Analysis of HIV-1 Viral Infectivity Factor-mediated Proteasome-dependent Depletion of APOBEC3G

Abstract

To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and non-functional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with ␥-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When coexpressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif C114S , which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif C114S from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile 9 disrupted the ability of Vif or Vif C114S to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo.

Nuclear Exclusion of the HIV-1 Host Defense Factor APOBEC3G Requires a Novel Cytoplasmic Retention Signal and Is Not Dependent on RNA Binding

“…Identification of the hA3G CRS is significant because the well studied antiviral activity of hA3G is a cytoplasmic phenomenon (40,(45)(46)(47)(48). The block to HIV-1 reverse transcription, hypermutation of ssDNA replicating HIV-1 proviral DNA and assembly with HIV-1 virions all occur in the cytoplasm (33, 34, 37, 57, 64 -66).…”

“…EGFP was selected because it has no subcellular localization determinants (diffuses freely throughout the cell), does not form multimers, and it, along with its derivatives (i.e. YFP and CFP), has been shown not to affect hA3G functionality or localization when attached to the N terminus of hA3G reporter constructs (45,47,64).…”

“…hA3G is the most potent inhibitor of vifdeficient HIV-1 infectivity in the APOBEC family (44). Of particular interest is that hA3G is restricted to the cytoplasm of T lymphocytes where it has diffuse as well as punctuate distributions (40,(45)(46)(47)(48). The localization of hA3G is functionally significant for each of its known activities.…”

APOBEC3 Cytidine Deaminases: Distinct Antiviral Actions along the Retroviral Life Cycle

“…It remains unclear whether these HMM complexes correspond to the A3G-containing cytoplasmic bodies that lack vimentin cages (82). Small molecule inhibitors of HMM A3G complex formation might be valuable, as this host protein-protein interface would not be expected to undergo rapid sequence variation like Vif.…”

APOBEC3G: an intracellular centurion

“…Deletional mutagenesis coupled with co-immunoprecipitation analyses reveals that the N-terminal region of Vif binds to the N-terminal region of A3G (amino acids 54-124; Conticello et al 2003;Marin et al 2003;Simon et al 2005;Wichroski et al 2005; figure 2). Further mechanistic insights have emerged from crossspecies studies.…”