12th Conference on Retroviruses and Opportunistic Infections Boston, Massachusetts - February 22-25, 2005

Conf Retrovir Opportunistic Infect 2005 Feb 22-25;12:abstract no. 31

Andrew Mehle¹, J Goncalves², M Santa-Marta², M McPike¹, and D Gabuzda^1
1Dana-Farber Cancer Inst, Boston, MA, USA and ²Univ of Lisbon, Portugal

BACKGROUND: The HIV Vif protein acts by overcoming the innate antiviral activity of APOBEC3G. Vif targets APOBEC3G for proteasomal degradation by associating with an E3 ubiquitin ligase containing Cullin5 and Elongins B and C (Cul5-EloB-EloC). Here, we investigate mechanisms that regulate the assembly and activity of Vif-Cul5 E3 ubiquitin ligase complexes.

METHODS: A SOCS (suppressor of cytokine signaling)-box-like motif was identified in Vif based on sequence alignment with other SOCS-box proteins. The association of wild type and mutant Vif proteins with components of the Cul5 complex was examined in transfected 293T cells and using recombinant proteins or peptides. In vitro ubiquitination reactions were performed using immunoprecipitated Cul5 complexes.

RESULTS: Vif contains a highly conserved motif with homology to the SOCS-box, a domain found in proteins that bind to EloC and function as specificity modules within Cul5 E3 ubiquitin ligases. Vif was shown to associate with Cul5-EloB-EloC through its SOCS-box motif, forming the Vif-Cul5 complex that recruits APOBEC3G for ubiquitination and degradation. The BC-box sequence within the SOCS-box motif mediated a direct interaction with EloC, similar to the function of BC-box motifs in other cellular and viral proteins. Mutational analysis demonstrated that the Vif BC-box has distinct sequence requirements when compared with other SOCS-box proteins. Vif binding to EloC was negatively regulated by serine phosphorylation in the BC-box motif. Ubiquitination of Vif was promoted by Cul5 in vitro and in vivo, and required an intact SOCS-box. Cellular factors that selectively recognize wild type or phosphorylated Vif were identified.

CONCLUSIONS: Vif targets APOBEC3G for degradation by forming a Cul5-EloBC E3 ubiquitin ligase through a novel SOC-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif. The finding that phosphorylation of Vif is important for productive HIV infection but not APOBEC3G degradation raises the possibility that Vif phosphorylation may also regulate another as yet unknown function important for HIV replication. Vif is autoubiquitinated by Cul5, analogous to F-box proteins that are autoubiquitinated within their SCF complex. These findings provide new insights into the mechanisms by which Vif counteracts the antiviral activity of APOBEC3G and also have implications for identifying new therapeutic targets.

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Copyright © 2005 - Foundation for Retrovirology and Human Health. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Foundation for Retrovirology and Human Health. Licensed (AIDSLINE) from National Library of Medicine.