Antiviral Mechanism and Biochemical Basis of the Human APOBEC3 Family

MINI REVIEW ARTICLE published: 09 July 2012 doi: 10.3389/fmicb.2012.00250 Antiviral mechanism and biochemical basis of the human APOBEC3 family

Mayumi Imahashi 1,2, Masaaki Nakashima1,3 andYasumasa Iwatani 1,2*

1 Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan 2 Graduate School of Medicine, Nagoya University, Nagoya, Japan 3 Graduate School of Engineering, Nagoya University, Nagoya, Japan

Edited by: The human APOBEC3 (A3) family (A, B, C, DE, F,G, and H) comprises host defense factors Atsushi Koito, Kumamoto University, that potently inhibit the replication of diverse retroviruses, retrotransposons, and the other Japan viral pathogens. HIV-1 has a counterstrategy that includes expressing the Vif protein to Reviewed by: Harold Charles Smith, University of abrogate A3 antiviral function. Without Vif, A3 proteins, particularly APOBEC3G (A3G) and Rochester, USA APOBEC3F (A3F), inhibit HIV-1 replication by blocking reverse transcription and/or integra- Hiroshi Matsuo, University of tion and hypermutating nascent viral cDNA. The molecular mechanisms of this antiviral Minnesota, USA activity have been primarily attributed to two biochemical characteristics common to A3 *Correspondence: proteins: catalyzing cytidine deamination in single-stranded DNA (ssDNA) and a nucleic Yasumasa Iwatani, Laboratory of Infectious Diseases, Department of acid-binding capability that is speciﬁc to ssDNA or ssRNA. Recent advances suggest that Microbiology and Immunology, unique property of A3G dimer/oligomer formations, is also important for the modiﬁcation Clinical Research Center, National of antiviral activity. In this review article we summarize how A3 proteins, particularly A3G, Hospital Organization Nagoya Medical inhibit viral replication based on the biochemical and structural characteristics of the A3G Center, 4-1-1 San-no-Maru, Naka-ku, Nagoya, Aichi 460-0001, Japan. protein. e-mail: [email protected] Keywords: antiviral, APOBEC3, APOBEC3G, cytidine deaminase, HIV, retrovirus, reverse transcription,Vif

INTRODUCTION the A3G of African green monkeys (Bogerd et al., 2004; Mangeat Productive infections of primary human lymphocytes,monocytes, et al., 2004; Schrofelbauer et al., 2004; Xu et al., 2004). Subsequent and certain T-cell lines by HIV-1 require a virally encoded gene mutational analyses have confirmed that the 128DPD130 motif of product, Vif (originally named “Sor” or “A”; Fisher et al., 1987; A3G,located near the zinc-coordinating residues of NTD,is crucial Strebel et al., 1987). In early work on Vif, vif-deficient virions for direct binding to HIV-1 Vif (Huthoff and Malim, 2007; Russell produced in non-permissive cells were found to be significantly et al., 2009; Lavens et al., 2010). This motif is just downstream of impaired in their ability to complete reverse transcription (Sova residues 124YYFW127, which are involved in A3G’s ability to bind andVolsky, 1993; von Schwedler et al., 1993), and they were 100- to nucleic acids (Huthoff and Malim, 2007). 1000-fold less infectious than wild type (WT) virions (Fisher et al., The primary mechanism by which A3G inhibits vif-deficient 1987; Strebel et al., 1987; Fouchier et al., 1996). Sheehy et al. (2002) HIV-1 replication requires its expression in virus producer cells identified A3G as the cellular enzyme that restricts the replication and its incorporation into virions (Mariani et al., 2003; Marin of vif-deficient HIV-1. et al., 2003; Sheehy et al., 2003; Stopak et al., 2003; Svarovskaia The human A3G protein is a cellular cytidine deaminase et al., 2004). During reverse transcription in the target cells, the that belongs to the APOBEC3 family, which comprises seven virion-packaged A3G deaminates cytidine to uridine in the viral members (A3A, B, C, DE, F, G, and H; LaRue et al., 2009). minus-strand DNA (Harris et al., 2003a; Lecossier et al., 2003; These proteins contain one (A3A, A3C, and A3H) or two (A3B, Mangeat et al., 2003; Zhang et al., 2003; Suspène et al., 2004; A3DE, A3F, and A3G) zinc-cluster domains with the consensus Yu et al., 2004). Subsequent incorporation of adenines instead sequence (H/C)XE(X)23–28CXXC (Wedekind et al., 2003). Among of guanines in the plus-strand results in extensive G-to-A hyper- the APOBEC3 family members, A3G is the most potent inhibitor mutation and inactivation of the viral genome. Shortly after A3G of HIV-1 but only in the absence of Vif. HIV-1Vif counteracts A3G was suggested as the key restriction factor against vif-deficient by promoting its polyubiquitination through the recruitment of a HIV-1, it was proposed that A3G-mediated deamination might be Cullin5-based E3 ubiquitin ligase complex (Yu et al., 2003), which a lethal trigger, eventually leading to the degradation of reverse- targets A3G proteins for rapid proteasomal degradation in infected transcribed viral DNA through a base-excision pathway and/or the cells. reduced replication of progeny viruses by introducing premature The specific A3G degradation is determined by the capability of stop codons and/or amino acid changes (Cullen, 2003; Goff, 2003; Vif to bind with A3G in the E3 ubiquitin ligase complex (reviewed Harris et al., 2003a,b; KewalRamani and Coffin, 2003). Indeed, the in Kitamura et al., 2011). The region in A3G responsible for HIV-1 catalytic center of the A3G protein is clearly essential for its antivi- Vif interaction was identified by the studies on the species speci- ral functions (Mangeat et al., 2003; Navarro et al., 2005; Iwatani ficity of Vif-mediated A3G degradation, which is determined by a et al., 2006; Browne et al., 2009). However, several lines of recent single amino acid difference in human A3G, D128 versus K128 in evidence have indicated that the catalytic activity of A3G is not

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sufficient to explain its full antiviral activity. What is the funda- 2008), as illustrated in Figure 1B. The apparent equilibrium dis- mental mechanism(s) of A3G antiviral activity that explains the sociation constant (Kd) for A3G (to ssDNA) is between 52 and observation by von Schwedler et al.(1993), that the reverse tran- 238 nM (Chelico et al., 2006; Iwatani et al., 2006, 2007), whereas scription of vif-deficient HIV-1 is impaired when produced from the Kd for HIV-1 nucleocapsid protein (NC) binding to RNA A3G-expressing “non-permissive” cells? is approximately 23–320 nM (Shubsda et al., 2002; Levin et al., 2005), suggesting that the nucleic acid-binding affinity of A3G BIOCHEMICAL PROPERTIES OF A3G is as high as that of NC. A3G binds preferentially to ssDNA or The zinc coordination of A3 family proteins is mediated by a ssRNA (Figure 1B; Yu et al., 2004; Iwatani et al., 2006; Shlyakht- histidine and two cysteines, which form a catalytic center for enko et al., 2011), especially dT or dU residues of ssDNA and cytidine deaminase activity. In A3G, the zinc-binding motif at AU-rich regions in ssRNA,respectively (Jarmuz et al.,2002; Iwatani the C-terminal domain (CTD) is primarily associated with cyti- et al., 2006). Interestingly, the substrate specificities and nucleotide dine deaminase catalysis whereas the N-terminal domain (NTD) preferences of the A3G protein differ for its deaminase and nucleic does not catalyze deamination (Figure 1A; Haché et al., 2005; acid-binding activities, as is the case for APO1 (Anant et al., 1995; Navarro et al., 2005; Iwatani et al., 2006; Friew et al., 2009). The Navaratnam et al., 1995; Anant and Davidson, 2000). Because A3G enzyme converts deoxycytidine (dC) residues to deoxyuri- mutations that disrupt zinc coordination at the NTD, such as dine (dU), and acts preferentially on residues that are preceded the substitution of the C100 residue with a serine, abrogate the by another dC, with a much higher preference for the 50-CCCA- nucleic acid-binding affinity of A3G (Navarro et al., 2005; Iwatani 30 sequence in single-stranded DNA (ssDNA; Beale et al., 2004; et al., 2006), some local conformation near the zinc coordination Suspène et al., 2004;Yu et al., 2004). During retroviral reverse tran- of NTD might be responsible for its recognition of single-stranded scription, A3G deaminates dC to dU in the viral minus-stranded nucleotides. DNA, and the subsequent incorporation of deoxyadenines (dA) The formation of an A3G homo-multimer is the third unique instead of deoxyguanines (dG) in the plus-strand results in G-to- feature of A3G. The intrinsic propensity of A3G multimerization A hypermutation of the nascent viral DNA (Harris et al., 2003a; has been verified by biochemical and structural studies (Jarmuz Lecossier et al., 2003; Mangeat et al., 2003; Zhang et al., 2003; et al., 2002; Navarro et al., 2005; Burnett and Spearman, 2007; Suspène et al., 2004; Yu et al., 2004). The ssDNA-specific deami- Bennett et al., 2008; Bulliard et al., 2009; Friew et al., 2009; nation by A3G appears to be determined by a structural groove, Huthoff et al., 2009; Chelico et al., 2010; McDougall et al., 2011). presumably accommodating ssDNA, that positions the cytosine Because the full-length A3G structure has not been determined, for deamination (Chen et al., 2008; Holden et al., 2008). the A3G interface for multimerization remain unclear. However, The nucleic acid-binding property of A3G is also a major bio- structural analyses by SAXS (small-angle X-ray scattering), co- chemical feature. The minimum unit of A3G for binding to ssDNA immunoprecipitation assays (Wedekind et al., 2006; Bennett et al., is a monomer (Chelico et al., 2010) and/or a dimer (Bennett et al., 2008), and X-ray crystallography (Shandilya et al., 2010) have

FIGURE 1 | Biochemical characteristics of A3G. (A) A3G consists of DNA (ssDNA) or RNA (ssRNA) but not to double-stranded nucleotides. an NTD and CTD. The NTD is responsible for the nucleic acid-binding A monomeric and/or dimeric A3G bind to ssDNA or ssRNA as the affinity of A3G and has no detectable deaminase activity. In contrast, minimum unit. (C) The A3G protein forms homodimers or higher-order the CTD is solely involved in deaminase activity and has less affinity for homo-oligomers through interactions between its NTDs (head–head) or nucleic acids than the NTD. (B) A3G specifically binds to single-stranded CTDs (tail–tail).

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demonstrated an interaction between the A3G CTDs (tail–tail), as “dead” zone for A3G-mediated deamination (Chelico et al., 2008, illustrated in Figure 1C. In addition, homo-dimerization through 2010). the NTDs also occurs (head–head),as shown in Figure 1C,and this It was hypothesized that the antiviral functions of A3G might interaction appears to depend on the presence of RNA (Friew et al., be associated with the uracilation of the nascent reverse transcripts 2009; Huthoff et al., 2009). These observations were supported by (Harris et al., 2003a; Zhang et al., 2003), resulting in their degrada- an analytical ultracentrifugation study that showed a predominant tion through the activity of cellular DNA glycosylases, e.g., UDG2 dimer form of A3G in equilibrium with minor monomeric and (uracil DNA glycosidase 2) and SMUG1 (single-strand selective tetrameric species under RNA-depleted conditions (Salter et al., monofunctional uracil DNA glycosylase). However, several groups 2009). have revealed that neither uracil DNA glycosidase affected the Chelico et al.(2006, 2008, 2010) have used an in vitro system antiviral effect of A3G (Kaiser and Emerman, 2006; Mbisa et al., to demonstrate that A3G has a 30 to 50 catalytic orientation speci- 2007; Langlois and Neuberger, 2008), although one study showed ficity for the deamination of naked ssDNA (Feng and Chelico, that UDG2 is involved in the degradation of nascent reverse tran- 2011). The preferred asymmetric direction for A3G catalysis likely scripts (Yang et al., 2007). In addition, we cannot exclude the yields an approximately 30-nt “dead” zone located at the 30 end possibility that other unidentified DNA repair enzymes might of ssDNA that is much less efficiently deaminated by A3G (Che- participate in the degradation mechanism. Therefore, further lico et al., 2008, 2010). However, we need further investigations studies will be required to elucidate the potential factors that pre- on how significant the length of dead zone is because the cen- cede the degradation of uracilated DNA following A3G-mediated tral CCC motif of ∼40-nt ssDNA can be deaminated efficiently deamination (“4” in Figure 2). by A3G in our or the other in vitro deamination assay systems Earlier studies on A3G suggested that G-to-A hypermutation (Beale et al., 2004; Yu et al., 2004; Iwatani et al., 2006). The for- resulting in lethal mutations was the sole basis of the A3G antivi- mation of tetramers and higher-order homo-oligomers of A3G ral mechanism. However, more recent studies have demonstrated on ssDNA is required for efficient deamination (McDougall et al., that the catalytic activity of A3G may not wholly determine its 2011). molecular mechanism, i.e., a deaminase–independent mechanism might also be involved in A3G antiviral activity: (i) mutations of ANTIVIRAL MECHANISMS OF A3G AGAINST VIF -DEFICIENT the catalytic center do not completely abolish antiviral activity HIV-1 against HIV-1 (Navarro et al., 2005; Iwatani et al., 2006; Holmes A3G exerts its inhibitory activity by being encapsidated into virus et al., 2007; Luo et al., 2007; Miyagi et al., 2008); (ii) A3G inhibits particles of vif-deficient HIV-1. During the subsequent infection replication of hepatitis B virus without detecting significant G-to- cycle, A3G has been proposed to interfere with reverse tran- A hypermutation (Turelli et al., 2004; Bonvin and Greeve, 2007; scription and/or integration through one or more molecular Nguyen et al.,2007; reviewed in Bonvin and Greeve,2008); and (iii) mechanisms (Figure 2). Based on whether the enzymatic activ- other A3 proteins block the replication of HIV-1 (Luo et al., 2007; ity is involved or not, there are two separable mechanisms, i.e., Miyagi et al., 2010), mouse mammary tumor virus (Okeoma et al., deaminase-dependent and -independent mechanisms. 2007), murine leukemia virus (Takeda et al., 2008), and parvovirus Although the catalytic center of A3G is clearly critical for its adeno-associated virus (Narvaiza et al., 2009) and retrotranspo- antiviral effect (Mangeat et al., 2003; Navarro et al., 2005; Iwatani sition of LINE-1 and Alu (Bogerd et al., 2006; Muckenfuss et al., et al., 2006; Browne et al., 2009), the precise molecular mech- 2006; Stenglein and Harris, 2006; Wissing et al., 2011) despite the anisms underlying the inhibition of the further processing of absence of editing activity. A3G-deaminated DNA products in cells remain unclear. A3G- Several groups have reported that the deaminase-independent mediated hypermutation of viral genomes is clearly detrimental mechanisms of reverse transcription inhibition would involve to further spreading the infection because mutations in the viral interference with tRNA primer annealing, initiation and elonga- structural and/or the regulatory genes may trigger defects in the tion of DNA synthesis, and minus-/plus-strand transfer reactions production of infectious progeny virus (“1” in Figure 2). For (Guo et al., 2006, 2007; Iwatani et al., 2007; Li et al., 2007; Luo example, because the preferred sequences of A3G include TGG et al., 2007; Mbisa et al., 2007; Anderson and Hope, 2008; Bishop (a codon for tryptophan within the viral orf), many G-to-A muta- et al., 2008; Zhang et al., 2008). Using enzymatically active recom- tions may incidentally produce premature stop codons, such as binant A3G and the in vitro reconstituted systems of HIV-1 reverse TAG (or TGA), resulting in viral inactivation (Simon et al., 2005; transcription, it has been demonstrated that A3G blocks all RT- Pace et al., 2006). catalyzed DNA elongation processes in a deaminase-independent In its second mechanism, A3G reduces the efficiency and speci- manner, although the protein does not significantly interfere with ficity of primer tRNA processing and removal, resulting in proviral tRNA primer placement (Iwatani et al.,2007). Moreover,the analy- DNA ends that are aberrant substrates for integration and plus- sis of endogenous reverse transcription in cell-free HIV-1 particles strand DNA transfer (Luo et al., 2007; Mbisa et al., 2007; “2” in also indicated that A3G reduces HIV-1 viral DNA levels by inhibit- Figure 2). In this mechanism, the presumed deamination sites are ing the elongation of reverse transcription rather than by inducing located at the plus primer-binding site (PBS), which is annealed the degradation of the reverse transcripts (Bishop et al., 2008). by the tRNA. Considering the biochemical characteristics of A3G, The block of RT elongation by A3G might be attributed to A3G’s it remains unclear how the A3G enzyme deaminates cytidine unique nucleic acid-binding ability (Iwatani et al., 2007). More residues on the DNA/RNA duplex and near the 30 end of the plus- recently, Wang et al.(2012) have observed physiological and func- strand transfer donor DNA, which is supposed to be a presumed tional interactions between RT and A3G, although our group

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FIGURE 2 | A3G blocks the reverse transcription and/or integration of A3G-mediated editing might create aberrant structures at viral DNA ends, vif -deficient HIV-1. Packaging of A3G proteins into vif -deficient virus which might be inefficient substrates for integration. (3) The reverse particles is prerequisite for the inhibition of viral replication by A3G. Upon the transcripts containing dU might induce DNA degradation by cellular DNA infection of target cells, A3G blocks the post-entry step of viral replication by repair pathways. (4) RT-mediated elongation could be blocked by the presence one or more of the following mechanisms: (1) Cytidine deamination of of A3G on RNA or DNA templates. A3G might exert both deaminase nascent reverse transcripts by A3G enzymes could prevent progeny virus activity-dependent (1–3) and deaminase activity-independent (4) functions to production due to inactivating mutations in viral genes and/or proteins. (2) inhibit vif -deficient HIV-1 replication. has been unable to detect direct interactions using recombinant is possible that the deaminase activity of A3G is largely required for A3G and RT proteins (Iwatani, Y., and Levin, J. G., unpublished its antiviral activity against vif-deficient HIV-1, although it is not observations). It might be interesting to know whether direct inter- known whether A3G-mediated deamination and/or the architec- action is applicable to other A3 proteins and/or retroviral RTs, i.e., ture of the catalytic center in A3G are intrinsically required for its how the broad range of A3G’s inhibitory effect can be linked to inhibitory activity. Further investigations will provide the funda- the specific interaction between RT and A3G. Further investiga- mental answers to explain the first observation by von Schwedler tions are required to understand the molecular mechanisms of the et al., made when A3G had not yet been discovered. deaminase-independent pathway in more detail. ACKNOWLEDGMENTS CONCLUSIONS We thank Dr Judith G. Levin (NIH, NICHD) for helpful dis- Studies over the past 10 years have established that human cussions. This work was supported in part by a grant-in-aid for APOBEC3 family proteins potently restrict retroviral replication. scientific research from the Ministry of Education, Culture, Sports, However, the molecular mechanisms of the A3 family’s antiviral Science, and Technology of Japan and by a grant for HIV/AIDS activities remain unclear. Recent biochemical studies of A3G may research from the Ministry of Health, Labor, and Welfare of provide a better understanding of these mechanisms. Currently, it Japan.

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Yu, X., Yu, Y., Liu, B., Luo, K., Kong, Zhang, L., Saadatmand, J., Li, X., Guo, commercial or financial relationships This article was submitted to Frontiers W., Mao, P., and Yu, X. F. (2003). F., Niu, M., Jiang, J., Kleiman, that could be construed as a potential in Virology, a specialty of Frontiers in Induction of APOBEC3G ubiquiti- L., and Cen, S. (2008). Function conflict of interest. Microbiology. nation and degradation by an HIV-1 analysis of sequences in human Copyright © 2012 Imahashi, Nakashima Vif-Cul5-SCF complex. Science 302, APOBEC3G involved in Vif- and Iwatani. This is an open-access arti- 1056–1060. mediated degradation. Virology 370, Received: 29 May 2012; accepted: 21 June cle distributed under the terms of the Zhang, H., Yang, B., Pomerantz, R. J., 113–121. 2012; published online: 09 July 2012. Creative Commons Attribution License, Zhang, C., Arunachalam, S. C., and Citation: Imahashi M, Nakashima M which permits use, distribution and Gao, L. (2003). The cytidine deami- and Iwatani Y (2012) Antiviral mecha- reproduction in other forums, provided nase CEM15 induces hypermutation Conflict of Interest Statement: The nism and biochemical basis of the human the original authors and source are cred- in newly synthesized HIV-1 DNA. authors declare that the research was APOBEC3 family. Front. Microbio. ited and subject to any copyright notices Nature 424, 94–98. conducted in the absence of any 3:250. doi: 10.3389/fmicb.2012.00250 concerning any third-party graphics etc.