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.
Author summary Restriction factors are cellular proteins that inhibit viral replication and represent a first line of defense against viral pathogens. The APOBEC3 (A3) family of restriction factors plays an important role in blocking retroviral infections. HIV-1 encodes the Vif protein to antagonize A3, which allows spread of virus in host and ultimately development of AIDS. Prior studies indicate that Vif hijacks host proteolysis pathways to degrade A3 restriction factors; however, our work demonstrates that Vif can neutralize A3s in a degradation-independent manner. These findings suggest viral suppressors of innate immunity work by multiple mechanisms to ensure robust replication. Knowledge of such mechanisms is critical for development of therapeutic strategies to restore the ability of the immune system to cripple viral infections.