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

ABSTRACTThe 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.

The Betaretrovirus Mason-Pfizer Monkey Virus Selectively Excludes Simian APOBEC3G from Virion Particles

ABSTRACT The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Schäfer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.

Foamy Virus Bet Proteins Function as Novel Inhibitors of the APOBEC3 Family of Innate Antiretroviral Defense Factors

ABSTRACT Foamy viruses are a family of complex retroviruses that establish common, productive infections in a wide range of nonhuman primates. In contrast, humans appear nonpermissive for foamy virus replication, although zoonotic infections do occur. Here we have analyzed the ability of primate and mouse APOBEC3G proteins to inhibit the infectivity of primate foamy virus (PFV) virions produced in their presence. We demonstrate that several APOBEC3 proteins can potently inhibit the infectivity of a PFV-based viral vector. This inhibition correlated with the packaging of inhibitory APOBEC3 proteins into PFV virions, due to a specific PFV Gag/APOBEC3 interaction, and resulted in the G to A hypermutation of PFV reverse transcripts. While inhibition of PFV virion infectivity by primate APOBEC3 proteins was largely relieved by coexpression of the PFV Bet protein, a cytoplasmic auxiliary protein of previously uncertain function, Bet failed to relieve inhibition caused by murine APOBEC3. PFV Bet bound to human, but not mouse, APOBEC3 proteins in coexpressing cells, and this binding correlated with the specific inhibition of their incorporation into PFV virions. Of note, both PFV Bet and a second Bet protein, derived from an African green monkey foamy virus, rescued the infectivity of Vif-deficient human immunodeficiency virus type 1 (HIV-1) virions produced in the presence of African green monkey APOBEC3G and blocked the incorporation of this host factor into HIV-1 virion particles. However, neither foamy virus Bet protein reduced APOBEC3 protein expression levels in virion producer cells. While these data identify the foamy virus Bet protein as a functional ortholog of the HIV-1 Vif auxiliary protein, they also indicate that Vif and Bet block APOBEC3 protein function by distinct mechanisms.