Functional analysis of the two cytidine deaminase domains in APOBEC3G

Virology 414 (2011) 130–136

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Virology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o

Functional analysis of the two cytidine deaminase domains in APOBEC3G Xiaoyu Li a,1, Jing Ma a,d,e,1, Quan Zhang a,1, Jinming Zhou a, Xiao Yin a, Congjie Zhai a, Xuefu You a, Liyan Yu a, Fei Guo b, Lixun Zhao a, Zelin Li c, Yi Zeng c, Shan Cen a,d,e,⁎ a

Institute of Medicinal Biotechnology, Beijing, China State Key Lab for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Science and Peking Union Medical School, Beijing, China The College of Life Science and Bio-engineering, Beijing University of Technology, Beijing, China d Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada e Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada b c

a r t i c l e

i n f o

Article history: Received 10 December 2010 Returned to author for revision 19 January 2011 Accepted 18 March 2011 Available online 13 April 2011 Keywords: HIV-1 APOBEC3G Vif Cytidine deaminase Encapsidation Degradation Antiviral

a b s t r a c t Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity. © 2011 Elsevier Inc. All rights reserved.

Introduction Lentiviruses, with the exception of equine infectious anemia virus, all encode a 23 kDa accessory protein Vif (virion infectivity factor) (Dettenhofer et al., 2000; Fisher et al., 1987; Gabuzda et al., 1992; Gibbs et al., 1994; Kan et al., 1986; Lee et al., 1986; Madani and Kabat, 1998; Simon et al., 1998; Simon and Malim, 1996; Sodroski et al., 1986). Vif is an important regulator of viral infection, which is required for the human immunodeficiency virus type 1 (HIV-1) to replicate in certain “non-permissive” cell types, such as primary T lymphocytes and H9, but is not required in other “permissive” cell types, such as SupT1 and Jurkat cells (Fisher et al., 1987; Gabuzda et al., 1992; Strebel et al., 1987). Non-permissive cells contain a host cellular factor called human APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G, hereafter referred to as hA3G), which prevents HIV-1 replication in the absence of Vif (Sheehy et al., 2002). hA3G is incorporated into HIV-1 virions during virus assembly and inhibits viral replication in the newly infected cells. Vif is able to bind to hA3G ⁎ Corresponding author at: Institute of Medicinal Biotechnology, Chinese Academy of Medical Science, Beijing 100050, China. Fax: + 86 10 63017302. E-mail addresses: [email protected], [email protected] (S. Cen). 1 These authors contributed equally to this work. 0042-6822/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2011.03.014

(Mariani et al., 2003) and can reduce both the cellular expression of hA3G and its incorporation into virions (Kao et al., 2003; Stopak et al., 2003). Several lines of evidence have demonstrated that Vif induces the rapid degradation of hA3G by a proteasome-dependent mechanism (Yu et al., 2003). APOBEC3G belongs to an APOBEC superfamily containing at least 11 members, including activation-induced deaminase (AID), APOBEC1, APOBEC2, APOBEC3A–H, and APOBEC4 (Conticello et al., 2005; Harris and Liddament, 2004; OhAinle et al., 2006; Rogozin et al., 2005). Each APOBEC protein contains one or two copies of cytidine deaminase domain (CD, a conserved His-X-Glu and Cys-X-X-Cys Zn2+ coordination motif). Seven APOBEC3 proteins have been identified as innate inhibitors of retrovirus replication and retrotransposon mobility (Cullen, 2006). In contrast to hA3A, hA3C and hA3H, which have single catalytic domain, hA3G contains two tandem CD domains and it is proven to be a more potent inhibitor of HIV-1 infectivity than other APOBEC3 proteins. While several reports have shown that the two CD sof hA3G display quite distinct functional properties—the N-terminal CD1 mediates RNA binding and virion encapsidation, and the C-terminal CD2 conducts deaminase catalytic activity (Hache et al., 2005; Iwatani et al., 2006; Navarro et al., 2005; Newman et al., 2005), there is still a debate on the role of the CDs in the biological function of hA3G. For instance, the Nterminal truncations of hA3G, in which the CD1 domain is missing, are

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able to be packaged into HIV-1 virions (Cen et al., 2004; Luo et al., 2004; Zennou et al., 2004), whereas mutations in conserved residues in CD1 prevent virion encapsidation of hA3G (Navarro et al., 2005). An intact CD2 domain retained deaminase activity in an Escherichia coli reporter system but failed to mutate viral genome (Chen et al., 2008). To further characterize the two CDs of hA3G, we have herein constructed three hA3G mutants: CD1-1 (containing duplicate CD1 domain), CD2-2 (containing duplicate CD2 domain) and CD2-1 (containing position switched CD1 and CD2 domain) and investigated the effect of CD domain switch or replacement upon viral incorporation, Vif-mediated degradation and antiviral function of hA3G. Result The incorporation of CD mutants into HIV-1 The CD1 and CD2 domains share high homology (Fig. 1A), and they are predicted to adopt similar structures (Jarmuz et al., 2002). Using fusion PCR, we have constructed three hA3G mutants containing a Cterminal HA tag (Fig. 1A): CD1-1, with the C-terminal CD domain (CD2) replaced with the identical N-terminal one (CD1); CD2-2, with the CD1 domain replaced with the identical CD2 domain; and CD2-1, with the position of CD1 and CD2 domain switched (Fig. 1A). We first checked the viral encapsidation of the three mutants. 293T cells were co-transfected with plasmids containing vif deficient HIV-1 proviral DNA and plasmids containing different wild type or mutant hA3G. The cell lysates of transfected cells were analyzed by Western blots (Fig. 1B), using antiHA (top panel) and anti-β-actin (bottom panel) antibodies as probes. Both wild type and mutant hA3G were expressed at similar levels in the cytoplasm. The viruses produced from these cells were purified and were also analyzed by Western blotting (Fig. 1C), using anti-HA (top panel) and anti-CAp24 (bottom panel). The result shows that all hA3G mutants were incorporated into the virion at levels similar to wild type hA3G. It indicates that the replacement or switch of CDs has no effect on hA3G viral incorporation. It is known that HIV-1 nucleocapsid (NC) domain within Gag is required for hA3G packaging likely through an RNA-mediated mechanism (Svarovskaia et al., 2004). To determine whether the mutated forms of hA3G were incorporated into HIV through the same mechanism as wild type hA3G does, we checked whether the packaging of hA3G mutants into virions requires the presence of NC domain and whether their interactions with HIV-1 Gag is RNA dependent. ZWt-p6.vif− encodes a Vif deficient full-length HIV-1 genome, in which the nucleocapsid sequence has been replaced with a yeast leucine zipper domain (Accola et al., 2000). It has been shown that the mutated HIV-1 Gag is able to assemble into virus like particle but fail to incorporate hA3G (Accola et al., 2000). Fig. 1D showed that, in contrast to BH10.Vif-, ZWt-p6.Vif− almost lost its ability to incorporate hA3G or its mutants. We next transfected 293T cells with the plasmid containing hGag and APOBEC3G and treated the cell lysates with RNase prior to immunoprecipitation with the anti-HA antibody. The precipitated proteins were analyzed by Western blotting using the anti-p24 antibody. The result shows that the RNase treatment diminished the co-immunoprecipitation of hA3G or its variants with Gag (Fig. 1E). These results indicate that virion encapsidation of the three mutants required the NC domain and that their interactions with HIV-1 Gag are sensitive to RNase, suggesting that the mutated forms of hA3G are incorporated into HIV through the same mechanism as wild type hA3G does.

Fig. 1. The incorporation of hA3G and its CD mutants into HIV-1. A. Illustration of the wild-type and mutated hA3G. The top drawing shows the sequence of two zinc coordination motif, CD1 and CD2; the lower drawing shows the wild-type and mutant hA3G, with numbers representing the amino acid positions. B. 293T cells were cotransfected with plasmids containing vif deficient HIV-1 proviral DNA and plasmids containing different wild type or mutant hA3G. The plasmids used are listed along the top of each panel and described under “Experimental procedures”. Western blots of cell lysates were probed with anti-HA (top panel), anti-β-actin (bottom panel) antibodies. C. Viruses produced by the 293T cells were purified and lysed in radioimmune precipitation assay buffer. The lysate was analyzed by Western blots and was probed with either anti-HA (upper panel) or anti-CA (lower panel) antibodies. D. 293T cells were co-transfected with wild type or mutant hA3G expression vector and plasmids BH10.vif− or ZWt-p6.Vif− proviral DNA. Viruses produced by the cells were purified, lysed and analyzed by Western blots. E. The cell lysates were subjected to RNase treatment, followed by immunoprecipitation with the anti-HA antibody. The precipitated proteins were analyzed by Western blotting using the anti-Gag antibody.

The interaction between HIV-1 Vif and hA3G CD mutants The current consensus view is that HIV-1 Vif protein interacts with hA3G as a part of a Vif–Cul5–SCF complex, thus resulting in polyubiquitination and proteosomal degradation of hA3G (Yu et al., 2003). Previous studies have shown that the Vif/hA3G interaction

involves the N-terminal region of the linker sequence within hA3G (Conticello et al., 2003; Marin et al., 2003; Zhang et al., 2008), while whether the motifs located outside of the region, especially the two zinc coordination motifs, affect Vif-mediated degradation is not known.

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To address the question, 293T cells were co-transfected with plasmids coding HIV-1 vif and plasmids containing either wild type or mutant hA3G. Western blot of cell lysates showed that the expression of wild type and variant CD1-1 was greatly reduced in the presence of Vif, while both variant CD2-2 and CD2-1 were relatively resistant to Vif (Fig. 2A). Since the hA3G/Vif interaction is a prerequisite for the degradation of hA3G, we next investigated if the resistance of CD2-2 and CD2-1 to Vif resulted from their inabilities to binding with Vif. 293T cells were transfected as described above and then treated with MG132, a proteasome inhibitor, at 24 h post-transfection. The lysates of transfected cells were analyzed by Western blots (Fig. 2B). The abilities of the different hA3G variants to bind to Vif were assessed by co-immunoprecipitation with mouse anti-HA and detected by rabbit anti-vif as shown in Fig. 2C. We found a similar amount of Vif present in the immunoprecipitate of the hA3G mutant, while CD2-2 and CD21 showed increasing cellular content, which may reflect weaker interaction of Vif with CD2-2 and CD2-1 than wild type hA3G. This

indicates that domain replacement or switch of CDs has no significant effect in hA3G/Vif interaction, whereas replacing CD1 with CD2 impairs the sensitivity of hA3G to Vif. Cytidine deamination activity of CD mutants Several groups previously reported that the CD2 of hA3G is responsible for its deamination activity and replacement of the conserved cystine, histidine or glutamic amino acid in CD2 domain did not affect its viral incorporation but greatly impaired its catalytic activity (Hache et al., 2005; Iwatani et al., 2006; Navarro et al., 2005; Newman et al., 2005). To determine whether the CD domain switch or replacement affect the editing activity of hA3G, SupT1 cells were infected by the virions containing wild type or CD mutated hA3G, and then the proviral DNA was extracted from these cells 48 h postinfection. PCR products representing the BH10 DNA sequence 492– 764 (containing the R, U5 and part of gag region) were sequenced and examined for mutations. Compared to hA3G which caused 1.76 G to A mutations per 100 bases in viral cDNA, the variant CD2-1 retains considerable deamination activity which caused 0.51 G to A mutations per 100 base bases (Fig. 3A), whereas the variants CD1-1 and CD2-2 completely lost their deamination activity. Interestingly, we found that the switch of the CD domain affected nucleotide sequence preference pattern of the deaminase. The preferred sequence context for wild type hA3G is GG (where the edited is underlined) which can be mutated at frequencies N91%, while the mutation frequency of other sequence context which differ in + 1 position such as GA, GT and GC is below 5%. However, in CD2-1 induced G to A mutations, although the sequence context of GG is still the most preferred context for CD2-1(N84%), the sequence context of GT is significantly increased to over 14% (Fig. 3B). We next investigated if the mutations affect sequence preference in −1 position of the edited guanosine. As shown in Fig. 3C, the result reveals that the sequence preference of either hA3G or CD2-1 did not correlated with frequency of the four kinds of GG context in HIV-1 genome (AGG (29.6%), TGG (25.9%), CGG (13.0%) and GGG (31.5%)). The TGG is preferred by wild type hA3G, which covers 56% of GG context mutations while other three kinds of GG context only cover 8.8%–23.5%. However, in CD2-1 variant induced mutations, both TGG and GGG are preferred, which covers approximately 45.5% and 49.3% respectively (Fig. 3C). These data demonstrated that switch of CD domain of hA3G greatly affected its sequence preference in −1 position of the edited guanosine. Anti-viral activity of zinc coordination motif mutants

Fig. 2. The interaction between HIV-1 Vif and hA3G CD mutants. 293T cells were cotransfected with wild type or CD mutant hA3G expression vectors and plasmid coding for HIV-1 Vif protein. Interaction between Vif and hA3G CD mutants was measured by co-immunoprecipitating these molecules from cell lysate with anti-HA. A. The cell lysates were probed with anti-HA (top panel), anti-Vif (bottom panel) antibodies. B. In the presence of MG132, Western blots of cell lysate were probed with anti-HA (top panel), anti-Vif (bottom panel) antibodies. C. The cell lysates were incubated with the mouse anti-HA antibody and then detected by rabbit anti-HA (upper panel) and antiVif (lower panel) antibodies.

Wild type hA3G and CD mutants were next examined for their inhibitory effect on vif-deficient HIV-1 in a single-round infection assay (Fig. 4A). Compared to the wild-type hA3G, the antiviral activities of the three CD mutants were significantly decreased: CD1-1 and CD2-2 retained about 27.3% and 10.0% of inhibition activity respectively, whereas CD2-1 completely lost its anti-HIV activity. Previous studies have shown that, in addition to mutate viral cDNA, hA3G inhibits HIV-1 replication through non-deaminating mechanism, e.g. inhibition of viral DNA synthesis and integration (Bishop et al., 2008; Li et al., 2007; Luo et al., 2007). Therefore, we next investigated whether anti-viral activity of CD1-1 and CD2-2, two deaminating activity deficient hA3G mutants, is derived from the inhibition of viral DNA synthesis. Real-time PCR quantitation of newly synthesized HIV-1 DNA shows that, in the presence of CD1-1 and CD2-2, the late DNA synthesis is decreased 71.0% and 54.3% respectively, whereas CD2-1 had little effect to viral DNA synthesis (Fig. 4B). These data suggested that the antiviral activity of CD1-1 and CD2-2 may result from the inhibition of viral DNA synthesis via a non-deaminating mechanism described previously (Bishop et al., 2008; Li et al., 2007; Luo et al., 2007).

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% sequences Fig. 3. Deamination activity of wild type and mutant hA3G. 293T cells were cotransfected with plasmids containing vif deficient HIV-1 proviral DNA and different wild type or mutant hA3G plasmids. DNA was extracted from these cells 16 h later, and PCR products representing the BH10 DNA sequence 469–699 (containing sequences starting form R region and ending in gag region) were sequenced and examined for mutations. A. The frequency of G to A mutation in the presence of wild type or mutant hA3G in Vif deficient proviral HIV-1. B. Comparison of the preferred sequence in + 1 position relative to the substrate G. C. Comparison of the preferred sequence in −1 position relative to the substrate G.

Discussion APOBEC3 genes are specific to mammals and are organized in a tandem array between two vertebrate-conserved flanking genes, CBX6 and CBX7 (LaRue et al., 2008). Seven human APOBEC3 genes are located chromosome 22 at q13.2 in a head-to-tail manner. One study based on phylogenetic analyses suggests that the ancestral members of APOBEC3 have only one CD which might be derived from AID or

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Fig. 4. Antiviral function of hA3G mutant proteins. A. Single cycle HIV-1 infectivity assay on TZM-bl reporter cell lines. 293T cells were co-transfected with plasmids containing vif deficient HIV-1 proviral DNA and different wild type or mutant hA3G plasmids. Culture supernatants were collected 48 h post-infection and quantified by measuring amounts of p24. The levels of infectious viruses were determined by infecting the TZMbl indicator cells. B. qPCR quantification of newly synthesized late viral DNA. DNA was extracted from SupT1 cells at 16 h post-infection with vif deficient HIV-1 containing wild type or mutant hA3G. Late (U5-gag) viral cDNA production was monitored by qPCR, as described in Experimental procedures.

APOBEC2 (Conticello et al., 2005) and then formed an APOBEC3 subfamily through gene duplication under significant selective pressure which might be related to prevention human endogenous retroviruses and retrotransposons from causing genomic instability (Sawyer et al., 2004; Wedekind et al., 2003; Zhang and Webb, 2004). While four APOBEC3 proteins (hA3B, hA3DE, hA3F, and hA3G) have two CD domains, hA3A, hA3C, and hA3H only have one deaminase motif. Whether duplication of the CD domain is related to its inhibitory activity has remained unclear. Indeed, some reports indicated that single domain APOBEC3 proteins show a very weak anti-viral activity of HIV (Yu et al., 2004). However, single domain APOBEC3 protein hA3A potently inhibits parvovirus adeno-associated virus (AAV) as well as endogenous retroelements (Chen et al., 2006), and recent findings also showed that single domain A3H is a potent inhibitor of HIV (Dang et al., 2008; Harari et al., 2009; Zhen et al., 2010).

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The CD1, but not CD2, of hA3G has been reported to mediate RNA binding and virion encapsidation (Iwatani et al., 2006; Navarro et al., 2005; Newman et al., 2005), and site mutation or deletion of conserved amino acids in CD1 prevents hA3G encapsidation (Friew et al., 2009; Navarro et al., 2005). However, our work showed here that the hA3G variants CD2-2 was able to be packaged efficiently into HIV-1 through a mechanism that wild-type hA3G dose. These data suggest that the CD1 domain is not required for viral incorporation of hA3G, or its role if any is not in a sequence specific manner. One explanation for this controversy is that the CD1 may be involved in viral incorporation of hA3G indirectly, e.g. maintain a proper conformation that is required for the hA3G/Gag interaction. It is possible that, in contrast to mutating conserved amino acids in CD1, switching structural homology CDs does not impact on the integral configuration of the protein, thereby having no effect upon its viral incorporation. In agreement with this hypothesis, several key residues of hA3G essential for its viral incorporation have been mapped in a linker region between the two CDs (Khan et al., 2009; Zhang et al., 2007), and several group also reported that various motifs outside of CD1 of APOBEC3 proteins have been reported to mediate RNA binding and virion targeting (Huthoff et al., 2009; Wang et al., 2007, 2008; Zhang et al., 2010). In addition, our previous study demonstrates that the linker region is required for hA3G viral incorporation, and removal of the N-terminal 104 amino acids (including the CD1 domain) does not affect the ability of hA3G to be packaged into Gag VLPs (Cen et al., 2004). One common feature of APOBEC family members is that they all possess single strand DNA/RNA deamination activity. Several groups previously reported that C-terminal CD2 domain of hA3G exhibits its deaminase catalytic activity (Hache et al., 2005; Iwatani et al., 2006; Navarro et al., 2005; Newman et al., 2005); it is therefore expected that the hA3G variant CD1-1 did not show any deamination activity (Fig. 4). Surprisingly, the h3G variant CD2-2 which contains two catalytic CD2 domains was also unable to mutate HIV-1 cDNA, even though its viral content was the same as wild type hA3G (Fig. 1). Our previous work has shown that hA3G C-terminal fragment (104–384) missing CD1 is unable to create G–A deamination mutations on viral cDNA (Guo et al., 2006). These results imply that, in addition to CD2, CD1 is also required for editing HIV-1 cDNA. In agreement with this, we did find that, among the three hA3G variants tested, CD2-1 is the sole mutant that possesses considerable deamination activity and contains both the CD domains. Furthermore, our data indicate that CD domain switch affected the local nucleotide sequence preferences of hA3G. Although GG context remains the most preferred hot mutation spot for CD2-1, the mutation created in GT sequence context increased significantly compared with wild type hA3G. The data also show that, in contrast to hA3G which prefers T in −1 positions relative to the substrate G, and 56% G–A mutations are shown in TGG context, CD2-1 variant prefers both GGG (49%) and TGG (46%). It has been reported that the sequence preference is determined by the CD2 domain of APOBEC proteins (Hache et al., 2005); our result demonstrates here that factors other than catalytic site itself of hA3G are also involved. Indeed, previous studies have shown subtle shifts in specificity of RNA editing when site-directed mutations at positions outside of CD2 in APOBEC3 proteins, e.g., D311Y mutation of hA3F (Kohli et al., 2009), D316R/ D317R double mutant of hA3G (Rausch et al., 2009). A recent work has identified a recognition loop of 9–11 amino acids located downstream of catalytic site of APOBEC proteins, which contributes significantly to the distinct sequence motifs of individual family members (Kohli et al., 2009). It is possible that this recognition loop, which is grafted from the downstream of CD2 into the corresponding sequence of CD1, causes the change in mutational signature of CD2-1. Although CD2-1 retains 30% of deamination activity of wild type hA3G, this variant completely lost its inhibition activity. On the contrary, the variant CD1-1 that has no editing activity retains about

27.3% anti-HIV-1 activity. This result suggests that although the deaminase activity of hA3G plays an important role in the inhibition of HIV-1 replication, the editing-independent antiviral mechanism is also required for the maximal potency of the innate immunity against HIV-1 infection. Experimental procedures Plasmid construction BH10 is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA. The construction of BH10.vif−, as well as wild-type hA3G has been previously described (Cen et al., 2004). ZWt-p6.vif− encodes a Vif-deficient full-length HIV-1 genome, in which the nucleocapsid sequence has been replaced with a yeast leucine zipper domain (Accola et al., 2000). The hGag plasmid, which encodes the HIV-1 Gag sequence, produces mRNA whose codons have been optimized for mammalian codon usage was a kind gift from G. Nabel, National Institutes of Health (Huang et al., 2001). The Vif expression plasmid pcDNA-HVif was kindly provided by Klaus Strebel, National Institutes of Health (Nguyen et al., 2004). The domain replaced and switched hA3G cDNAs were generated by overlapping extension PCR, using wild type hA3G expression plasmids as template (Cen et al., 2004). Each cDNA fragments were amplified separately, by using a primer specific for the overlap region; for replacement of CD2 with CD1, the following intermediate primers were used: (A1) 5′ TCT CAT CTC TGG GTG GCG GCC TTC AAG GAA 3′; (A2) 5′ TTC CTT GAA GGC CGC CAC CCA GAG ATG AGA 3′; (A3) 5′ AGC CAT TTC CTG GGC ACA CTT TGT GCA GGG 3′; (A4) 5′ CCC TGC ACA AAG TGT GCC CAG GAA ATG GCT 3′; for replacement of CD1 with CD2, the following intermediate primers were used: (B1) 5′ ACA GCT CTG CAT GGT ACT TAA GTT CG GA 3′; (B2) 5′ TCC GAA CTT AAG TAC CAT GCA GAG CTG T 3′; (B3) 5′ GCC ATA TCC CTT GTA CAG CTG AAG CAG G 3′; (B4) 5′ CCT GCT TTC AGC TGT ACA AGG GAT ATG GC 3′; the N-terminal and Cterminal primer were same as wild type hA3G: 5′-TAA GCG GAA TTC ATG AAG CCT CAC TTC AGA (forward primer) and 5′-TAG AAG CTC GAG TCA AGC GTA ATC TGG AAC (reverse primer). The amplified PCR products were digested with EcoRI and XhoI, whose sites were placed in each of the PCR primers. These fragments were cloned into the EcoRI and XhoI sites of the pcDNA3.1V5/His vector. Cells, transfections, and viruses purification HEK-293T cells and TZM-bl cells were grown in complete Dulbecco's modified Eagle's medium plus 10% fetal calf serum, 100 units of penicillin, and 100 μg of streptomycin/ml. HEK-293T cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. In Vif/hA3G interaction assay (Vif and hA3G/mutants plasmid co-transfected 293 cells with a ratio of 1:1), MG132 was added to the transected cells 24 h post-transfection (final concentration of 5 μM). Cells were collected 48 h post-transfection for protein or immunoprecipitation assay. For the production of viruses or Gag VLP (293 cells were co-transfected hGag and hA3G/mutants plasmid with a ratio of 1: 1), supernatant was collected 48 h posttransfection. Viruses were pelleted from culture medium by centrifugation in a Beckman Ti-45 rotor at 35,000 rpm for 1 h. The viral pellets were then purified by centrifugation in a Beckman SW 41 rotor at 26,500 rpm for 1 h through 15% sucrose onto a 65% sucrose cushion. The band of purified virus was removed and pelleted in 1× TNE (20 mM Tris, pH 7.8, 100 mM NaCl, 1 mM EDTA) in a Beckman SW 41 rotor at 40,000 rpm for 1 h. Protein analysis Cellular and viral proteins were extracted with radioimmune precipitation assay buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1%

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sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 2 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A, 100 mg/ml phenylmethylsulfonyl fluoride). The cell and viral lysates were analyzed by SDS– PAGE (10% acrylamide), followed by blotting onto nitrocellulose membranes (GE HealthCare). Western blots were probed with monoclonal antibodies that are specifically reactive with HIV-1 capsid (Zepto Metrocs Inc.), hA3G (NIH AIDS Research and Reference Reagent Program), hemagglutinin (HA, Santa Cruz Biotechnology, Inc.), and β-actin (Sigma), or with Vif-specific polyclonal antiserum 2221 (NIH AIDS Research and Reference Reagent Program). Detection of proteins was performed by enhanced chemiluminescence (PerkinElmer Life Sciences), using secondary anti-mouse (for hA3G, capsid, HA, and β-actin) and anti-rabbit (for Vif) antibodies, both obtained from GE Health Care. Bands in Western blots were quantitated using the Image J 1.35s automated digitizing system (National Institutes of Health). Immunoprecipitation assay 293T cells from 100-mm plates were collected 48 h posttransfection and lysed in 500 μl of CytoBuster Protein Extraction Reagent (novagen). Insoluble material was pelleted at 1800g for 30 min. The supernatant was used as the source of immunoprecipitated complexes. Equal amounts of protein were incubated with 5 μl of specific antibody for 16 h at 4 °C. In Vif/hA3G mutants complexes assay, specific anti-HA mouse antibody (Santa Cruz Biotechnology) was used and for Gag/hA3G complexes assay, specific anti-HA rabbit antibody was used (Santa Cruz Biotechnology), followed by the addition of protein A-Sepharose (Amersham Biosciences) for 2 h. The immunoprecipitate was then washed three times with TNT buffer and twice with phosphate-buffered saline. After the final supernatant was removed, 30 μl of 2× sample buffer (120 mM Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, 2% β-mercaptoethanol, and 0.02% bromphenol blue) was added, and the precipitate was then boiled for 5 min to release the precipitated proteins. After microcentrifugation, the resulting supernatant was analyzed using Western blots. In the RNase treatment assay, the cell lysates were pretreated with 100 μg RNase before the immunoprecipitation, as described previously (Khorchid et al., 2002). Sequencing of viral genomic DNA The sequencing of viral genomic DNA was performed as follows. Viral supernatants from transfected 293 cells were filtered through 0.45-μm filters and treated with DNase at 100 IU/ml for 1 h at 37 °C to prevent proviral DNA carryover. A total of 10 ng viral p24 was used to infect 2 × 105 SupT1 cells in 24 well plate using spin infection; add polybrene (Sigma H9268) to a final concentration of 4 μg/ml; spin at 1000g (Beckman centrifuge GPH, Rotor 3.7, 2500 rpm) for 120 min at room temperature. After spinning, the infected cells were washed twice with phosphate-buffered saline and plated into six-well plates. Complete RPMI 1640 medium prewarmed to 37 °C was added to the infection mixture. Cultured cells were collected 24 h post-infection, and DNA was extracted using a DNeasy Blood and tissue kit (QIAGEN Inc.). PCR was performed with Platinum Taq polymerase (Invitrogen Life Technologies). The primers were as follows: forward (469 to 492), 5′-TTAGACCAGATCTGAGCCTGGGAG-3′; reverse (679 to 699), 5′GAGTCCTGCGTCGAGA GAGCT-3′. The PCR products were cloned into pCR4-TOPO vector (Invitrogen Life Technologies), and individual clones were sequenced. Single cycle HIV-1 infectivity assay on TZM-bl reporter cell lines 293T cells were co-transfected with plasmids containing vif deficient HIV-1 proviral DNA and different wild type or mutant hA3G plasmids. Culture supernatants were collected 48 h post-

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infection and quantified by measuring the amounts of p24 using HIV-1 p24 ELISA kit (Xpressbio, Thurmont, MD, USA) following the manufacturer's instructions. TZM-bl cells (4 × 104) were infected with equal amounts (20 ng p24 antigen) of Vif deficient HIV-1 containing wild-type or mutant hA3G. Forty-eight hours post-infection, cells were harvested in 200 μl 1× Luciferase Cell Culture Lysis Reagent (Promega) and assayed for luciferase activity with the luciferase assay kit (Promega). Real-time PCR quantitation of newly synthesized HIV-1 DNA Equal amounts of DNase-treated virions (100 ng p24) were used to infect 1 × 106 SupT1 cells using spin infection as previously described. Cultured cells were collected 16 h post-infection, and DNA was extracted using a DNeasy Blood and tissue kit (QIAGEN Inc.). Using equal amounts of cellular genomic DNA (determined spectrophotometrically at an optical density of 260 nm), late (U5-gag) minusstrand reverse transcripts were quantitated by the Light Cycler Instrument (Roche Diagnostics GmbH) using the following primers: late RT forward (560 to 578), 5′-TGTGTGCCCGTCTGTTGTGTGA-3′; late RT reverse (679 to 699), 5′-GAGTCCTGCGTCG AGAGAGCT-3′. Acknowledgments This work was supported in part by the National S&T Major Special Project on Major New Drug Innovation 2009ZX09303-005 (X.F.Y. and C.S.), the National S&T International Collaboration 2010DFA31580 (J.D.J) and 2010DFB30870 (Q.J.), Nature Science Foundation of China 30973569 (S.C.), the State Key Laboratory for Molecular Virology and Genetic Engineering grant (S.C.), and the Canadian Institutes of Health Research grant (S.C.). References Accola, M.A., Strack, B., Gottlinger, H.G., 2000. Efficient particle production by minimal Gag constructs which retain the carboxy-terminal domain of human immunodeficiency virus type 1 capsid-p2 and a late assembly domain. J. Virol. 74, 5395–5402. Bishop, K.N., Verma, M., Kim, E.Y., Wolinsky, S.M., Malim, M.H., 2008. APOBEC3G inhibits elongation of HIV-1 reverse transcripts. PLoS Pathog. 4, e1000231. Cen, S., Guo, F., Niu, M., Saadatmand, J., Deflassieux, J., Kleiman, L., 2004. The interaction between HIV-1 Gag and APOBEC3G. J. Biol. Chem. 279, 33177–33184. Chen, H., Lilley, C.E., Yu, Q., Lee, D.V., Chou, J., Narvaiza, I., Landau, N.R., Weitzman, M.D., 2006. APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons. Curr. Biol. 16, 480–485. Chen, K.M., Harjes, E., Gross, P.J., Fahmy, A., Lu, Y., Shindo, K., Harris, R.S., Matsuo, H., 2008. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G. Nature 452, 116–119. Conticello, S.G., Harris, R.S., Neuberger, M.S., 2003. The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G. Curr. Biol. 13, 2009–2013. Conticello, S.G., Thomas, C.J., Petersen-Mahrt, S.K., Neuberger, M.S., 2005. Evolution of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Mol. Biol. Evol. 22, 367–377. Cullen, B.R., 2006. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J. Virol. 80, 1067–1076. Dang, Y., Siew, L.M., Wang, X., Han, Y., Lampen, R., Zheng, Y.H., 2008. Human cytidine deaminase APOBEC3H restricts HIV-1 replication. J. Biol. Chem. 283, 11606–11614. Dettenhofer, M., Cen, S., Carlson, B.A., Kleiman, L., Yu, X.F., 2000. Association of human immunodeficiency virus type 1 Vif with RNA and its role in reverse transcription. J. Virol. 74, 8938–8945. Fisher, A.G., Ensoli, B., Ivanoff, L., Chamberlain, M., Petteway, S., Ratner, L., Gallo, R.C., Wong-Staal, F., 1987. The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science 237, 888–893. Friew, Y.N., Boyko, V., Hu, W.S., Pathak, V.K., 2009. Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA. Retrovirology 6, 56. Gabuzda, D.H., Lawrence, K., Langhoff, E., Terwilliger, E., Dorfman, T., Haseltine, W.A., Sodroski, J., 1992. Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes. J. Virol. 66, 6489–6495. Gibbs, J.S., Regier, D.A., Desrosiers, R.C., 1994. Construction and in vitro properties of SIVmac mutants with deletions in “nonessential” genes. AIDS Res. Hum. Retroviruses 10, 607–616. Guo, F., Cen, S., Niu, M., Saadatmand, J., Kleiman, L., 2006. Inhibition of formula-primed reverse transcription by human APOBEC3G during human immunodeficiency virus type 1 replication. J. Virol. 80, 11710–11722.

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