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Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection

  • Author Footnotes
    † These authors contributed equally to this work.
    Lu Sun
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Author Footnotes
    † These authors contributed equally to this work.
    ,
    Author Footnotes
    Current address: School of Medicine, Chongqing University, Shapingba, Chongqing 400044, China
    Hui Feng
    Footnotes
    † These authors contributed equally to this work.
    Current address: School of Medicine, Chongqing University, Shapingba, Chongqing 400044, China
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Ichiro Misumi
    Affiliations
    Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 USA
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  • Takayoshi Shirasaki
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Lucinda Hensley
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Author Footnotes
    § Current address: BioAgylitix, Durham, North Carolina 27713, USA
    Olga González-López
    Footnotes
    § Current address: BioAgylitix, Durham, North Carolina 27713, USA
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Itoe Shiota
    Affiliations
    Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 USA
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  • Wei-Chun Chou
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Jenny P.-Y. Ting
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 USA
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  • John M. Cullen
    Affiliations
    College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27607, USA
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  • Dale O. Cowley
    Affiliations
    Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 USA

    Animal Models Core Facility, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Jason K. Whitmire
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599 USA

    Department of Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Stanley M. Lemon
    Correspondence
    Corresponding author. Address: Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
    Affiliations
    Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Department of Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

    Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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  • Author Footnotes
    † These authors contributed equally to this work.
    Current address: School of Medicine, Chongqing University, Shapingba, Chongqing 400044, China
    § Current address: BioAgylitix, Durham, North Carolina 27713, USA
Published:September 21, 2022DOI:https://doi.org/10.1016/j.jhep.2022.09.013

      Highlights

      • HAV undergoes limited replication in B6 mice, inducing an antiviral transcriptional response that restricts infection.
      • Genetically engineered Mavsvs/vs mice express murine MAVS protein (mMAVS-VS) that is cleaved by HAV 3ABC protease.
      • 3ABC cleavage of mMAVS-VS enhances virus replication and reduces liver inflammation in HAV-infected Ifnar1-/- mice.
      • By itself, 3ABC cleavage of MAVS is not sufficient to overcome the host species barrier to HAV infection in B6 mice.

      Background & Aims

      Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range.

      Methods

      Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS (‘mMAVS-VS’), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice.

      Results

      The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice.

      Conclusions

      MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice.

      Impact and implications

      The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed in vivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

      Graphical abstract

      Keywords

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