Biochemical and Biophysical Investigation of the Molecular Determinants of HIV-1 Vif Packaging

Abstract

HIV-1 virion infectivity factor (Vif) is an accessory protein that is packaged into virions and is essential for viral replication of HIV. In the absence of Vif, a host cytidine deaminase, APOBEC3G, is incorporated into virions and delivered to target cells where it mutates viral cDNA. Vif recruits a cullin 5-based ubiquitin ligase that targets APOBEC3G for proteosomal degradation. It has been demonstrated that Vif is packaged into the viral core through interactions with the viral genomic RNA. The mechanistic role of packaged Vif has not been determined, but mutations that disrupt Vif packaging, abolish HIV-1 infectivity.We have made several Vif mutations in a basic region spanning residues 75-115 that inhibit packaging. Deletion mutagenesis further confirms that residues beyond 115 are dispensable for RNA binding. An RNA binding assay was developed to look at the correlation between packaging and RNA binding. Stem-loop RNAs derived from the 5’-untranslated region of the HIV-1 genome were chemically synthesized and labeled with fluorescein. The binding of Vif to labeled RNA was measured by changes in fluorescence anisotropy. Binding data showed that some packaging mutants had a reduced affinity for RNA while for other mutants, binding affinity was unaffected, suggesting that other factors may contribute to the specificity of packaging. Evidence suggests HIV-1 Nucleocapsid (NC) may assist in the proper packaging of Vif. Our results demonstrate a RNA dependent interaction between Vif and NC that may be relevant to intravirion packaging of these proteins.Since in the absence of functional Vif, APOBEC3G is packaged into the viral core in an RNA dependent manner, we propose that Vif packaging may help to exclude APOBEC3G from the viral core by competing or interfering with APOBEC3G packaging.