African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion Before Core Release From Multivesicular Endosomes

PLoS Pathog. 2016 Apr 25;12(4):e1005595. doi: 10.1371/journal.ppat.1005595. eCollection 2016 Apr.

Abstract

African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • African Swine Fever / virology*
  • African Swine Fever Virus / pathogenicity*
  • Animals
  • Blotting, Western
  • Capsid / metabolism
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Endocytosis
  • Endosomes / ultrastructure
  • Endosomes / virology
  • Flow Cytometry
  • Gene Knockdown Techniques
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Multivesicular Bodies / metabolism
  • Multivesicular Bodies / ultrastructure
  • Swine
  • Viral Envelope Proteins / metabolism
  • Virus Internalization*
  • Virus Uncoating / physiology*

Substances

  • Viral Envelope Proteins

Grant support

This work was supported by grants BFU2009-08085 and AGL2013-48998-C2- 2-R from the Spanish Ministerio de Economía y Competitividad. GA is supported by the ‘Amarouto Program for senior scientists from the Comunidad Autónoma de Madrid’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.