Human APOBEC3G incorporation into murine leukemia virus particles

Virology 337 (2005) 175 – 182 www.elsevier.com/locate/yviro

Human APOBEC3G incorporation into murine leukemia virus particles Melanie Kremera,b, Alexandra Bittnera, Barbara S. Schnierlea,b,* a

Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt am Main, Germany b Paul-Ehrlich Institute, Abt. 2/01, Paul-Ehrlich Strasse 51-59, D-63225 Langen, Germany Received 21 January 2005; returned to author for revision 8 February 2005; accepted 1 April 2005 Available online 28 April 2005

Abstract The human APOBEC3G protein exhibits broad antiretroviral activity against a variety of retroviruses. It is packaged into viral particles and executes its antiviral function in the target cell. The packaging of APOBEC3G into different viral particles requires a mechanism that confers this promiscuity. Here, APOBEC3G incorporation into murine leukemia virus (MLV) was studied using retroviral vectors. APOBEC3G uptake did not require either its cytidine deaminase activity or the presence of a retroviral vector genome. Results from immunoprecipitation and co-localization studies of APOBEC3G with a MLV Gag – CFP (cyan fluorescent protein) fusion protein imply an interaction between both proteins. RNase A treatment did not inhibit the co-precipitation of Gag – CFP and APOBEC3G, suggesting that the interaction is RNA independent. Like human immunodeficiency virus (HIV) Gag, the MLV Gag precursor protein appears to interact with APOBEC3G, indicating that Gag contains conserved structures which are used to encapsidate APOBEC3G into different retroviral particles. D 2005 Elsevier Inc. All rights reserved. Keywords: APOBEC3G; MLV; Vif; Retroviral vector

Introduction The human cytidine deaminase APOBEC3G (CEM15) is an antiviral host factor that restricts replication of retroviruses (Mangeat et al., 2003; Sheehy et al., 2003) and hepadnaviruses (Turelli et al., 2004). APOBEC3G function has been mainly studied in the context of human immunodeficiency virus (HIV) infection. In the absence of the HIV Vif (viral infectivity factor) protein, APOBEC3G deaminates dC to dU in the first-strand cDNA intermediate of the reverse transcription reaction (Marin et al., 2003). This results in a severe reduction in functional viral genomes integrated into host cells and impairs viral replication. The HIV-1 accessory protein Vif is required by HIV for replication in non-permissive cells, such as primary T cells, which express APOBEC3G. Vif-deficient HIV variants and murine leukemia virus (MLV), which does not encode Vif or a functional homolog, incorporate APOBEC3G into viral * Corresponding author. Paul-Ehrlich Institute, Paul-Ehrlich Strasse 51-59, D-63225 Langen, Germany. Fax: +49 6103 77 1273. E-mail address: [email protected] (B.S. Schnierle). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.04.006

particles. The outcome is the delivery of the mutagen to the target cell, making it available to act on the viral cDNA. MLV has been described to be resistant to the murine homologue of APOBEC3G, which explains the absence of a Vif-like activity in MLV (Kobayashi et al., 2004). Vif neutralizes APOBEC3G by preventing its encapsidation into HIV virions by a mechanism that is not entirely understood. It has been suggested that Vif induces the ubiquitin-dependent degradation of APOBEC3G (Marin et al., 2003; Rose et al., 2004; Yu et al., 2003) and/or Vif affects the translation of APOBEC3G (Kao et al., 2003; Stopak et al., 2003). The mechanism of APOBEC3G incorporation into retroviral particles is also not well understood. It has been suggested that APOBEC3G is encapsidated by direct interaction with the HIV Gag precursor protein (Alce and Popik, 2004; Cen et al., 2004; Douaisi et al., 2004), the nucleocapsid (NC) (Luo et al., 2004) or RNA (Schafer et al., 2004; Svarovskaia et al., 2004; Zennou et al., 2004). Given that APOBEC3G affects distantly related retroviruses, its packaging should involve a common mechanism. We have studied the incorporation of APOBEC3G into MLV vector

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particles and show here that APOBEC3G is encapsidated by interaction with MLV Gag and does not require the presence of genomic RNA.

Results The cytidine deaminase activity of APOBEC3G is required to reduce retroviral vector titers We analyzed the consequence of APOBEC3G expression on MLV infectivity using MLV-based retroviral vectors. Vectors were transiently produced in 293T cells, either in the presence or absence of APOBEC3G expression. Infectivity of the vectors was monitored by transduction of a retroviral vector genome encoding the green fluorescent protein (GFP) into NIH3T3 cells. In addition to wild-type (wt) APOBEC3G, we used an APOBEC3G variant carrying a single amino acid exchange at position 259 (APO-E259Q). This mutation affects the conserved zinc binding domain (H-XE-(X)24-30-PCXXC) found in the active site of all cytidine deaminases and the mutated glutamate is involved in proton shuttling (Jarmuz et al., 2002). Two aromatic residues in this (X)24-30 domain are predicted to be involved in RNAbinding (Jarmuz et al., 2002). Although not experimentally shown here, it is very unlikely that RNA binding is affected by the E259Q mutation. It has been previously described to have almost completely lost its antiviral effect on HIV infectivity due to impaired cytidine deaminase activity (Shindo et al., 2003). Fig. 1 summarizes the titers obtained. Titers obtained from vectors produced by co-transfection of

Fig. 1. Retroviral vector titers determined by monitoring GFP-positive cells. 293T cells were transfected with 7 Ag pHIT60 (Gag/Pol), 3 Ag pEnv wt(HX) (Env) 10 Ag pSFG-EGFP (vector encoding GFP) and 5 Ag pcDNA – APOBEC3G – Myc (APOBEC3G), pcDNA – E259Q (APO – E259Q) or pRC – CMV (empty vector as control) expression plasmids using a standard calcium phosphate precipitation technique. Transductions were carried out by incubating NIH3T3 target cells for 2 days with 1 ml, 100 Al and 10 Al of supernatants from transfected 293T cells containing retroviral particles. Viral titers were determined from the percentage of GFP-positive cells. The titers are expressed in infectious units per ml (IU/ml).

the empty expression plasmid (pRC-CMV) were set to 100%. In the presence of an APOBEC3G expression vector, the titer declined to 7.8%. The APOBEC3G variant APOE259Q, however, only slightly interfered with the transduction rate (78%) indicating that, in agreement with previous observation with HIV, this mutant had lost its antiviral activity against MLV. APOBEC3G and APO-E259Q are incorporated into MLV particles independently of vector mRNA Encapsidation of APOBEC3G and APO-E259Q into MLV-based vector particles was analyzed by Western blot analysis. Viral particles were prepared from supernatants of transfected 293T cells by ultracentrifugation through a 30% sucrose cushion. The human APOBEC3G gene was expressed as a C-terminal fusion with the Myc-tag (APOBEC3G-Myc), which enabled APOBEC3G detection by an antibody directed against the Myc epitope (Marin et al., 2003). APOBEC3G expression was first analyzed in 293T cell lysates and equal gel loading was confirmed by detection of h-actin (Fig. 2A). Analysis of viral particles showed that both the wt and the mutant APOBEC3G proteins were incorporated into particles (Fig. 2B, lanes 1, 2 and 4, 5). Slight variations in the amount of APOBEC3G protein detected were due to loading differences, as indicated by levels of the MLV capsid (CA) protein p30. APOBEC3G was encapsidated in the absence of vector mRNA (Fig. 2B, lanes 2 and 5). We next addressed whether co-expression of the HIV Vif gene also affects APOBEC3G incorporation into MLV particles. Retroviral vectors were produced in the presence of APOBEC3G and HIV-1 Vif expression. The co-expression of Vif diminished the amount of APOBEC3G in cell lysates (Fig. 2C, lane 1) and resulted in its exclusion from MLV vector particles (Fig. 2D, lane 1). However, Vif expression during vector production resulted in Vif incorporation into MLV particles, independent of APOBEC3G co-expression, which might result from overexpression of Vif (Fig. 2D, lanes 1 and 3). Vector titers were higher when Vif was co-expressed: transduction rates in the presence of APOBEC3G alone were only 35% of those obtained in the additional presence of Vif (data not shown). This indicates that Vif also restored APOBEC3G-restricted MLV infectivity. MLV Gag partially co-localizes with APOBEC3G and can be co-precipitated with APOBEC3G Packaging of APOBEC3G into HIV particles is mediated by the Gag protein (Alce and Popik, 2004; Cen et al., 2004; Luo et al., 2004; Schafer et al., 2004; Svarovskaia et al., 2004; Zennou et al., 2004) and an interaction of APOBEC3G with MLV Gag has recently been observed following overexpression of both proteins by vaccinia virus-expressed T7 polymerase (Douaisi et al., 2004). We

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Fig. 2. APOBEC3G incorporation into MLV particles. (A) Western blot analysis of transfected 293T cells lysates. The amount of expression plasmid used for transfections is indicated in Ag. The APOBEC3G protein was detected via its Myc-tag with an anti-Myc antibody and equal loading was analyzed, after stripping the blot, with an antibody directed against h-actin. (B) Western blot analysis of viral particles obtained by ultracentrifugation of cell culture supernatants from transfected 293T cells. The APOBEC3G protein was detected via its Myc-tag with an anti-Myc antibody and equal loading was analyzed, after stripping the blot, with an antibody directed against the MLV Gag p30 CA protein. (C) Western blot analysis of transfected 293T cells lysates. The amount of expression plasmid used for transfections is indicated in Ag. The APOBEC3G-Myc protein was detected with an anti-Myc antibody, Vif was detected with an anti-Vif antibody and equal loading was analyzed, after stripping the blot, with an antibody directed against h-actin. (D) Western blot analysis of viral particles obtained by ultracentrifugation of cell culture supernatants from transfected 293T cells. The APOBEC3G-Myc protein was detected with an anti-Myc antibody, Vif with an anti-Vif antibody and equal loading was analyzed, after stripping the blot, with an antibody directed against the MLV Gag p30 CA protein. Vif was detected after running an additional SDS-PAGE and Western blot analysis with twice the amount of viral particles as that used for APOBEC3G and Gag.

studied the co-localization of APOBEC3G and MLV Gag by using a Gag expression plasmid encoding a chimeric Gag molecule which had the cyan fluorescent protein (CFP) fused to the C terminus of the NC domain. Expression of this construct leads to the formation of fluorescently labeled, non-infectious viral particles (generous gift of M. Collins and Y. Takeuchi) (Andrawiss et al., 2003). This fusion protein has been used previously to detect particle production by measuring fluorescence. Gag –CFP protein releases virus-like particles upon transfection into 293T cells, which are detectable by microscopy as fluorescent spots (Andrawiss et al., 2003) (Fig. 3B). Plasmids encoding APOBEC3G-Myc and Gag –CFP were transfected into 293T cells. After 2 days, cells were fixed, stained with an antiMyc antibody (9E10) and analyzed by confocal laser scanning microscopy. APOBEC3G expression could be detected in the cytoplasm (Fig. 3A) and Gag – CFP showed cytoplasmic and dotted membrane staining (Fig. 3B). Some of the Gag –CFP spots were enriched with APOBEC3G, appearing as white dots in the overlay in Fig. 3D (indicated by arrows). We further studied the interaction of APOBEC3G with MLV Gag. After transfection of APOBEC3G-Myc and

Gag – CFP or wt Gag (pHIT60) expression plasmids into 293T cells, viral particles were harvested by ultracentrifugation and the material was analyzed by Western blot. Particle formation was analyzed by detection of Gag – CFP using an anti-GFP antibody or detection of Gag using an anti-p30 CA antibody (Fig. 4A). In addition, APOBEC3GMyc was immunoprecipitated from pelleted viral particles with an antibody directed against the Myc epitope. The precipitated material was analyzed by Western blot using an anti-GFP or an anti-p30 CA antibody. Gag is usually cleaved by the viral protease into matrix (MA), CA and NC (Fig. 4A, lane 2), however, the Gag – CFP protein remains as a precursor and is not cleaved (Andrawiss et al., 2003) (Fig. 4A, lanes 1 and 4). Co-precipitation of Gag – CFP with APOBEC3G could be observed (Fig. 4B, lane 1) and interaction with wt Gag precursor and CA p30 could be observed after longer exposure of the Western blot (Fig. 4B, lanes 2 and 5). A minor background binding of the Gag –CFP precursor protein to the beads alone could be observed (Fig. 4B, lane 4); however, the interaction was always more prominent in the presence of APOBEC3G, indicating that this protein interacts with MLV Gag components.

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The interaction of APOBEC3G with Gag – CFP interaction is not affected by RNase A treatment

Fig. 3. Co-localization of APOBEC3G and Gag – CFP. The Gag – CFP expression plasmid, encoding a chimeric Gag molecule containing CFP fused to the C terminus of the NC domain and the APOBEC3G – Myc fusion constructs were transfected in equal amounts into 239T cells. Cells were fixed with methanol/acetone (1/1) and incubated with an anti-Myc antibody, followed by an R-PE-labeled anti-mouse antibody. Cells were examined with a Zeiss Laser-Scanning Microscope LSM510 equipped with a 63 objective. Enhanced cyan fluorescent protein (ECFP) was excited with a krypton 413 laser line and R-PE was excited with a HeNe 543 laser line. (A) APOBEC3G, (B) Gag – CFP, (C) bright field, (D) overlay.

We have shown that APOBEC3G is incorporated into MLV vector particles in the absence of a viral genome (Fig. 2B), suggesting that APOBEC3G incorporation does not require viral genomic RNA. Therefore, the interaction of Gag – CFP and APOBEC3G was analyzed in the presence or absence of 1 mg/ml RNase A (Qiagen, Hilden, Germany). APOBEC3G-Myc, Gag – CFP and a retroviral vector encoding GFP or an empty expression plasmid (pRC-CMV) were transfected into 293T cells and viral particles were concentrated by ultracentrifugation 3 days later. Particle formation was confirmed by Western blot analysis using an anti-GFP antibody (Fig. 5A). APOBEC3G-Myc was immunoprecipitated from these viral particles using an anti-Myc antibody. The samples were divided into two equal groups, one of which was treated with RNase A during the immunoprecipitation procedure. Western blot analysis confirmed immunoprecipitation of APOBEC3G-Myc from viral particles (Fig. 5B, lower band) and Gag –CFP could be co-immunoprecipitated from samples containing APOBEC3G-Myc. Neither the presence of the vector RNA (Fig. 5C compare lanes 1 and 5 with 4 and 8) nor the treatment with RNase A (Fig. 5C, lanes 5 and 8) influenced the co-immunoprecipitation of Gag –CFP. This indicates that the interaction of APOBEC3G with MLV Gag is RNA-independent or only

Fig. 4. Co-immunoprecipitation of Gag – CFP with APOBEC3G. Virus-like particles were generated by transfection of Gag/Pol expression plasmids in the presence or absence of APOBEC3G – Myc. The plasmid amount is given in Ag. Viral particles were prepared as described before and analyzed by Western blot. (A) Detection of Gag either with an antibody directed against GFP to identify the Gag – CFP fusion protein or with an anti-p30 CA antibody. The Gag – CFP protein is not cleaved by the viral protease (lanes 1 and 4) unlike the wt Gag protein (lane 2). (B) Viral particles were also used for immunoprecipitations with an anti-Myc antibody. Gag – CFP in the precipitate was detected with an anti-GFP antibody and p30 CA with an anti-p30 CA antibody. The presence of APOBEC3G – Myc allowed the co-precipitation of Gag – CFP and very low amounts of wt Gag, only detectable after longer exposure of the blot (lane 5).

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Fig. 5. The APOBEC3G – Gag – CFP interaction is not affected by RNase A treatment. Virus-like particles were generated in 293T cells, purified by ultracentrifugation and either directly analyzed for particle formation by Western blot analysis with an anti-GFP antibody (A) or used for immunoprecipitations using an anti-Myc antibody. Successful immunoprecipitation was controlled by Western blot analysis of the precipitate with an anti-Myc antibody (B) and coprecipitation of Gag – CFP was detected with an anti-GFP antibody (C).

dependent on small RNAs not accessible to cleavage by RNase A. In addition, we verified the APOBEC3G/Gag – CFP interaction in cell lysates of transfected 293T cells to exclude that RNA is involved in the initial APOBEC3G/ Gag – CFP interaction in the cytoplasm, but not required later in the viral particle. APOBEC3G-Myc was immunoprecipitated from cell lysates and the samples were either treated with RNase A or left untreated during the immunoprecipitation procedure. As shown in Fig. 6, RNase A did not significantly inhibit the APOBEC3G/Gag – CFP interaction and the slightly lower amount of precipitated Gag – CFP is due to loading differences (Figs. 6A, B, lanes 1 and 3). Due to unspecific binding of Gag – CFP to sepharose

Fig. 6. The APOBEC3G – Gag – CFP interaction in cell lysates is not affected by RNase A treatment. Cell lysates were generated from 293T cells and either directly analyzed for Gag – CFP expression by Western blot analysis with an anti-Gag antibody (C) or used for immunoprecipitations using an anti-Myc antibody. Successful immunoprecipitation was controlled by Western blot analysis of the precipitate with an anti-Myc antibody (B) and co-precipitation of Gag – CFP was detected with an anti-Gag antibody (A).

beads, we had to use lower concentrations of RNase A with cell lysates in contrast to viral particles, however, 1 Ag/ml RNase A did not affect this interaction and indicates that RNA is not essential for the APOBEC3G/Gag – CFP interaction.

Discussion The effect of human APOBEC3G on MLV infection has been controversially discussed. While some publications have described a clear antiviral function (Harris et al., 2002; Kobayashi et al., 2004; Mangeat et al., 2003), others did not observe a strong inhibition of MLV infectivity by APOBEC3G (Mariani et al., 2003). Using MLV-based retroviral vectors, we did observe a strong reduction of their transduction efficiency in the presence of APOBEC3G. An APOBEC3G variant with impaired deaminase activity did not significantly interfere with vector transduction, which further demonstrates that deaminase catalytic activity is required to confer the antiretroviral effect of APOBEC3G. The human APOBEC3G protein is incorporated into HIV and MLV particles. We confirmed incorporation into MLV particles and could further demonstrate that neither APOBEC3G’s deaminase activity nor the presence of vector RNA is required for packaging into MLV particles. To study the encapsidation mechanism, we used a variant of the MLV Gag precursor protein including an NC –CFP fusion protein (Gag– CFP). This is not processed into the separate Gag domains, but forms virus-like particles in the absence of envelope protein and genomic RNA (Andrawiss et al., 2003). Using confocal laser scanning microscopy and coimmunoprecipitations, we observed that APOBEC3G interacts with Gag – CFP. Co-localization was observed in punctuated areas resembling budding viral particles. Co-

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immunoprecipitation with APOBEC3G could be shown for the precursor protein Gag – CFP and a weak interaction could be observed with wt Gag. This weak interaction with wt Gag might be due to lower amounts of wt Gag protein in 293T cells (Fig. 4A, lane 2). The APOBEC3G –Gag – CFP interaction resisted RNase A treatment, arguing for an RNA-independent interaction. However, RNA has been described to be essential for APOBEC3G binding to HIV Gag (Schafer et al., 2004). The amino acid sequences of retroviral Gag proteins have only a low degree of sequence homology, and packaging of non-specific RNA by Gag as well as APOBEC3G binding to RNA has been described (Jarmuz et al., 2002). As APOBEC3G has been reported to bind to RNA, binding to Gag via RNA cannot be entirely excluded. However, all retroviral Gag precursor proteins are organized in the same order, that is, MA, CA and NC. Gag proteins share common features, for example, membrane association and myristylation of MA. NC is highly conserved among retroviruses. It contains zinc finger motifs which are surrounded by basic regions and binds the genomic RNA. These common functions make it a good candidate for a ubiquitous interface and a direct binding of APOBEC3G to the HIV NC domain has been described (Cen et al., 2004). Co-expression of APOBEC3G and Vif during MLV vector production in 293T cells resulted in an elimination of the APOBEC3G protein from cell lysates and, as expected, its absence from viral particles. In addition, Vif expression reversed the inhibitory effect of APOBEC3G on viral titers. However, the experiments were carried out by overexpression of Vif in the presence of low amounts of APOBEC3G, which might account for the absence of APOBEC3G protein in cell lysates. Incomplete protein degradation of APOBEC3G by Vif has been described (Kao et al., 2004). We also found Vif protein incorporated into MLV particles, an observation described previously by others (von Schwedler et al., 1993), which makes it tempting to speculate that another Vif function might include masking a domain on Gag that mediates the interaction with residual APOBEC3G protein, however, this might as well only reflect an unspecific uptake of Vif due to its overexpression. APOBEC3G encapsidation into HIV and MLV particles involves a common interaction with the Gag precursor protein. The HIV Vif protein also interferes with the antiviral function of APOBEC3G against MLV. This makes MLV an attractive model system to be used to identify and study inhibitors of the APOBEC3G – Vif interaction, which could potentially be used in HIV therapy.

Myc-tagged human APOBEC3G (Marin et al., 2003). Gag –CFP was constructed based on the pHIT60 plasmid (Soneoka et al., 1995) and the Pol coding region between nucleotides 2235 and 5733 (corresponding to the Mo – MLV sequence) was replaced by the CFP coding sequences resulting in expression of Gag with a NC CFP fusion protein (Andrawiss et al., 2003). Transfection of this plasmid into 293T cells results in the production of fluorescent, non-infectious particles. The plasmid pHIT60 encodes the MLV Gag/Pol region (Soneoka et al., 1995); pEnv wt(HX) encodes the ecotropic MLV envelope protein (Erlwein et al., 2002); and pSFG – EGFP is a MLV-based retroviral vector encoding GFP (Lindemann et al., 1997). Transfection of pHIT60, pEnv wt (HX) and pSFG – EGFP into 293T cells result in the production of infectious vector particles, which are able to transduce the GFP encoding vector sequences into target cells. PNLA1(CD4 ) encodes the HIV-1 Vif, Tat, Rev, Vpu and Nef, allowing very high expression of Vif in 293T cells (Willey et al., 1992). The point mutation in the APOBEC3G coding sequences (E259Q) was introduced using the Quick Change Site-directed Mutagenesis Kit (Stratagene, Heidelberg), according to the manufacturer’s protocol and using the oligonucleotides with the sequences Am-1: 5V-gcc gcc atg cac agc tgt gct tcc and Am-2: 5V-gga agc aca gct gtg cat ggc ggc. The variant was sequenced over the entire coding region. Cell culture, transfections, viral infection and determination of titers 293T cells and NIH3T3 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO/BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS; GIBCO/BRL, Eggenstein, Germany). A day before transfection, 293T cells were seeded at a density of 3  106 cells in a 10-cm tissue culture plate. The cells were transfected with 10 –20 Ag expression plasmids using a standard calcium phosphate precipitation technique. After 2 days of culture, serial dilutions of viral supernatants from transfected 293T cells were passed through 0.45-Am filters (Greiner, Frickenhausen, Germany) and incubated with 2  105 NIH 3T3 cells. 48 –72 h after transduction, the numbers of GFP-expressing cells were detected by FACS analysis. The titers are given in infectious units per ml (IU/ml) and are the average of three independent experiments. Western blot analysis

Materials and methods Plasmids The following plasmids were used for transfections: pcDNA – APOBEC3G – Myc encoding a C-terminally

Cell lysates were obtained and Western blot analyses were performed as described previously (Schnierle et al., 1996). Western blot analysis was performed with the following antibodies: mouse anti-Myc antibody (BDBiosciences, Heidelberg, Germany), mouse anti-h-actin

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antibody (Sigma-Aldrich, Munich, Germany), polyclonal rabbit antiserum directed against MLV CA p30 (kind gift of J. Schmidt, GSF, Munich, Germany), mouse anti-Vif antibody (NIH AIDS Research and Reference Reagent Program, #319), polyclonal rabbit antiserum directed against GFP (BD-Biosciences-Clontech, Heidelberg, Germany), which also recognizes CFP and horseradish peroxidase-coupled sheep anti-mouse IgG antibodies or protein A (Amersham Biosciences, Freiburg, Germany). Detection was performed using an enhanced chemiluminescence Western blot detection kit (Amersham Biosciences, Freiburg, Germany). Isolation of viral particles Two days after transfection of 293T cells with expression plasmids, the cell supernatants were filtered through a 0.45-Am filter and viral particles were pelleted through a 30% sucrose cushion for 90 min at 35,000 rpm in a SW41 rotor. Concentrated viral particles were analyzed by Western blot as described for cell lysates or were used for immunoprecipitations. Immunoprecipitation Viral particles, partially purified by ultracentrifugation, were resuspended in 500 Al TNT buffer [20 mM Tris – HCl (pH 7.5), 200 mM NaCl, 1% Triton X-100, 1 mM Pefabloc (Roche, Mannheim, Germany)] and 2 Al anti-Myc-tagantibody (BD-Bioscience, Heidelberg, Germany) were added and incubated for 2 h at 4 -C. Immune complexes were collected with protein A/G-sepharose (Santa Cruz, Biotechnology, Heidelberg, Germany) and washed three times with TNT buffer. Bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE, blotted onto nitrocellulose membranes and were subjected to Western blot analysis. Immunocytochemistry Cells were fixed with methanol/acetone (1/1) and APOBEC3G was detected after successive incubations with an anti-Myc antibody and an R-PE-coupled goat anti-mouse IgG antibody (DAKO, Hamburg, Germany). Cells were examined with a Zeiss Laser-Scanning Microscope LSM510 equipped with a  63 objective. Enhanced cyan fluorescent protein (ECFP) was excited with a krypton 413 laser line and R-PE was excited with a HeNe 543 laser line.

Acknowledgments We thank C. Haynes for helpful discussions and critically reading the manuscript. We are grateful to D. Kabat, M. Collins, A.J. Kingsman, D. Lindemann and V. Bosch for

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kindly providing the plasmids pcDNA-APOBEC3G-Myc, Gag – CFP, pHIT60, pSFG-EGFP and pNL-A1(CD4 ).

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