HIV-1 Vif: Counteracting Innate Antiretroviral Defenses
Authors: Bryan R. Cullen
Journal: Molecular Therapy (2003)

Retroviruses and the distantly related retrotransposons are ancient genetic parasites that can harm cells and host organisms in at least two ways. Retroviruses can of course cause direct pathogenic effects. However, both retroviruses and retrotransposons can also cause genetic damage due to their potential to disrupt the expression of genes that flank proviral integration sites. Although human immunodefi- ciency virus type 1 (HIV-1) does not target germ cells, other retroviruses can infect germ cell precursors, which are also subject to genetic damage caused by endogenous retrotrans- posons. In germ cells, proviral integrations have the poten- tial to cause genetic lesions that are then passed onto the progeny of the infected host. It is therefore reasonable to suggest that eukaryotes are under selective pressure to devel- op innate mechanisms that can prevent, or at least reduce, the efficiency of retrovi- ral infection and/or retrotransposition. Similarly, retroviruses must have evolved mechanisms that circumvent these cellular defense mechanisms, at least in their nor- mal host species. Recent evidence demon- strates the existence of an antiretroviral defense mechanism in mammals, includ- ing humans, that relies on the action of a DNA editing enzyme termed APOBEC3G. Unfortunately, in human cells this defense is effectively neutralized by the HIV-1 Vif (virion infectivity factor) protein [1Sheehy, A. M., Gaddis, N. C., Choi, J. D., and Malim, M. M. (2002). Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418: 646-650.Google Scholar, 2Lecossier, D., Bouchonnet, F., Clavel, F., and Hance, A. J. (2003). Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 300: 1112.Google Scholar, 3Harris, R. S., et al. (2003). DNA deamination mediates innate immunity to retroviral infection. Cell 113: 803-809.Google Scholar, 4Zhang, H., et al. (2003). The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424: 94-98.Google Scholar, 5Mangeat, B., et al. (2003). Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99-103.Google Scholar, 6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar] Vif is an HIV-1 auxiliary protein that is expressed late in the viral replication cycle and is critical for viral replication in pri- mary cells. In the absence of Vif, HIV-1 infected primary cells are able to produce high levels of apparently normal HIV-1 virions, which nevertheless are unable to initiate productive infections [7von Schwedler, U., Song, J., Aiken, C., and Trono, D. (1993). Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67: 4945-4955.Google Scholar, 8Gaddis, N. C., et al. (2003). Comprehensive investigation of the molecular defect in vif-deficient human immunodeficiency virus type 1 virions. J. Virol. 77: 5810-5820.Google Scholar]. Although infection occurs and reverse transcription initiates, very few integrated proviruses result. While Vif is required for HIV-1 replication in all primary human cells, the protein is dispensable in a small number of human T-cell lines, which are therefore referred to as "permissive" cells [7von Schwedler, U., Song, J., Aiken, C., and Trono, D. (1993). Vif is crucial for human immunodeficiency virus type 1 proviral DNA synthesis in infected cells. J. Virol. 67: 4945-4955.Google Scholar, 9Gabuzda, D. H., et al. (1992). Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes. J. Virol. 66: 6489-6495.Google Scholar]. Interestingly, Vif-deficient viruses made in permissive cells are fully competent to undergo one round of virus replication in "nonpermissive" primary cells, while Vif-deficient viruses from nonpermissive cells remain defective even when used to infect permissive cells. In other words, the phenotype of the viral producer cell is key. Fusion of nonpermissive and permissive cells was found to result in nonpermissive heterokaryons, demonstrating that the nonpermissive phenotype is dominant [10Madani, N., and Kabat, D. (1998). An endogenous inhibitor of human immunode- ficiency virus in human lymphocytes is overcome by the viral Vif protein. J. Virol. 72: 10251-10255.Google Scholar, 11Simon, J. H. M., Gaddis, N. C., Fouchier, R. A. M., and Malim, M. H. (1998). Evidence for a newly discovered cellular anti-HIV-1 phenotype. Nature Med. 4:1397- 1400.Google Scholar]. This led to the proposal that nonpermissive cells express an innate antiviral factor that blocks the infectivity of progeny, Vif-deficient HIV-1 virions. Based on this hypothesis, work- ers looked for an mRNA present only in nonpermissive cells that, when expressed in permissive cells, could confer the nonpermissive phenotype. A gene, now termed APOBEC3G, was subsequently identified that satisfied these criteria [1Sheehy, A. M., Gaddis, N. C., Choi, J. D., and Malim, M. M. (2002). Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418: 646-650.Google Scholar]. Interestingly, APOBEC3G belongs to a family of proteins that can edit nucleic acids. Specifically, APOBEC3G displays homology to proteins that can either deaminate cytidine (C) to uridine (U) on mRNAs, or deoxycytidine (dC) to deoxyuri- dine (dU) on single-stranded DNA molecules [1Sheehy, A. M., Gaddis, N. C., Choi, J. D., and Malim, M. M. (2002). Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418: 646-650.Google Scholar]. This find- ing suggested that APOBEC3G might block HIV-1 replication by editing RNA or DNA produced by Vif-deficient, but not by wild type, HIV-1. Efforts to demonstrate editing of HIV-1 mRNAs in nonpermissive cells, or of the genomic RNAs pres- ent in virions produced from these cells, did not reveal any C to U mutations. However, reverse transcripts produced by these HIV-1 virions in target cells were found to bear large numbers of dG to dA mutations in the sense strand of the provirus [2Lecossier, D., Bouchonnet, F., Clavel, F., and Hance, A. J. (2003). Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 300: 1112.Google Scholar, 4Zhang, H., et al. (2003). The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424: 94-98.Google Scholar, 5Mangeat, B., et al. (2003). Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99-103.Google Scholar, 6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar]. It is therefore believed that APOBEC3G edits dC to dU on the minus DNA strand during the initial stages of reverse transcription (Fig. 1). As predicted by this hypoth- esis, APOBEC3G is efficiently packaged into virions pro- duced in the absence of Vif [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar]. Strikingly, APOBEC3G also blocks the infectivity of HIV-1 virions that lack all viral pro- teins except Gag and Pol, thus suggesting that APOBEC3G may interact with regions of the Gag and/or Gag-Pol polyprotein that have been conserved during retroviral evo- lution [5Mangeat, B., et al. (2003). Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99-103.Google Scholar]. Consistent with this hypothesis, APOBEC3G is also packaged into retroviral virions produced by the dis- tantly related retrovirus, murine leukemia virus (MLV), and can inhibit MLV replication by the same mechanism [3Harris, R. S., et al. (2003). DNA deamination mediates innate immunity to retroviral infection. Cell 113: 803-809.Google Scholar, 5Mangeat, B., et al. (2003). Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99-103.Google Scholar]. It remains unclear how other retroviruses that do not encode obvious Vif homologs normally circumvent this innate antiviral defense. While APOBEC3G clearly inhibits HIV-1 replication by extensive editing of reverse transcripts, the major phenotype observed is a marked reduction in the formation of integrat- ed proviruses. To explain this result, it has been suggested that dU may be excised from HIV-1 reverse transcripts in the cytoplasm by the cellular DNA repair enzyme uracil DNA glycosylase, thereby generating abasic DNA sites that serve as targets for endonucleolytic cleavage [3Harris, R. S., et al. (2003). DNA deamination mediates innate immunity to retroviral infection. Cell 113: 803-809.Google Scholar, 6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar] (Fig. 1). Proviral intermediates produced during infection by Vif-deficient HIV-1 have in fact been shown to be quite unstable [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar, 12Simon, J. H. M., and Malim, M. H. (1996). The human immunodeficiency virus type 1 Vif protein modulates the postpenetration stability of viral nucleoprotein com- plexes. J. Virol. 70: 5297-5305.Google Scholar]. Moreover, the small number of integrated proviruses that are observed upon infection by Vif-deficient HIV-1 are exten- sively mutated and are therefore unable to mount a produc- tive infection [5Mangeat, B., et al. (2003). Broad antiretroviral defense by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424: 99-103.Google Scholar]. Efforts to determine how Vif interferes with this innate antiretroviral pathway led to the demonstration that Vif specifically interacts with human APOBEC3G and blocks incorporation of the latter into HIV-1 virions [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar] (Fig. 1). Vif also significantly reduces the steady-state level of expression of the human APOBEC3G protein. At present, it remains unclear whether this reduction in APOBEC3G expression is critical to the ability of Vif to block APOBEC3G function or whether it is secondary to an inhibition that results primari- ly from formation of the complex between Vif and APOBEC3G. It is important to note that Vif can be packaged into HIV-1 virions [13Liu, H., et al. (1995). The Vif protein of human and simian immunodeficiency virus is packaged into virions and associates with viral core structures. J. Virol. 69: 7630- 7638.Google Scholar] (Fig. 1). Although this suggests that Vif might prevent human APOBEC3G packaging by blocking access to intra-virion binding sites, the inability of Vif to inhibit virion incorporation and biological activity of close- ly related APOBEC3G proteins from other species (see below) renders this hypothesis unlikely. The replication pattern of HIV-1 is highly species specific. HIV-1 is only able to replicate in humans and closely related apes such as chimpanzees. In contrast, HIV-1 does not repli- cate in monkeys or in any other mammal tested thus far. In part, this pattern of species tropism reflects the absence, par- ticularly in rodent cells, of key cellular cofactors for HIV-1 replication. However, this phenomenon could also result from the inability of HIV-1 to overcome innate antiretroviral resistance pathways present in nonhuman cells. In fact, APOBEC3G orthologs present in rodents and in both African green monkeys (agm) and rhesus macaques are able to effec- tively block HIV-1 replication by the same mechanism as human APOBEC3G. However, the mouse and monkey orthologs are not responsive to HIV-1 Vif [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar]. In the case of mouse APOBEC3G, this lack of responsiveness correlates with the inability of HIV-1 Vif to interact with and destabi- lize the mouse APOBEC3G protein and to block its incorpo- ration into HIV-1 virions [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar]. While agm and rhesus macaque APOBEC3G are nonre- sponsive to HIV-1 Vif, thus at least partly explaining why these monkeys are unable to support a productive HIV-1 infection, it is interesting to note that the Vif protein encod- ed by the agm form of Simian immunodeficiency virus (SIV) is also unable to block human APOBEC3G function [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar]. In contrast, the Vif protein encoded by the form of SIV that can replicate in rhesus macaques is fully competent to inhibit human APOBEC3G. This latter SIV is closely related to the second lentiviral pathogen of humans, termed HIV-2, while no relative of SIVagm has been detected in humans. These data suggest that the ability of individual SIVs to cause zoonotic infections of humans may be largely determined by whether the encoded Vif protein can block human APOBEC3G function [6Mariani, R., et al. (2003). Species-specific exclusion of APOBEC3G from HIV-1 virions by Vif. Cell 114:21-31.Google Scholar, 14Simon, J. H. M., et al. (1998). The regulation of primate immunodeficiency virus infectivity by Vif is cell species restricted: a role for Vif in determining virus host range and cross-species transmission. EMBO J. 17: 1259-1267.Google Scholar]. Thus, while APOBEC3G has unfortunately failed to protect us from HIV-1 and HIV-2, it has likely prevented additional lentiviral zoonoses that could have been as bad or worse. Many questions about the APOBEC3G antiretroviral pathway remain to be addressed. How is APOBEC3G target- ed to retroviral virions? Given that HIV-1 only recently became a significant human pathogen, did APOBEC3G evolve to protect humans against other retroviruses or per- haps even against retrotransposons, endogenous genetic parasites that share many features in common with retro- viruses? How do retroviruses such as MLV, which lack a Vif homolog, prevent APOBEC3G from inhibiting their replica- tion? And lastly, how does Vif interact with APOBEC3G and how does this interaction prevent APOBEC3G function? Research on the topic of host innate immunity to retroviral infection has made rapid progress in the last few months, not only in the area of Vif and APOBEC3 function but also in understanding a second innate antiretroviral pathway, encoded by a human factor termed Ref1 [15Bieniasz, P. D. (2003). Restriction factors: a defense against retroviral infection. Trends Microbiol. 11: 286-291.Google Scholar]. Future discoveries may suggest ways to enhance the effectiveness of these endogenous defense mechanisms, for example by blocking the ability of Vif to inhibit APOBEC3G function or by enhancing the activity of APOBEC3G. Finally, these studies also have potentially significant implications for efforts to use retroviruses in general, and lentiviruses in particular, as vectors for human gene therapy.