Abstract
BackgroundThe human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.ResultsIn this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.ConclusionsOur results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions.
Highlights
The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions
The conversion of cytidine to deoxyuridine on the minus-strand cDNA either results in guanine to adenine changes on the viral plus-strand cDNA to yield highly mutated viral genomes or triggers the degradation of the deaminated minus strand cDNA through the action of a DNA repair mechanism that involves removal of the uracil base by uracil DNA glycosylase and subsequent endonucleolytic cleavage at the abasic sites by apyrimidinic endonuclease
HeLa cells were transfected with pcDNA-APO3G together either with wild type pNL-A1 (Fig. 1A, lane 2) or its vif-defective variant, pNL-A1vif(-)
Summary
The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. The conversion of cytidine to deoxyuridine on the minus-strand cDNA either results in guanine to adenine changes on the viral plus-strand cDNA to yield highly mutated viral genomes or triggers the degradation of the deaminated minus strand cDNA through the action of a DNA repair mechanism that involves removal of the uracil base by uracil DNA glycosylase and subsequent endonucleolytic cleavage at the abasic sites by apyrimidinic endonuclease (reviewed in [13,14]) While both mechanisms are detrimental to virus replication, the reported inability of vif-defective viruses grown in restrictive cells to reverse transcribe the viral genome into fulllength cDNA is more consistent with the latter mechanism involving the degradation of deaminated viral cDNA [15,16,17,18,19]. Only 10% of cells expressing wild type Vif were double-positive while 95% of cells expressing an inactive Vif variant contained APOBEC3G [6]
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