Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors

PLoS Pathog. 2018 Jan 5;14(1):e1006830. doi: 10.1371/journal.ppat.1006830. eCollection 2018 Jan.

Abstract

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

Publication types

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

MeSH terms

  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology*
  • Cullin Proteins / chemistry
  • Cullin Proteins / metabolism
  • Cytidine Deaminase
  • Cytosine Deaminase / metabolism*
  • HEK293 Cells
  • HIV Infections / immunology
  • HIV Infections / therapy
  • HIV Infections / virology
  • HIV-1 / immunology
  • HIV-1 / metabolism
  • Humans
  • Immunoglobulin Fab Fragments / chemistry*
  • Ubiquitin / metabolism
  • Ubiquitination
  • Virus Assembly
  • vif Gene Products, Human Immunodeficiency Virus / chemistry
  • vif Gene Products, Human Immunodeficiency Virus / immunology*

Substances

  • Antiviral Agents
  • Cullin Proteins
  • Immunoglobulin Fab Fragments
  • Ubiquitin
  • vif Gene Products, Human Immunodeficiency Virus
  • vif protein, Human immunodeficiency virus 1
  • Cytosine Deaminase
  • APOBEC3 protein, human
  • Cytidine Deaminase