR-binding antibodies therefore lead to enhanced entry and SR-B1 independence?

    Lastly, one presumes that the antibody selected long term culture failed to select any notable mutations? Was this assessed?

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