HIV-1 Vif protein blocks the cytidine deaminase activity of B-cell specific AID in E. coli by a similar mechanism of action

Molecular Immunology 44 (2007) 583–590

HIV-1 Vif protein blocks the cytidine deaminase activity of B-cell specific AID in E. coli by a similar mechanism of action Mariana Santa-Marta, Frederico Aires da Silva, Ana Margarida Fonseca, Sylvie Rato, Joao Goncalves ∗ URIA-Centro de Patog´enese Molecular, Faculdade de Farm´acia, Universidade de Lisboa, 1649-019 Lisboa, Portugal Received 30 December 2005; received in revised form 24 January 2006; accepted 7 February 2006 Available online 31 March 2006

Abstract HIV-1 Vif protein protects viral replication in non-permissive cells by inducing degradation of APOBEC3G via ubiquitination and proteasomal pathway, although new studies indicate a putative role in Vif’s direct inhibition of APOBEC3G. APOBEC3G is member of a homologous family of proteins with cytidine deaminase activity expressed with characteristic tissue specificity, that in humans consist of APOBEC1, APOBEC2, APOBEC3A-H, APOBEC4 and the activation-induced deaminase (AID), a B lymphoid protein necessary for somatic hypermutation, gene conversion and class switch recombination. In this work we show that Vif can counteract AID’s activity in E. coli in absence of specific eukaryotic co-factors necessary for AID induced somatic hypermutation, gene conversion and to stimulate class switch recombination in B-cells. We show that AID inhibition is mediated by a direct protein–protein interaction via unique amino acid D118 an homologous mutant responsible for the species-specific restriction of HIV-1 Vif protein existent for APOBEC3G. These results raise the hypothesis that Vif related proteins can act as a broad inhibitor of deaminase activity. Moreover as AID and Vif evolved in different cellular environments, these results may indicate that Vif related proteins might mimic cellular factors that interact with a structural conserved domain of cytidine deaminases during evolution. © 2006 Elsevier Ltd. All rights reserved. Keywords: Vif; AID; Activation-induced deaminase; APOBEC3G; Cytidine deaminases

1. Introduction Human immunodeficiency virus type-1 (HIV-1) Vif protein is essential for virus replication in non-permissive cells such as macrophages, primary human T-cells, and some restrictive T-cell lines (Gabuzda et al., 1992; Sova and Volsky, 1993). Non-permissive cells expresses APOBEC3G, a cellular protein that actively blocks retroviral infection which is counterAbbreviations: HIV-1, human immunodeficiency virus, type 1; Vif, viral infectivity factor; AID, activation-induced cytidine deaminase; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; APOBEC1, apolipoprotein (apo) B editing cytidine deaminase; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; AHT, anhydrotetracycline; IPTG, isopropyl-1-thio-␤-dgalactoside; PBS, phosphate-buffered saline; HA, hemagglutinin ∗ Corresponding author at: URIA-Centro de Patog´ enese Molecular, Av. das Forc¸as Armadas, 1649-019 Lisboa, Portugal. Tel.: +351 21 7946489; fax: +351 21 7946491/34212. E-mail address: [email protected] (J. Goncalves). 0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2006.02.005

acted by the HIV-1 Vif (Wiegand et al., 2004; Zheng et al., 2004). APOBEC3G is a member of a homologous cytidine deaminase family that in humans consist in APOBEC1, APOBEC2, APOBEC3A-H, APOBEC4 and the activation-induced deaminase (AID) (Jarmuz et al., 2002; Wedekind et al., 2003; Conticello et al., 2005). The APOBEC family members are expressed in specific tissues: APOBEC 3G is expressed primarily in lymphoid and myeloid cell lineages (Jarmuz et al., 2002); AID is a B lymphoid protein that acts on DNA, catalyzing C-U deamination of immunoglobulin genes to induce somatic hypermutation, gene conversion and to stimulate class switch recombination (Muramatsu et al., 1999, 2000; Arakawa et al., 2002; Revy et al., 2000; Okazaki et al., 2002; Harris et al., 2002b). Vif counteracts APOBEC3G by inhibiting its translation or intracellular half-life (Stopak et al., 2003; Kao et al., 2003) and inducing its polyubiquitination and proteasomal degradation by a specific interaction with APOBEC3G. The deaminase is recruit to degradation as part of a Vif-Cul5-SCF complex through a

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novel SOCS (suppressor of cytokine signaling)-box and an HCCH motif present in Vif, essential for the recruitment of Cul5 to the complex (Mehle et al., 2004a,b; Yu et al., 2003, 2004; Conticello et al., 2003; Marin et al., 2003; Luo et al., 2005). The increase degradation and/or reduced level of APOBEC3G expression by Vif excludes APOBEC3G from incorporation into virions (Kao et al., 2003; Liu et al., 2004) and consequent absence during reverse transcription in the target cell, allowing the virus to replicate. Nevertheless, recent works showed that Vif-induced APOBEC3G degradation may not be the only mechanism for the production of fully infectious virus (SantaMarta et al., 2004; Goncalves and Santa-Marta, 2004; Kao et al., 2004). The species-specificity of Vif to inhibit APOBEC3G function, is correlated with its ability to physically associate with APOBEC3G using a single amino acid D128 (Mangeat et al., 2004; Bogerd et al., 2004; Schrofelbauer et al., 2004; Mariani et al., 2003; Xu et al., 2004). Recent advances in cytidine deaminases showed that other members of the APOBEC3 family (APOBEC3F and APOBEC3B) (Bishop et al., 2004; Wiegand et al., 2004; Zheng et al., 2004; Liddament et al., 2004) also have the ability to restrict HIV-1 Vif defective virus. APOBEC3G is closely related to AID and both can cause C/G to T/A mutations in DNA (Petersen-Mahrt and Neuberger, 2003; Harris et al., 2003a). AID is widely express in B-lymphoid cells and the mechanism used to induce vertebrate affinity maturation of antibodies is very similar to APOBEC-mediated anti-viral defense (Harris and Liddament, 2004). Nevertheless, as shown by Zheng et al. (2004) when AID is expressed in 293T cells it does not inhibit HIV-1 infectivity. Thus as AID is closely related to APOBEC3G and mutates dC residues in E. coli DNA and single-stranded-DNA in vitro (Petersen-Mahrt et al., 2002; Dickerson et al., 2003; Chaudhuri et al., 2003; Bransteitter et al., 2003; Ramiro et al., 2003) we wanted to evaluate if the HIV-1 Vif protein specifically recognizes and interferes with the homologous but distinct AID cytidine deaminase. As our goal was to evaluate deaminase activity alone in absence of specific eukaryotic co-factors we used a similar E. coli system described previously by Ramiro et al. (2003). Our results showed that Vif counteracts AID’s activity in E. coli. Deaminase activity inhibition by Vif is mediated by a direct protein–protein interaction that is dependent on a single amino acid D118 in AID. Moreover, as AID and Vif evolved in a different cellular environment, these results raise the hypothesis that Vif (and potentially other Vif-like proteins) might mimic a cellular factor that interacts with a structural conserved domain of cytidine deaminases inhibiting or modulating the activity of a broad range of host cell cytidine deaminases. 2. Results 2.1. AID induces cytidine deamination in E. coli and HIV-1 Vif expression inhibits its deaminase activity To examine if HIV-1 Vif protein inhibits the AID induced mutation in E. coli, an initial study was performed based on the system previously described by Ramiro et al. The mutator activity of AID protein (kindly provided by Dr. Nussenzweig)

and co-expressed AID and HIV-1 Vif (Santa-Marta et al., 2004) proteins was first monitored by assessing the frequency of rifampicin-resistant (RifR ) colonies (Ramiro et al., 2003). To control the mutator efficiency of AID deaminase, the previously described putative active-site mutant AID-E58Q (Ramiro et al., 2003; Harris et al., 2002a) was constructed.

Fig. 1. AID induces rapid mutation in E. coli DNA and co-expression HIV-1 Vif inhibits its deaminase activity. Ten to 12 individual colonies of the BH156 UDGdeficient strain sequentially transformed with ptacKanL94P, plus pASKAID, or pASKAID-E58Q combined with pDHC29 or pDHC29Vif were grown to exponential phase and protein expression was induced during 3 h to express deaminases, target kanL94P gene and Vif constructs. (A) RifR frequency resulting from the expression of AID, AID-E58Q and AID plus Vif to rpoB gene target. Circles represent the RifR mean frequency of individual starting colonies; horizontal bars represent mean values that are given under x-axis. (B) KanR frequency resulting of the expression of AID, AID-E58Q and AID plus Vif to kanL94P gene target. Circles represent the KanR mean frequency of individual starting colonies; horizontal bars represent mean values that are given under x-axis. (C) Expression of AID, AID-E58Q and AID plus Vif proteins in BH156 UDGdeficient cells. After 3 h of induction with AHT and IPTG, cells were disrupted and whole extracts were resolved by standard SDS and analyzed by western-blot with correspondent antibodies. Statistical analysis indicated in Section 2.

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All experiments were performed in BH156 UDG-deficient (ung-1) E. coli (kindly provided by Dr. Nussenzweig) to increase the mutation frequency. A population of 10–12 individual colonies of bacteria alone, and expressing AID plus pDHC29, AID plus pDHC29Vif, AID-E58Q plus pDHC29 and AID plus pDHC29Vif-C114F were assayed in the presence of tet inducer (AHT) and lac inducer (IPTG) as described in materials and methods. Mutation frequencies were calculated as the ratio of RifR colonies to the total number of viable cells for each individual colony. As shown in Fig. 1A, when AID is expressed in E. coli the mean RifR frequency (mean frequency: 399 × 10−8 ; p = 0.5) is about 10 times higher than the bacterial cells alone (mean frequency: 37.3 × 10−8 ; p = 0.00021) and the putative active site mutant AID-E58Q (mean frequency: 67.5 × 10−9 ; p = 0.00039) as previously described (Ramiro et al., 2003; Harris et al., 2002a). In Fig. 1A, the concomitant expression of Vif protein with AID abolished the mutation frequency caused by the deaminase (AID plus Vif mean frequency: 186.2 × 10−8 ; p = 0.00701). As AID-mediated cytidine deamination activity in E. coli is linked to transcription (Ramiro et al., 2003), we tested whether HIV-1 Vif protein could also interfere with AID-induced transcription phenotype. We used an inactive allele of the kanamycin resistance gene (kanL94P), in which the TTG codon encoding a leucine is altered to a proline (CCA) (Ramiro et al., 2003). Deaminase activity was measured by reversion to kanamycin resistance (KanR ) resulting from single C-to-T mutation in codon 94 to either CTA (leucine) or TCA (serine) when transcription is induced by IPTG. The BH156 containing ptacKanL94P, pDHC29 or pDHC29Vif, together with pASK-AID were induced with IPTG and AHT, after cells reached an optical density of 0.6 at 660 nm. As shown in Fig. 1B, when AID

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is expressed (BH156 transformed with ptacKanL94P, pDHC29 and pASK-AID), the frequency of reversion by cytidine deamination in the kan resistance gene (ratio of KanR colonies to the total number of viable cells for each individual colony) was near 40 times higher (mean frequency: 957.6 × 10−6 ; p = 0.5) than the background level (mean frequency: 63.5 × 10−6 ; p = 0.00042) and that of the putative active-site mutant AID-E58Q (mean frequency: 26.4 × 10−6 ; p = 0.00042). As described previously for APOBEC3G (Santa-Marta et al., 2004), co-expression of AID with HIV-1 Vif protein reduced the AID-induced transcription phenotype to background level (mean frequency: 89.5 × 10−6 ; p = 0.00084) (Fig. 1B). In all independent assays, the number of viable cells observed was maintained, indicating that the protein expression did not affect cell viability (data not shown). The reduction of cytidine deaminase activity when Vif is concomitantly expressed with AID is consistent with the hypothesis of Vif can act as a direct inhibitor of AID deaminase activity. Sequencing of KanR colonies showed specific deamination by C-to-T mutations in codon 94, in average half to CTA (leucine) and half to TCA (serine) (data not shown). 2.2. A single amino acid substitution renders AID resistant to Vif inhibition It was previously shown that a single D128K substitution rendered human APOBEC3G resistant to Vif-induced depletion and consequently maintained its capacity to inhibit HIV-1 replication in the presence of Vif (Mangeat et al., 2004; Xu et al., 2004; Schrofelbauer et al., 2004; Mariani et al., 2003; Bogerd et al., 2004). The alignment of AID and APOBEC3G amino acid sequences show that AID D118 is homologous to the APOBEC3G D128 (Fig. 2) (Santa-Marta et al., 2004; Cascalho,

Fig. 2. CLUSTALW alignment of the human APOBEC3G (shown amino acid 1–200), human AID (shown amino acid 1–182) and human APOBEC1 (shown amino acid 1–191). CLUSTAL W multiple sequence alignment shows identities in dark shadow. APOBEC3G contains a duplicated active site, a linker and a pseudoactive site. The different domains are identified based on homology to APOBEC1. The properties common to cytidine deaminases are indicated as follow: zinc ligand amino acids (
), residues that mediate proton shuttling during catalysis (* ) (Cascalho, 2004; Harris et al., 2003b).

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small increase in KanR mutants may result from Vif-induced stabilization of AID-D118K. Future studies are necessary to evaluate this hypothesis. These results are in contrast with coexpression of the viral protein with AID where a dramatic reduction in cytidine deamination is observed (mean frequency: 73.6 × 10−6 ; p = 0.00084). As shown in Fig. 3B all AID constructs were expressed similarly. These results indicate that differences in AID and AID-D118K deaminase activity in presence or absence of Vif are not due to altered levels of proteins. To assess if the expression of HIV-1 Vif protein was essential for the inhibition of deaminase activity of AID in E. coli, a functional inactive mutant of Vif protein (Vif-C114F) was used as a negative control (Ma et al., 1994; Yang et al., 1996). As shown in Fig. 3A, co-expression of Vif-C114F and AID did not affect significantly its deaminase activity (mean frequency: 340 × 10−6 ; p = 0.03240). Therefore, these results strongly suggest that expression of Vif can block AID-mediated cytidine deaminase activity in absence of specific eukaryotic co-factors, and this effect may be influenced by the single amino acid substitution (D118K) in the enzyme. 2.3. AID inhibition by Vif involves a strong protein interaction Fig. 3. The single amino acid substitution D118K renders AID resistant to Vif blocking. (A) Expression of AID induces a KanR mutator phenotype that is abolished when Vif is co-expressed and not with co-expression of Vif inactive mutant, Vif-C114F. In contrast, expression of AID-D118K induces a KanR mutator phenotype that is not inhibited by the co-expression of Vif. The BH156 UDGdeficient strain with ptacKanL94P was sequentially transformed with pDHC29, pDHC29-Vif, pDHC29 plus AID, pDHC29-Vif plus AID, pDHC29 plus AIDD118K, pDHC29-Vif plus AID-D118K. Individual colonies were grown to exponential phase and protein expression was induced during 3 h with AHT and IPTG. Circles represent the KanR mean frequency of individual starting colonies; horizontal bars represent mean values that are given under x-axis. (B) Protein expression of AID alone or co-expressed with Vif or Vif-C114F and AID-D118K alone or co-expressed with Vif in BH156 UDG-deficient cells. After 3 h of induction with AHT and IPTG for AID and Vif constructs respectively, cells were disrupted and whole extract were resolved by standard SDS and analyzed by western-blot with correspondent antibodies. Statistical analysis indicated in Section 2.

2004). Therefore a question remained if the D118K mutation could be sufficient to overcome the block of AID deaminase activity by HIV-1 Vif as reported to APOBEC3G, or instead if the single mutation affects itself the enzymatic activity of AID. To answer this question AID D118K mutant was constructed and expressed in presence of pDCH29 or pDHC29Vif. As shown in Fig. 3A, AID-D118K has significant deaminase activity as shown by statistical analysis (p = 0.09764), although the mean frequency of KanR mutants obtained (mean frequency: 485.5 × 10−6 ; p = 0.09764) was reduced when compared to the wild type AID (mean frequency: 957.6 × 10−6 ) reflecting a possible structural alteration that affects its enzymatic activity. When Vif was co-expressed with AID-D118K in bacteria, it loses the ability to inhibit AID-D118K deaminase activity, as mutant AID still deaminases its target with statistical significance (p = 0.11063) and, instead, it is responsible for a small increase in the rate of cytidine deamination as observed (mean frequency: 558 × 10−6 ; p = 0.11063) compared with AID D118K expression alone (mean frequency: 485 × 10−6 ). This

To determine whether Vif-mediated block of AID cytidine deaminase activity was due to a specific direct interaction, we performed co-immunoprecipitation assays (Fig. 4). Briefly, BH156 cells alone or BH156 transformed with pDHC29 and pASK-AID; or pDHC29-Vif and pASK-AID; or pDHC29 and pASK-AID–E58Q; or pDHC29Vif and pASK-AID–E58Q; or pDHC29 and pASK-AID–D118K; or pDHC29Vif and pASKAID–D118K; or pDHC29-VifC114F and pASK-AID were grown and induced with IPTG and AHT to express Vif and AID constructs, respectively. Each sample was disrupted by sonication and cellular lysates were analyzed by standard SDS-PAGE. Proteins were transferred to nitrocellulose membrane and probed with HRP-conjugated anti-FLAG monoclonal antibody (Sigma) for AID detection and HRP-conjugated high affinity anti-HA (Roche) for Vif detection. To assess the presence of an AID-Vif complex and gaining insight into possible interaction forces involved in the putative AID-Vif complex, cell lysates were immunoprecipitated with anti-HA affinity matrix (Roche). Immunoprecipitated samples were washed in a low-ionic and high-ionic strength buffer and eluted with SDS-containing buffer. After gel electrophoresis and transfer onto nitrocellulose membranes, eluted proteins were probed with HRP-conjugated high affinity antiHA antibody (Roche), for Vif detection. After stripping, the membrane was probed with HRP-conjugated anti-FLAG monoclonal antibody (Sigma) to determine if AID was effectively co-immunoprecipitated. As demonstrated in lane 8 of Fig. 4, pull down by the anti-HA matrix of BH156 cells alone showed no unspecific protein immunoprecipitation. Conversely, immunoprecipitation of HA-tagged proteins constantly demonstrated the presence of Vif-HA proteins (Fig. 4A and B). Immunoprecipitation of AID, AID-E58Q or AID-D118K with anti-HA beads in samples without Vif expression, did not retrieved AID when

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Fig. 4. Vif binds to AID and substitution mutation D118K is not essential to protein-protein interaction. Protein expression was performed after 3 h of induction with AHT and IPTG for AID and Vif constructs, respectively. Proteins were immunoprecipitated using anti-HA affinity matrix (Roche) o.n. at 4 ◦ C and washed with a high-ionic buffer or a low-ionic buffer. Proteins were resolved by standard SDS and analyzed by western-blot with HRP-conjugated anti-HA monoclonal antibody 3F10 (Roche). (A) Co-immunoprecipitation of AID constructs in presence or absence of Vif constructs with anti-HA matrix (Roche) and washes with low-ionic buffer. AID constructs were analyzed by western-blot with HRP-conjugated anti-FLAG (Sigma), and Vif constructs using HRP conjugated-anti-HA (Roche). (B) Co-immunoprecipitation of AID proteins in presence or absence of Vif constructs using anti-HA matrix (Roche) and washes with high-ionic buffer. AID constructs were analyzed by western-blot with HRP-conjugated anti-FLAG (Sigma), and Vif constructs using HRP conjugated-anti-HA (Roche).

low-ionic or high-ionic strength buffers were used (Fig. 4A and B). In contrast, co-immunoprecipitation of AID, AID-E58Q or AID-D118K with anti-HA beads (Roche) in presence of Vif, constantly retrieved all protein variants in presence both lowionic or high-ionic strength buffers (Fig. 4A and B). To evaluate if the non-functional Vif-C114F protein can interact with AID, both proteins were co-expressed and coimmunoprecipitated. As shown in Fig. 4, absence of AID’s inhibition by the Vif-C114F mutant is not due to loss of interaction as Vif-C114F immunoprecipitated similarly at both salt concentrations when compared to wild-type Vif. These results indicated the presence of a stable complex between Vif and AID. This complex is not altered by the catalytic mutation E58Q in AID or by the activity blocking mutation D118K. The reported interaction of Vif with D118K mutant of deaminase was consistent in all experiments, indicating an alternative mechanism for Vif/AID binding. Nevertheless, we cannot exclude the hypothesis of a related problem to the bacterial system used, since a putative eucariotic factor capable of regulating its activity is not present. 3. Discussion Results of this study demonstrate that the catalytic activity of AID cytidine deaminase is inhibited by the HIV-1 Vif protein in an E. coli system. AID is a B lymphoid protein that deaminates DNA, inducing somatic hypermutation, gene conversion and stimulating class switch recombination in target cells (Muramatsu et al., 1999, 2000; Arakawa et al., 2002; Harris et al., 2002b; Okazaki et al., 2002; Revy et al., 2000). AID is a cytosine deaminase with genetic and functional homology to APOBEC3G (Fig. 2), the T-cell restriction factor for HIV-1 Vif defective virus (Cascalho, 2004; Santa-Marta et al., 2004). Similarly to APOBEC3G, AID is also known to be active in an E.

coli system (Petersen-Mahrt et al., 2002; Harris et al., 2002a; Ramiro et al., 2003). We previously shown that the HIV-1 Vif protein was able to inhibit the APOBEC3G protein in E. coli system firstly described by Ramiro et al. (Santa-Marta et al., 2004). This system proved to be very useful in studies regarding catalytic deaminase activity alone, as there is no protein regulation by post-translational modifications or degradation via ubiquitinatin-proteasome pathway. We used the same system, to evaluate if Vif could interact with AID and inhibit its deaminase activity. Our results show that AID was active in the system, as the frequency of rifampicin-resistant (RifR ) colonies resulted from the deamination of the rpoB gene was significantly higher when compared to cells alone or to the inactive catalytic mutant, AIDE58Q (Ramiro et al., 2003; Harris et al., 2002a). Concomitant expression of AID and HIV-1 Vif protein, resulted in RifR values similar to background levels of mutagenesis (cells alone) and to AID non-catalytic mutant (AIDE58Q) (Ramiro et al., 2003; Harris et al., 2002a). These results show that Vif actively blocks the deaminase activity of AID. We also evaluated the ability of Vif to inhibit the induced transcription phenotype of AID. As shown in the KanR assay, concomitant expression of Vif protein also inhibits the induced transcription phenotype of AID as it retrieves KanR values to background levels of mutagenesis (cells alone and non-active AIDE58Q). This is somewhat remarkable since AID, a B-cell cytidine deaminase and HIV-1 Vif protein do not co-evolved or encountered during viral infection in T-cells. These results prove the need for a better understanding of the similarities between AID and APOBEC3G proteins. AID and APOBEC3G are members of the APOBEC-cytidine deaminase family of proteins that share a large homology (Jarmuz et al., 2002; Wedekind et al., 2003; Harris et al., 2002a; Harris and Liddament, 2004). APOBEC3G appears to be a geneduplication of the AID protein (Jarmuz et al., 2002; Conticello et al., 2005). In the context of target site preference, AID is quite

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distinct from the APOBEC3 proteins (Vartanian et al., 1991, 2002), presumably due to a co-evolution of AID with the variable and switch regions of the immunoglobulin loci that allow both optimal targeting of somatic mutations during antibody affinity maturation and efficient class switch recombination (Beale et al., 2004; Pham et al., 2003). The structural basis for major difference in target specificity of AID and APOBEC3 proteins is currently unknown. As in APOBEC3G, a single amino acid substitution from D128 (aspartate) to K (lysine) can render this protein resistant to depletion by HIV-1 Vif (Bogerd et al., 2004; Mangeat et al., 2004; Mariani et al., 2003; Schrofelbauer et al., 2004), we sought to evaluate if the homologous AID-D118 was also important for Vif ability to inhibit AID deaminase activity. Similarly to the mutant phenotype of APOBEC3G (APOBEC3G-D128K), the AID-D118K mutant suffered a small reduction in its deaminase activity during the kanamycin induced transcription. In APOBEC3G, the D128 amino acid appears to represents a direct contact point for Vif (Santa-Marta et al., 2004), or alternatively the amino acid change at this position influences the global conformation of the enzyme. Previous studies support the notion that this amino acid is positioned in a protein loop and is suitable for protein contact in APOBEC3G (Santa-Marta et al., 2004). After alignment and identification of the D118 residue in AID as homologous to D128 of APOBEC3G, the mutant AID-D118K showed no Vif-dependent inhibition of its deaminase activity. These results hypothesize that similar patterns of Vif activity towards AID and APOBEC3G involve a direct inhibition of deaminase activity by a protein–protein interaction. It has been suggested that evolution of APOBEC3G in primates has been driven by positive selection (Sawyer et al., 2004; Zhang and Webb, 2004). Given the role demonstrated by some APOBEC3 members in viral restriction, it may well be that issues pertaining to host/virus interaction have provided the driving force for the rapid expansion of the entire APOBEC3 locus in primates (Conticello et al., 2005). As APOBEC3 locus is derived from AID, trough amplification and reassortment that is likely to have occurred through unequal crossover/recombination facilitated by retroviral elements (Conticello et al., 2005) we speculate that an ancestor of HIV-1 Vif protein might be involved in this process and might have co-evolved with the APOBEC3 locus maintaining the initial structural basis of interaction. 4. Materials and methods 4.1. Plasmids Plasmid ptacKanL94P and plasmid pASK-AID were a generous gift from Dr. Michel C. Nussenzweig, ptacKanL94P has a mutation at codon 94 that produces a kanamycin-sensitive (Kans ) phenotype. Mutating CCA to TCA or CTA restores resistance. Transcription of kanL94P is controlled by the IPTG-inducible tac promoter (Ramiro et al., 2003). Plasmid pASK-AID, plasmid expresses mouse AID, modified according to the E. coli codon preference to produce AIDE. coli . pASK-IBA7 constructs are under the control of tet promoter, which is repressed in the absence of AHT by tet repressor

(Ramiro et al., 2003). Constructs AID-E58Q and AID-D118K were derived from pASK-AID and generated by site-directed mutagenesis according to manufacturer’s protocol (Stratagene). The AID-E58Q mutant was obtained using the oligonucleotides 5 -GGCTGCCACGTGCAGCTGCTCTTCCTCCG3 , 5 -CGGAGGAAGAGCAGCTGCACGTGGCAGCC-3 , the AID-D118K mutant was obtained using the oligonucleotides 5 CCTCTACTTCTGTGAGAAGCGCAAGGCTGAGCC-3 , 5 GGCTCAGCCTTGCGCTTCTCACAGAAGTAGAGG-3 . All mutations were confirmed by sequencing using an external primer 5 -AAATCGAAGGGCGCCGAG-3 . The pDHC29-Vif and Vif-C114F are described (Fujita et al., 2002; Santa-Marta et al., 2004). All Vif constructs are tagged with hemaglutinin epitope and all AID constructs were tagged with FLAG epitope. The plasmid replication origins of ptacKanL94D, pASKIBA-7 and pDHC29 are pSC101, pUC and pST19, respectively, for compatibility in E. coli. 4.2. Monitoring cytidine deamination activity and protein expression This procedure has been previously described by Ramiro et al. and Santa-Marta et al. Briefly, BH156 UDG-deficient strain (dcm-6, thr1, hisG4, leuB6, rpsL, ara14, supE44, lacY1, tonA31, tsx78, galK2, xyl5, thi1, mtl1, unh-1), a kind gift from Dr. Michel C. Nussenzweig, were transformed with ptacKanL94D, followed by independent transformation of the subsequent plasmid combinations; pASK-AID/pDHC29;pASKAID/pDHC29-Vif; pASK-AID/pDHC29-Vif-C114F; pASKAID–E58Q/pDHC29; pASK-AID–E58Q/pDHC29-Vif; pASKAID -D118K/pDHC29; pASK-AID-D118K/pDHC29-Vif. For selection of ptacKanL94P, cell cultures were grown in selective medium containing 100 ␮g/ml spectinomycin (Spc). For selection of pDHC29 derived constructs, cell cultures were grown in selective medium containing 34 ␮g/ml chloramphenicol (Clo). For selection of pASK derived constructs, cell cultures were grown in selective medium containing 100 ␮g/ml ampicillin (Amp). For the RifR assay, 10–12 independent colonies were grown in selective SOB medium supplemented with 20 mM MgCl2 to an optical density of 0.6 at 660 nm and induced with 0.2 ␮g/ml of AHT (Sigma) plus 1 mM IPTG for 3 h at 37 ◦ C. Cell cultures were centrifuged, washed and spread in LB plates containing 100 ␮g/ml Amp, 100 ␮g/ml Spc and 34 ␮g/ml Clo (cell viability plates) or 100 ␮g/ml Amp, 100 ␮g/ml Spc, 34 ␮g/ml Clo and 100 ␮g/ml Rifampicin (Rif) (RifR plates). The mutation frequencies were calculated as the ratio of RifR colonies to the total number of viable cells, identified for each individual colony. Student t-test was performed between BH156 AID population (positive control) and all others in order to establish statistical significance of mutagenesis results. For the KanR assay, we proceeded in a similar manner. Cultures were spread in LB plates containing 100 ␮g/ml Amp, 100 ␮g/ml Spe and 34 ␮g/ml Clo (cell viability plates) or 100 ␮g/ml Amp, 100 ␮g/ml Spc, 34 ␮g/ml Clo and 50 ␮g/ml Kanamycin (KanR plates). The mutation frequencies were calculated as the ratio of KanR colonies to the total number of viable cells, identified

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for each individual colony. Three independent assays were performed for each RifR and KanR induced mutation assay. Student t-test was performed between BH156 AID population (positive control) and all others in order to establish statistical significance of mutagenesis results. For protein expression of AID and Vif constructs, 10 ml of cell cultures were grown for 3 h, to reach an OD of 0.6 at 660 nm and were induced with 0.2 ␮g/ml of AHT (Sigma) plus 1 mM IPTG. Cells were disrupted by sonication in PBS, plus complete protease inhibitor cocktail (Roche). Samples were separated on SDS-PAGE gels under reducing conditions, transferred to a Hybond-C Extra membrane (Amersham Biosciences) and blotted with HRP-conjugated anti-HA monoclonal antibody 3F10 (Roche) and HRP-conjugated anti-FLAG monoclonal antibody (Sigma). 4.3. Co-immunoprecipitation assays To evaluate protein–protein interactions in vivo, we performed co-immunoprecipitation assays. BH156 cells were transformed with seven different plasmid combinations; pDHC29/ pASK-AID, pDHC29-Vif/pASK-AID; pDHC29/pASK-AIDE58Q; pDHC29-Vif/pASK-AID-E58Q; pDHC29/pASK-AIDD118K; pDHC29-Vif/pASK-AID-D118K; pDHC29-VifC114F/pASK-AID. Cells were grown in SOB medium supplemented with 20 mM MgCl2 and respective antibiotics for plasmid selection. Protein expression was induced at OD660nm ∼ 0.6 with 1 mM IPTG and 0.2 ␮g/ml AHT during 3 h. Cells were disrupted by sonication in PBS and supplemented with complete protease inhibitor cocktail (Roche). Protein expression of all constructs was analyzed by SDS-PAGE gels under reducing conditions, transferred to a Hybond-C Extra membrane (Amersham Biosciences) and blotted with HRP-conjugated anti-HA monoclonal antibody 3F10 (Roche) and HRP-conjugated anti-FLAG monoclonal antibody (Sigma). After confirmation of protein expression, samples were incubated o.n. at 4 ◦ C with anti-HA affinity matrix (Roche). When applicable samples were washed four times with low-ionic buffer (0.15 M NaCl, 0.05 M Tris pH 7.5, 0.1% NP-40, and 1× with 0.15 M NaCl, 0,05 M Tris pH 7,5) or high-ionic buffer (0.5 M NaCl, 0.05 M Tris pH 7.5, 0.1% NP-40, 1% Triton X-100 and 1× with 0.5 M NaCl, 0.05 M Tris pH 7.5). Beads were boiled in Laemmli buffer and proteins were resolved by standard SDS-PAGE techniques, transferred to a Hybond-C Extra membrane (Amersham Biosciences) and blotted with HRP-conjugated anti-FLAG monoclonal antibody (Sigma) and HRP-conjugated anti-HA monoclonal antibody 3F10 (Roche). Acknowledgements We thank Michel Nussenzweig, Gregory Phillips and Michael Neuberger for the kind gift of reagents. We thank Marilia Cascalho for helpful discussion and Dinis Pestana for helpful guidelines for statistical analysis. This work was supported by grants from the Fundac¸a˜ o para a Ciˆencia e Tecnologia (POCTI/33096/MGI/2000 and PSIDA/MGI/49729/2003). A.M.F. was supported with a BI from Fundac¸a˜ o para a Ciˆencia e Tecnologia. M.S.M. and F.A.S. are recipients

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