Binding of host factors to stabilized HIV-1 capsid tubes

Virology. 2018 Oct:523:1-5. doi: 10.1016/j.virol.2018.07.019. Epub 2018 Jul 26.

Abstract

The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders.

Keywords: Binding; CPSF6; Capsid; Cyp A; HIV-1; Human TRIM5α-R332P; MxB; Stabilized tubes; TRIMCyp.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry*
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Biological Assay*
  • Capsid Proteins / chemistry*
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • HIV-1 / chemistry*
  • HIV-1 / metabolism
  • HeLa Cells
  • Host-Pathogen Interactions
  • Humans
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Mutation
  • Myxovirus Resistance Proteins / chemistry*
  • Myxovirus Resistance Proteins / genetics
  • Myxovirus Resistance Proteins / metabolism
  • Nucleocapsid / chemistry
  • Nucleocapsid / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • mRNA Cleavage and Polyadenylation Factors / chemistry*
  • mRNA Cleavage and Polyadenylation Factors / genetics
  • mRNA Cleavage and Polyadenylation Factors / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Capsid Proteins
  • MX2 protein, human
  • Membrane Proteins
  • Myxovirus Resistance Proteins
  • Recombinant Proteins
  • TRAT1 protein, human
  • cleavage factor Im, human
  • mRNA Cleavage and Polyadenylation Factors