APOBEC3G and HIV-1: Strike and Counterstrike Vanessa B. Soros, PhD, and Warner C. Greene, MD, PhD

Corresponding author Warner C. Greene, MD, PhD Gladstone Institute of Virology and Immunology, Departments of Medicine, Microbiology, and Immunology, University of California, San Francisco, 1650 Owens Street, CA 94158-2261, USA. E-mail: [email protected] Current Infectious Disease Reports 2006, 8:317–323 Current Science Inc. ISSN 1523-3847 Copyright © 2006 by Current Science Inc.

APOBEC3G (A3G), a deoxycytidine deaminase, is a powerful host antiretroviral factor that can restrict HIV-1 infection. This restriction is counteracted by the HIV-1 virion infectivity factor (Vif) protein, whose activity culminates in depletion of A3G from infected cells. In the absence of Vif, viruses encapsidate A3G, which acts in part to mutate viral DNA formed during reverse transcription upon subsequent infection of a new cell. Cellular A3G also functions as a postentry restriction factor for HIV in resting CD4 T cells, where it resides in a low molecular mass form. Unfortunately, this barrier is forfeited when CD4 T cells are activated because A3G is recruited into inactive high molecular mass ribonucleoprotein complexes. In addition to restricting HIV, A3G and related deaminases may counter other retroviruses and protect the cell from endogenous mobile retroelements. Understanding A3G complex assembly and its interplay with HIV Vif may make possible future development of a new class of HIV therapeutic agents.

Introduction HIV principally infects CD4 T cells and macrophages despite their expression of APOBEC3G (A3G), a cellular deoxycytidine deaminase with potent antiviral activity against HIV. The fact that more than 40 million people are infected with HIV worldwide indicates that this virus can circumvent the antiviral effects of A3G. Recent studies demonstrate that the HIV virion infectivity factor (Vif) protein counteracts the antiviral effects of A3G by depleting intracellular stores of this factor, thereby blocking A3G encapsidation into budding virions. Conversely, HIV viruses lacking Vif are effectively checked by A3G, revealing why Vif is critical for viral infectivity in vivo. To more fully appreciate this remarkable antiviral factor, it is illustrative to describe how A3G was first identi-

fied. Vif is not required for HIV infection of all cells; select laboratory-adapted T-cell lines were shown to support the growth of HIVΔVif viruses, prompting their classification as “permissive” cells. Conversely, other closely related cell lines termed “nonpermissive” required Vif for HIV replication. Strikingly, nonpermissive cells supported full production of ΔVif viruses, but the progeny virions lacked the ability to infect the next target cell [1,2]. Expression of Vif in the nonpermissive virus-producing cells fully rescued production of infectious HIVΔVif virions, but expression of Vif in the target cell did not [3]. Insights into the nature of Vif action emerged when heterokaryons formed between permissive cells and nonpermissive cells were tested for the ability to produce infectious HIVΔVif viruses. These viral progeny proved to be noninfectious, suggesting that nonpermissive cells produce an antiviral factor that is somehow defeated when Vif is expressed in the virus-producing cell [4•,5•]. Through subtractive hybridization of mRNA from nonpermissive and permissive cells, Sheehy et al. [6••] identified this antiviral factor as A3G, a member of a family of cytidine deaminases [7]. Intriguingly, these enzymes catalyze deamination of cytidine residues in nucleic acid, creating uridines in their stead [8•]. Expression of A3G in permissive cells rendered these cells nonpermissive for HIVΔVif infection, indicating that this single gene is sufficient to generate the nonpermissive state [6••]. Together, these findings highlight the presence of a potentially formidable defense against HIV-1 mounted by the cytoplasmic enzyme A3G. Unfortunately, this defense is readily countered by the HIV-1 Vif protein.

How A3G Exerts its Antiviral Effects In the absence of Vif, A3G is recruited into budding virions through its interaction with the nucleocapsid component of the Gag polyprotein and/or HIV genomic RNA, as depicted in Figure 1A (number 1) and reviewed elsewhere [9–11]. These viruses are consequently rendered noninfectious because virion-encapsidated A3G undermines an integral subsequent step in the viral life cycle: reverse transcription. During reverse transcription, the RNA genome is first converted into a minus single-stranded DNA (ssDNA), which in turn serves as the template for the synthesis of a complementary plus (coding) strand to form a double-

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Update on AIDS Figure 1. A, HIV-1 restriction by APOBEC3G (A3G). Incorporation of cellular A3G into HIV-1Δvirion infectivity factor (Vif) virions (1) restricts the ability of the virus to establish an infection in a new target cell. Deoxycytidine deamination by A3G during reverse transcription in the new target cell results in either degradation of the minus singlestranded DNA (2) or dG→dA hypermutation (3). Low molecular mass (LMM) A3G in resting CD4 T cells from peripheral blood can restrict HIV post-entry into a new target cell (4) in a manner that is unchecked by Vif and may be independent of its deaminase activity. The HIV protein Vif counteracts A3G in virusproducing cells by targeting it for degradation (5). (Continued on page 321)

stranded DNA copy of the RNA genome. It is the minus ssDNA that A3G targets for deamination, converting deoxycytidines (dC) to deoxyuridines (dU) primarily at dCdC sites [9–11], with the frequency of mutation influenced by the duration of single strandedness [12••]. Although not fully understood, the presence of these dU residues may initiate nontemplate DNA repair by uracil DNA glycosylase [13] in an attempt to eliminate the unwanted uracil residues. Successful action of the glycosylase produces abasic sites, which in turn are targeted by host cell endonucleases for strand cleavage. Thus, as depicted in Figure 1A (number 2), one possible outcome of A3G deamination of deoxycytidines is the degradation of the minus ssDNA, thereby abrogating the HIV life cycle. This process is apparently imperfect, because some deaminated minus ssDNA can escape degradation and serve as a template for plus-strand synthesis. Because of the presence of deoxyuridines instead of deoxycytidines, dG→dA hypermutations are introduced into the plusstrand viral DNA (Fig. 1A, number 3). Massive dG→dA hypermutation is, in fact, a hallmark of A3G anti-HIV activity [9–11]. Such mutations likely alter the amino acid sequence of multiple viral gene products and introduce lethal stop codons, compromising HIV production. Whether low levels of deoxycytidine deaminase activity play a role in expanding HIV-1 sequence diversity is a matter of some debate. It is notable that the HIV genome is remarkably rich in A residues [14,15]. In some patients, dG→dA hypermutated viral genomes appear but usually only late in infection [16–20]. However, these hypermutated species are virtually never the predominant genotype that is propagated; indeed, they likely correspond to crippled or even dead-end viruses.

The Vif Counterstrike Despite the potent antiviral activity of A3G, HIV-1 effectively infects and spreads within its natural A3Gexpressing cellular targets in vivo. This reflects the ability of HIV Vif to block the action of A3G. Vif acts by depleting cells of A3G (Fig. 1A, number 5), both by inhibiting its synthesis [21•] and stimulating its accelerated degradation by the 26S proteasome [9–11]. Consequently, no A3G is available for recruitment into the budding virions. Thus, these viruses remain fully infectious, and HIV reverse transcription proceeds unchallenged in the next target cell. Although it is currently unclear how Vif partially inhibits the translation of A3G mRNAs, the mechanism by which Vif stimulates A3G proteolysis has largely been deciphered [9–11]. It should be noted that there remains some controversy as to whether complete depletion of cellular pools of A3G is required to prevent incorporation of A3G into viral particles [22,23]. It is possible that Vif could also impair A3G incorporation into virions by sequestering the enzyme from sites of virion assembly. Unraveling the mechanism by which Vif counteracts A3G revealed why expression of Vif in nonpermissive virus-producing cells “rescued” ΔVif virions, whereas Vif expression in the target cell did not. Vif expression in nonpermissive cells destroyed the A3G that was rendering the emerging viruses noninfectious. However, this observation raises another conundrum: If the activated CD4 T cells and macrophages that HIV-1 naturally infects all express A3G, why does cellular A3G not attack reverse transcripts from infecting viruses? How does HIV ever establish an infection in cells expressing this antiviral factor?

APOBEC3G and HIV-1: Strike and Counterstrike

The Complexities of A3G Complexes Unexpectedly, the answer emerged through the analysis of cellular A3G. When cell lysates were analyzed by molecular “sieving,” A3G was detected almost exclusively in high molecular mass (HMM) complexes larger than 2 megadaltons [24••]. Of note, treatment of these lysates with RNase produced a sharp shift of A3G to a low molecular mass (LMM) form consistent in size with monomers or dimers of the enzyme. Strikingly, when the deaminase activity of these two complexes was analyzed, the HMM complex proved inactive, whereas LMM A3G exhibited readily detectable deoxycytidine deaminase activity. Thus, A3G in activated CD4 T cells is negatively regulated by its assembly into enzymatically inactive HMM ribonucleoprotein complexes. This finding likely explains why these cytoplasmic HMM A3G complexes are unable to interrupt reverse transcription of HIV virions entering activated CD4 T cells. Similarly, macrophages, which support productive HIV infection, express the inactive HMM form of A3G. In an interesting biological twist, these studies also shed new light on the long-standing mystery of why blood-derived resting CD4 T cells and monocytes are such poor hosts for HIV-1 infection despite supporting binding and entry of virions into cells. In notable contrast to activated CD4 T cells and macrophages, which express HMM A3G, both resting CD4 T cells and monocytes express the LMM form of A3G. Importantly, when LMM A3G expression was “knocked down” with specific siRNAs, the resting CD4 T cells were rendered permissive for HIV infection. These findings indicated that the LMM form of cellular A3G functions as a potent post-entry restriction factor for HIV depicted in Figure 1A (number 4). Furthermore, this restricting activity is unchecked by Vif, as insufficient quantities of Vif are delivered by the infecting virion, and the virus has not progressed far enough into its life cycle to produce new Vif. In contrast to resting CD4 T cells circulating in the bloodstream, resting naïve CD4 T cells in lymphoid tissue are permissive to infection with CXCR4-tropic strains of HIV. Why does cellular A3G not restrict HIV replication in these resting T cells? In these cells, A3G forms HMM ribonucleoprotein complexes akin to the blood-derived activated CD4 T cells permissive to HIV infection. This shift of A3G from LMM into HMM complexes in resting CD4 T cells is induced by cytokines, including interleukin-2 and interleukin-15, present in the milieu of lymphoid tissues [25]. Another surprising development concerning the mechanism of HIV restriction by LMM A3G complexes emerged when the reverse transcripts formed in resting CD4 T cells from blood were examined for evidence of A3G-induced dG→dA hypermutations [24••]. In fact, only 8% of the transcripts contained such mutations. These results suggest that LMM A3G may exert its antiviral activity independent of its deoxycytidine deaminase activity. This potential nonenzymatic form of antiviral

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activity by A3G has also been observed in A3G mutagenesis studies [26•] and in other viral contexts as discussed below, and it is fast emerging as a recurrent theme in A3G biology. However, it is important to note that rapid destruction of heavily deaminated reverse transcripts in the resting CD4 T cells could also account for the paucity of hypermutations detected.

A3G: But One Weapon in the Armory Although A3G was initially shown to be sufficient to render permissive cells nonpermissive, subsequent studies revealed that factors other than A3G could similarly convert cells to the nonpermissive state. A3G is but one member of a family of eight deoxycytidine deaminase genes (A3A to A3H) clustered within a 130-kb region of chromosome 22. This gene family arose as a result of tandem duplication with unequal crossover after the genetic radiation of mice, which contain a single APOBEC3 gene [7]. Several other members of the APOBEC3 family have since been demonstrated to also restrict HIV and other retroviral infections in vitro. Like A3G, A3F and A3C are expressed in peripheral blood lymphocytes, spleen, ovary, and testes. A3B is not coexpressed with A3F and A3G but, like A3C, is found in a variety of cancer cell lines. All display some level of deoxycytidine deaminase activity in experimental systems, each targeting distinctive dC residues in a sequence-specific manner [9–11]. A3F also restricts HIV-1 infection in vitro, albeit somewhat less efficiently than A3G, and is also effectively targeted by HIV-1 Vif for proteasome-mediated degradation. Because resting CD4 T cells and monocytes express A3F, it is likely that A3F can also exert a post-entry restriction block to HIV-1 infection in these cells. A3B displays some anti-HIV activity in vitro, yet its function is not antagonized by HIV-1 Vif. However, because A3B is not expressed in the natural targets of HIV-1 infection, it is unlikely that A3B forms a formidable barrier to HIV-1 spread in vivo. It is plausible to speculate that A3B may protect cells in which it is expressed against other retroviruses. A3C displays, at best, weak anti-HIV activity in vitro and is sensitive to the Vif counterattack.

A3G: A Broad-range Defense Because the APOBEC3 deaminases attack the minus ssDNA reverse transcript of HIV-1, the possibility that these enzymes may also restrict other human viruses that replicate through reverse transcription—such as HIV-2, human T-cell leukemia virus (HTLV), and hepatitis B virus (HBV)—attracted great attention. Similarly, the ability of human APOBEC3s to restrict nonhuman retroviruses from entering the human population has been explored [9–11]. HIV-2 infects the same activated CD4 T cells and macrophages as HIV-1, namely cells expressing A3G and A3F. HIV-2 encodes a Vif gene whose counterattack against APOBEC3 proteins resembles that of HIV-1

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Vif [27]. HIV-2 Vif can target both A3G and A3F for proteasome-mediated degradation [27,28], although A3G may not pose as formidable a restriction against HIV-2 as it does against HIV-1 [29]. HTLV-1 also infects the same A3G- and A3F-expressing target cells as HIV-1 but does not encode a Vif homologue. Naturally occurring dG→dA hypermutations in HTLV-1 sequences are extremely rare, suggesting that HTLV effectively resists hypermutation by A3G and A3F [30]. Experimental investigations thus far have yielded disparate and conflicting results regarding A3G restriction of HTLV-1 [30–32], but several groups report incorporation of A3G into HTLV-1 virions [31,32]. The relative resistance of HTLV to A3G and A3F could reflect an infrequent encounter with these cytoplasmic enzymes. HTLV principally replicates by host cell division and only rarely through reverse transcription. Because A3G and A3F do not appear to affect somatic nuclear DNA during cell division, it may be that they simply have less opportunity to attack HTLV-1 than HIV-1. HBV infection of hepatocytes involves reverse transcription of a pregenomic RNA into DNA, but unlike HIV-1, this process occurs in subviral cores found in the virus-producing cells, not upon infection of new target cells. Because dG→dA mutations can be detected in HBV sequences from a few patients [33–35], the potential for A3G anti-HBV activity was explored using in vitro systems. Overexpression of A3G and A3F in hepatoma cell lines could restrict HBV replication [34,36,37,38•]. However, examination of dG→dA hypermutations in HBV DNA have yielded conflicting results, depending on the cell line used to support replication and the method of DNA analysis employed. Equivalent restriction by wild-type and catalytic mutants of A3G suggest that A3G potentially can restrict HBV replication by blocking pregenomic RNA packaging or by destabilizing reverse transcription complexes [38•]. This mechanism again highlights a potential nonenzymatic mechanism of A3G antiviral action. Does A3G play a role in combating natural HBV infection? Endogenous expression of the APOBEC3s in primary human hepatocytes is barely detectable [39]. However, treatment of primary hepatocytes with interferon α (IFN-α) upregulates the expression of endogenous A3B, A3C, A3F, and A3G [39]. This finding is potentially relevant clinically, because IFN-α is widely used as a treatment for HBV infection. Could the antiviral effect of IFN-α be mediated through the action of an upregulated APOBEC3 factor? It remains to be determined whether clearance of HBV core antigen following IFN-α treatment correlates with increased expression of any of the APOBEC3 family members. Similarly, observation of increased expression of the APOBEC3s in tissues in which HBV is effectively suppressed and observation of negligible expression in acutely infected tissue would be consistent with a role for APOBEC3s in fighting HBV. Finally, it will be of interest to determine whether IFN-α treatment increases dG→dA hypermutation of HBV DNA.

APOBEC3s and the “Enemy Within” Because the APOBEC3 proteins effectively attack exogenous retroviruses during reverse transcription, the notion that these enzymes also impair retrotransposition of endogenous retroelements is attractive. Transposable genetic elements [40] and host genomes are in constant conflict. In humans, 45% of the genome derives from mobile genetic elements, including more than 230,000 endogenous retroviruses, essentially all of which are now defective. However, other retrotransposons lacking long terminal repeats (LTRs) are also present in the genome, including long interspersed nuclear elements (LINEs) and short interspersed nuclear elements, including Alu; both elements depend on the reverse transcription machinery of LINE. It has been estimated that Alu effectively retrotransposes with a frequency between one in 30 and one in 300 live births. Because retrotransposition culminates in integration into the genome, such events can be damaging to the host if the integration occurs within an open reading frame. An open question is how such retrotransposition events are normally controlled. Although A3G has been reported to have no effect on LINE retrotransposition [41], studies have not yet been reported on evaluation of its effects on Alu elements, the most successful retrotransposons in the human genome. In contrast to humans, mice contain several active families of LTR retrotransposons, including MusD and intracisternal A-type particle (IAP). In vitro studies have shown that various human APOBEC3s (notably A3A, A3B, and A3G) can restrict the retrotransposition of both MusD and IAP endogenous retroviruses [42,43•]. Inhibition involves dG→dA mutation with A3B and A3G but not A3A [43•]. Thus, A3A may use a nonenzymatic mechanism to control retrotransposition. Remarkably, expression of human A3G or A3F in yeast cells inhibits retrotransposition of an endogenous yeast LTR retrotransposon [44•,45•]. Therefore, it is plausible that the dramatic evolutionary expansion of the APOBEC3 family of genes from one in mice to eight in humans may have occurred to deal with endogenous retroelements. Because this expansion appeared before emergence of the primate lentiviruses, it is difficult to implicate these exogenous retroviruses as a driving force behind APOBEC3 expansion. For more information on the evolutionary expansion and positive selection of APOBEC3 genes in primates, readers are referred to fascinating recent papers [7,46–48].

Clinical Implication of A3G Polymorphisms Initially, seven single nucleotide polymorphisms (SNPs) were detected in the A3G gene, although only one of these altered the amino acid sequence of A3G: H186R [49]. The 186R allele was found more frequently in African Americans but was rare in individuals of European descent. In a cohort of more than 3000 subjects (949 African-Americans), the presence of the 186R allele was associated with

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Figure 1. (Continued) B, Potential therapeutic targets aimed at inducing an APOBEC3G (A3G)-mediated antiviral restriction. Inhibition of the interaction between A3G and virion infectivity factor (Vif) (1) or Vif and the proteasome (2) could shelter A3G from Vif-mediated degradation. Induction of LMM A3G expression in cells that do not express A3G (3) or disassembly of inactive high molecular mass (HMM) A3G complexes into LMM A3G forms (4) could induce a post-entry restriction.

a more rapid decline in CD4 T-cell counts and accelerated disease progression. However, in vitro analysis of 186R did not reveal changes in protein stability or increased sensitivity to HIV-1 Vif. Our analysis indicates that the A3G-186R allele also readily forms HMM complexes in vitro (Soros, Unpublished data). Thus, the mechanism by which A3G-186R may affect HIV infection remains to be determined. In an analysis of a Caucasian cohort (n > 600) of slow progressors (n > 240), 14 novel SNPs were identified in A3G, including an additional codon-changing variant: Q275E [50]. However, none of the SNPs, including 186R, correlated with progression to AIDS. In another approach, A3G mRNA levels were quantified in 25 HIV-infected subjects, including eight long-term nonprogressors (LTNPs), 17 progressors, and six HIV-uninfected subjects [51]. In this small cohort, a statistically significant inverse correlation was found between A3G mRNA levels and viral load, and a positive correlation between A3G mRNA levels and CD4 count. A3G mRNA levels were highest in the LTNPs and the lowest in the progessors. Although limited in scope, these observations suggest that higher levels of A3G expression may correlate with lower replication of HIV, increased survival of CD4 T cells, and lower rate of HIV disease progression. It remains to be determined whether higher levels of A3G mRNA correlate with excess protein that escapes the Vif counterattack and/or increased levels of LMM A3G protein. Sequencing analysis for evidence of dG→dA hypermutation in the LTNPs also merits investigation. Of note, another study did not find any significant correlation among A3F or A3G mRNA levels, plasma viremia, and CD4 T-cell counts [52]. Whether differences in the study designs or cohorts account for these conflicting

results, both studies did demonstrate higher levels of A3G mRNA expression in HIV-uninfected subjects compared with HIV-infected subjects. Finally, it may be rewarding to study polymorphic variation in other proteins whose activities intersect with A3G-mediated restriction of HIV. Namely, factors that mediate the assembly of HMM complexes and the degradation of A3G in the proteasome could also affect A3G antiviral activity and clinical outcome of HIV infection.

Vif and HMM A3G as Therapeutic Targets One attractive new strategy for HIV drug development could involve developing small molecules that inhibit Vif binding to A3G (Fig. 1B, number 1). The observation that a single-point mutation in a species A3G can render it insensitive to the cognate Vif of the infecting virus [27,53,54] suggests that such a strategy is reasonable. Similarly, drugs aimed at disrupting Vif ’s interaction with the cellular factors that enable degradation may neutralize its ability to counteract A3G (Fig. 1B, number 2). However, given the rapid rate at which HIV-1 sequence varies within an infected host, drug-resistant mutants will likely evolve. These inhibitors would be most effective as a component of a multidrug strategy. LMM A3G forms an effective post-entry block to HIV-1 infection in nonactivated CD4 T cells. Thus, therapeutic strategies aimed at inducing the formation of LMM A3G complexes (Fig. 1B, number 3) or disrupting HMM A3G complexes (Fig. 1B, number 4) could render a cell resistant to productive HIV-1 infection. The correlation between IFN-α treatment, increased expression of A3G, and control of HBV infection suggests that treatments aimed at upreg-

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ulating A3G may also be beneficial. In support of this strategy, increased expression of A3G in dendritic cells as they mature coincides with the appearance of LMM A3G; mature dendritric cells are less permissive to HIV infection. However, simple upregulation of A3G may not exclusively lead to increased LMM A3G expression. Expression of A3G in CD4 T cells increases upon activation, but HMM complex formation is simultaneously induced.

Conclusions In the war against HIV, the APOBEC3 family of deoxycytidine deaminases has taken the field of modern retrovirology by storm. Although much has been learned in the past 4 years since the identification of A3G as an antiviral factor, even more remains to be deciphered. The nature of the HMM A3G complexes remains unknown, as do their functions. How the APOBEC3s potentially exert antiviral effects in the absence of deoxycytidine deamination is unclear. Moreover, very little is known about the interplay of the APOBEC3s and different retroelements. Finally, the potential of new antiviral drugs based on blocking Vif action or enabling LMM A3G function remains to be determined. What is certain, however, is that the next 4 years will be as exciting as the previous 4.

Acknowledgments Due to space limitations, the authors were unable to cite all the outstanding contributions from various laboratories in the Vif-APOBEC3 field. In lieu, they have cited only the three most recently published review papers.

References and Recommended Reading Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance

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APOBEC3G and HIV-1: Strike and Counterstrike Kao S, Miyagi E, Khan MA, et al.: Production of infectious human immunodeficiency virus type 1 does not require depletion of APOBEC3G from virus-producing cells. Retrovirology 2004, 1:27. 23. Mehle A, Goncalves J, Santa-Marta M, et al.: Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation. Genes Dev 2004, 18:2861–2866. 24.•• Chiu YL, Soros VB, Kreisberg JF, et al.: Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 2005, 435:108–114. This is the first demonstration that cellular A3G is regulated by assembly into large RNase-sensitive complexes, and moreover, that LMM A3G forms a potent post-entry restriction to HIV infection in resting CD4 T cells. 25. Kreisberg JF, Yonemoto W, Greene WC: Endogenous factors enhance HIV infection of tissue naive CD4 T cells by stimulating high molecular mass APOBEC3G complex formation. J Exp Med 2006, 203:865–870. 26.• Newman EN, Holmes RK, Craig HM, et al.: Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity. Curr Biol 2005, 15:166–170. The authors demonstrate that A3G mutants lacking wild-type deaminase activity retain some antiviral activity. This highlights a potential nonenzymatic mechanism of A3G antiviral activity. 27. Xu H, Svarovskaia ES, Barr R, et al.: A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion. Proc Natl Acad Sci U S A 2004, 101:5652–5657. 28. Wiegand HL, Doehle BP, Bogerd HP, Cullen BR: A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins. EMBO J 2004, 23:2451–2458. 29. Ribeiro AC, Maia e Silva A, Santa-Marta M, et al.: Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G. J Virol 2005, 79:823–833. 30. Mahieux R, Suspene R, Delebecque F, et al.: Extensive editing of a small fraction of human T-cell leukemia virus type 1 genomes by four APOBEC3 cytidine deaminases. J Gen Virol 2005, 86:2489–2494. 31. Navarro F, Bollman B, Chen H, et al.: Complementary function of the two catalytic domains of APOBEC3G. Virology 2005, 333:374–386. 32. Sasada A, Takaori-Kondo A, Shirakawa K, et al.: APOBEC3G targets human T-cell leukemia virus type 1. Retrovirology 2005, 2:32. 33. Gunther S, Sommer G, Plikat U, et al.: Naturally occurring hepatitis B virus genomes bearing the hallmarks of retroviral G→A hypermutation. Virology 1997, 235:104–108. 34. Suspene R, Guetard D, Henry M, et al.: Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci U S A 2005, 102:8321–8326. 35. Noguchi C, Ishino H, Tsuge M, et al.: G to A hypermutation of hepatitis B virus. Hepatology 2005, 41:626–633. 36. Rosler C, Kock J, Kann M, et al.: APOBEC-mediated interference with hepadnavirus production. Hepatology 2005, 42:301–309. 37. Rosler C, Kock J, Malim MH, et al.: Comment on “Inhibition of hepatitis B virus replication by APOBEC3G”. Science 2004, 305:1403. Author reply 1403. 38.• Turelli P, Mangeat B, Jost S, et al.: Inhibition of hepatitis B virus replication by APOBEC3G. Science 2004, 303:1829. This was the first report on the antiviral effects of A3G on a human retrovirus other than HIV and again raises the potential for a nonenzymatic mechanism of restriction. 22.

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