Protein composition of the hepatitis A virus quasi-envelope

Proc Natl Acad Sci U S A. 2017 Jun 20;114(25):6587-6592. doi: 10.1073/pnas.1619519114. Epub 2017 May 10.


The Picornaviridae are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins (n = 105) were significantly enriched for components of the endolysosomal system (>60%, P < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.

Keywords: ESCRT; exosome; extracellular vesicle; multivesicular body; picornavirus.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / metabolism
  • Capsid Proteins / metabolism*
  • Cell Line
  • Cell Line, Tumor
  • Dipeptidyl Peptidase 4 / metabolism
  • Endosomal Sorting Complexes Required for Transport / metabolism
  • Endosomes / metabolism
  • Exosomes / metabolism
  • Hepatitis A virus / metabolism*
  • Humans
  • Multivesicular Bodies / metabolism
  • Proteomics / methods
  • Tetraspanins / metabolism
  • Virion / metabolism
  • Virus Release / physiology


  • Amino Acids
  • Capsid Proteins
  • Endosomal Sorting Complexes Required for Transport
  • Tetraspanins
  • Dipeptidyl Peptidase 4