Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4 ⁺ T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

Abstract

bThe Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4؉ T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4 ؉ T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3= termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4 ؉ T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

Anti-APOBEC3G Activity of HIV-1 Vif Protein Is Attenuated in Elite Controllers

“…At the least, we found that Vifinduced intracellular degradation of APOBEC3G could be one of the possible mechanisms of attenuated Vif function in EC. Although we did not evaluate Vif's interactions with other APOB EC3G proteins, the intrinsic potencies of other APOBEC3 proteins (such as APOBEC3DE/3F/3H) appear to be lower than those of APOBEC3G (46,(72)(73)(74)(75). Moreover, we did not observe interpatient differences in Vif-mediated infectivity in control experiments where the APOBEC3G expression plasmid was not delivered into 293 T cells (data not shown).…”

“…First, as a marker for anti-APOBEC3G activity of Vif in vitro, we analyzed Vif-mediated infectivity that is mediated by multiple mechanisms, including promotion of the degradation of APOBEC3G (24-26, 31, 32), prevention of encapsidation of APOBEC3G (69)(70)(71), and interaction with other APOBEC3G proteins (APOBEC3DE/3F /3H) (72)(73)(74). We failed to evaluate the levels of virion encapsidation of APOBEC3G, since encapsidated APOBEC3G was not detected in the presence of Vif protein under our physiologically optimized in vitro conditions.…”

HIV-1 and HIV-2 Vif Interact with Human APOBEC3 Proteins Using Completely Different Determinants

“…In addition, while it seems that the levels of A3D, A3F, and A3G expression are sufficient to inhibit Vif-deficient HIV-1 in primary CD4 ϩ T cells (41,68), it is unknown which A3 proteins can inhibit replication of Vif-deficient HIV-2 in this cell type and whether Vif2 can counteract these antiviral proteins. The results of our studies showed that A3A was insensitive to Vif2 and A3B, which is Vif1 insensitive (43), displayed partial sensitivity to Vif2-induced degradation.…”

Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex

“…The packaged APOBEC3 proteins inhibit infection in target cells by deaminating deoxycytidine residues on the DNA minus strand during reverse transcription, inducing hypermutation in newly synthesized HIV-1 DNA. APOBEC3 proteins also inhibit replication by cytidine deaminase-independent mechanism(s), such as blocking reverse transcription (18)(19)(20)(21). Vif binds several APOBEC3 proteins and targets them for ubiquitinylation and degradation in the proteasome in virus producer cells, thereby blocking the antiviral activity (22).…”