The interferon-inducible GTPase MxB promotes capsid disassembly and genome release of herpesviruses
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Evaluation Summary:
This paper uses an innovative cell-free system to identify antiviral factors that interact with HSV-1 in cells. In addition to cataloging many capsid-interacting factors, the paper probes the antiviral mechanism of one of these, MxB. The data provide strong support for an intriguing model in which MxB "punches" holes in HSV-1 capsids, releasing viral DNA and potentially triggering host DNA sensors. However, the binding of a variety of factors to the capsid appears able to bind to and shield the capsids from MxB attack, suggesting a new perspective on how viruses might evade some host defenses.
(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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Abstract
Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid-host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.
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Author Response:
Evaluation Summary:
This paper uses an innovative cell-free system to identify antiviral factors that interact with HSV-1 in cells. In addition to cataloging many capsid-interacting factors, the paper probes the antiviral mechanism of one of these, MxB. The data provide strong support for an intriguing model in which MxB "punches" holes in HSV-1 capsids, releasing viral DNA and potentially triggering host DNA sensors. However, the binding of a variety of factors to the capsid appears able to bind to and shield the capsids from MxB attack, suggesting a new perspective on how viruses might evade some host defenses.
While we focused here on the role of the IFN-inducible proteins, and in particular on MxB restricting herpesviruses by disassembling their capsids, we would like to add that our experimental system has the …
Author Response:
Evaluation Summary:
This paper uses an innovative cell-free system to identify antiviral factors that interact with HSV-1 in cells. In addition to cataloging many capsid-interacting factors, the paper probes the antiviral mechanism of one of these, MxB. The data provide strong support for an intriguing model in which MxB "punches" holes in HSV-1 capsids, releasing viral DNA and potentially triggering host DNA sensors. However, the binding of a variety of factors to the capsid appears able to bind to and shield the capsids from MxB attack, suggesting a new perspective on how viruses might evade some host defenses.
While we focused here on the role of the IFN-inducible proteins, and in particular on MxB restricting herpesviruses by disassembling their capsids, we would like to add that our experimental system has the potential to identify both, antiviral as well as proviral proteins interacting with HSV-1 capsids.
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Evaluation Summary:
This paper uses an innovative cell-free system to identify antiviral factors that interact with HSV-1 in cells. In addition to cataloging many capsid-interacting factors, the paper probes the antiviral mechanism of one of these, MxB. The data provide strong support for an intriguing model in which MxB "punches" holes in HSV-1 capsids, releasing viral DNA and potentially triggering host DNA sensors. However, the binding of a variety of factors to the capsid appears able to bind to and shield the capsids from MxB attack, suggesting a new perspective on how viruses might evade some host defenses.
(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 …
Evaluation Summary:
This paper uses an innovative cell-free system to identify antiviral factors that interact with HSV-1 in cells. In addition to cataloging many capsid-interacting factors, the paper probes the antiviral mechanism of one of these, MxB. The data provide strong support for an intriguing model in which MxB "punches" holes in HSV-1 capsids, releasing viral DNA and potentially triggering host DNA sensors. However, the binding of a variety of factors to the capsid appears able to bind to and shield the capsids from MxB attack, suggesting a new perspective on how viruses might evade some host defenses.
(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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Reviewer #1 (Public Review):
This manuscript by Serrero et al reports on a very clever series of experiments that identified the dynamin-like GTPase resistance protein B (MxB) as an interferon-inducible protein that interacts with and disassembles herpesvirus capsids. The herpesvirus virion is complex consisting of viral structural proteins in hundreds of copies that make up the capsid and an additional tegument layer consisting of proteins that interact with the capsid and link it with the viral envelope. In these studies, extracts prepared from human macrophages were mixed with various forms of HSV capsids and the capsid-host proteins complexes were characterized by quantitative mass spectrometry (MS), immunoblot, and electron microscopy analysis. Using this novel assay the authors identified MxB as an interferon-inducible protein …
Reviewer #1 (Public Review):
This manuscript by Serrero et al reports on a very clever series of experiments that identified the dynamin-like GTPase resistance protein B (MxB) as an interferon-inducible protein that interacts with and disassembles herpesvirus capsids. The herpesvirus virion is complex consisting of viral structural proteins in hundreds of copies that make up the capsid and an additional tegument layer consisting of proteins that interact with the capsid and link it with the viral envelope. In these studies, extracts prepared from human macrophages were mixed with various forms of HSV capsids and the capsid-host proteins complexes were characterized by quantitative mass spectrometry (MS), immunoblot, and electron microscopy analysis. Using this novel assay the authors identified MxB as an interferon-inducible protein that had the remarkable property to bind and dissemble capsids resulting in the release of the viral genome. Interestingly, capsid assembly did not require proteases but was dependent on the ability of MxB to hydrolyze GTP and dimerize. Studies with capsids stripped of various tegument proteins indicated that MxB binding is localized to the capsid vertices and likely requires the capsid vertex specific proteins for this interaction.
This is an excellent study. I found all the experiments, including the supplemental data, to be complete and convincingly interpreted. The paper is well written, very concisely presented considering the large amount of data, and will be important to those studying the antiviral mechanisms of interferon-induced proteins. The many experiments all contribute to the detailed analysis of MxB's role in the antiviral innate immune response to herpesviruses. The conclusions of this paper are well supported by data. One of the bonuses of these experiments is that the MS studies identified many interferon-induced proteins that will need further characterization to determine their antiviral mechanism.
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Reviewer #2 (Public Review):
Serrero et al. set out to identify the interactions between herpes simplex virus I (HSV-1) and host proteins by conducting proteomics analyses of cellular factors that bind to cell-free capsids. They prepared the capsids under various salt conditions and after trypsin digestion treatment to mimic potentially different states that might occur during viral entry or exit. They used cellular proteins found in control or interferon-treated macrophages. Following incubation of the capsids and cellular factors, they pelleted the capsids and identified many capsid-binding proteins by mass spectrometry. They reproduced the results by repeating the experiment several times and a few of the proteins have been previously reported to bind to viral capsid or tegument, but for the most part, the authors did not …
Reviewer #2 (Public Review):
Serrero et al. set out to identify the interactions between herpes simplex virus I (HSV-1) and host proteins by conducting proteomics analyses of cellular factors that bind to cell-free capsids. They prepared the capsids under various salt conditions and after trypsin digestion treatment to mimic potentially different states that might occur during viral entry or exit. They used cellular proteins found in control or interferon-treated macrophages. Following incubation of the capsids and cellular factors, they pelleted the capsids and identified many capsid-binding proteins by mass spectrometry. They reproduced the results by repeating the experiment several times and a few of the proteins have been previously reported to bind to viral capsid or tegument, but for the most part, the authors did not independently validate the results, nor did they explore the significance of the interactions. However, these proteomics data could prove to be useful as a reference for future studies.
Because the authors detected the host restriction factor MxB as one of the capsid-interacting factors and because it has been reported to restrict herpesviruses, they then used their binding assays along with quantitative electron microscopy (EM) to investigate the mechanism by which MxB inhibits HSV-1. Most interestingly, they find that MxB destroys HSV-1 capsids, seemingly by punching holes at the vertices. Expression of MxB by interferon treatment or, more convincingly, by transgene expression, results in a greater number of 'punched' capsids. MxB mutants lacking the N-terminus or deficient in GTP binding or hydrolysis are less able or unable to damage the capsids. The concordance of these results with prior studies of MxB restriction of HSV-1 replication supports the idea that the way MxB acts is by damaging capsids. Binding of these mutants to capsids seems to be necessary but is not sufficient for damaging the capsids. They also show that the effect of MxB on capsids is generalizable to other alphaherpesviruses (HSV-2 and VZV).
Perhaps the most intriguing result is that MxB acts most efficiently on naked capsids; its binding to and destruction of capsids is inhibited under conditions where more viral tegument proteins should be associated with capsids and may shield the capsids from targeting by MxB. The authors also show that MxB damage to capsid leads to viral DNA leakage and they hypothesize, but do not test, that the released DNA might trigger of DNA sensors and promote immune responses. Overall, this work reveals useful insights into the MxB anti-herpesviral mechanism and provides an original model of how viruses might evade capsid-targeting host defenses.
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