Specific Packaging of APOBEC3G Into HIV-1 Virions Is Mediated by the Nucleocapsid Domain of the Gag Polyprotein Precursor

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Virology 328 (2004) 163–168 www.elsevier.com/locate/yviro Rapid Communication Specific packaging of APOBEC3G into HIV-1 virions is mediated by the nucleocapsid domain of the gag polyprotein precursor

Alexandra Sch7fer, Hal P. Bogerd, Bryan R. Cullen*

Howard Hughes Medical Institute and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA Received 25 June 2004; accepted 4 August 2004 Available online 9 September 2004

Abstract

In cells infected by HIV-1 mutants lacking a functional Vif protein, APOBEC3G is specifically packaged into progeny virions and then interferes with the process of virus infection. Here, we show that incorporation of APOBEC3G into HIV-1 virions is mediated by the specific interaction of APOBEC3G with the carboxy-terminal nucleocapsid/p6 domain of the Gag polyprotein precursor. As a result, HIV-1 virus-like particles that lack the nucleocapsid domain fail to package APOBEC3G. Surprisingly, RNA was also found to be essential for formation of the nucleocapsid–APOBEC3G complex in vitro, thus raising the possibility that RNA may form a bridge between these two proteins. D 2004 Elsevier Inc. All rights reserved.

Keywords: HIV-1; APOBEC3G; Virion incorporation; RNA binding

Introduction leukemia virus (Harris et al., 2003; Mangeat et al., 2003), one would predict that the virion binding sites for In cells infected with wild-type HIV-1, the host APOBEC3G are likely to be highly conserved. Here, we antiretroviral defense protein APOBEC3G is bound by the demonstrate that APOBEC3G incorporation into HIV-1 viral Vif protein and then targeted for degradation by the virions is mediated by the conserved nucleocapsid (NC) proteasome (Conticello et al., 2003; Marin et al., 2003; domain of Gag acting in concert with RNA. Sheehy et al., 2003; Stopak et al., 2003; Yu et al., 2003). In contrast, in APOBEC3G-expressing cells infected by HIV-1 mutants lacking a functional Vif protein, APOBEC3G is Results specifically packaged into progeny HIV-1 virions and then interferes with the process of reverse transcription by Although it is well established that virion packaging of massively editing the nascent proviral DNA minus strand APOBEC3G represents a key step in the mechanism of (Harris et al., 2003; Mangeat et al., 2003; Zhang et al., action of this host antiretroviral defense factor (Mariani et 2003). A key question is how APOBEC3G is selectively al., 2003; Marin et al., 2003; Sheehy et al., 2003; Stopak et incorporated into HIV-1 virions, a step that is critical for the al., 2003), it has been unclear how APOBEC3G is able to antiretroviral activity of APOBEC3G. However, as APO- specifically associate with progeny HIV-1 virions. To BEC3G can also inhibit the activity of a range of other address this question, we first asked whether APOBEC3G retroviruses, including the only very distantly related murine would be able to inhibit the infectivity of pseudotyped HIV- 1 virions produced in the absence of six of the nine HIV-1 gene products, that is, in the absence of Env, Tat, Vpu, Vpr, * Corresponding author. Howard Hughes Medical Institute and Depart- ment of Molecular Genetics and Microbiology, Duke University Medical Vif, and Nef, as well as in the absence of the bulk of the Center, PO Box 3025, Durham, NC 27710. Fax: +1 919 681 8979. viral RNA genome. For this purpose, we used the pLL3.7 E-mail address: [email protected] (B.R. Cullen). lentiviral vector (Fig. 1A), which retains only the minimal

Fig. 1. Inhibition of the infectivity of pseudotyped minimal HIV-1 virions by hAPOBEC3G. (A) Schematic structure of the LL3.7 lentiviral vector. CMV, cytomegalovirus promoter; C, RNA packaging sequence; cPPT, central polypurine tract; EGFP, enhanced green fluorescent protein gene; WRE, woodchuck hepatitis virus response element; SIN LTR, self-inactivating long terminal repeat retaining R and U5 but lacking almost all of U3. (B through G) Fluorescence images of cells infected with LL3.7 virions produced in the presence or absence of hAPOBEC3G, mAPOBEC3, and/or HIV-1 Vif, as indicated. cis-acting sequence elements required for HIV-1 genome produced in the presence of HIV-1 Gag, Pol, Rev, and Tat packaging (i.e., the C sequence) and reverse transcription but in the absence of Env, Vpu, Vif, Vpr, and Nef (Mangeat (i.e., the primer binding site, the polypurine tracts (PPT) and et al., 2003). part of the LTR) (Fig. 1A) (Rubinson et al., 2003). In As hAPOBEC3G is able to inhibit the infectivity of the addition, this vector encodes enhanced green fluorescent LL3.7 virions analyzed in Fig. 1, these should share the protein (EGFP) transcribed under the control of a cytome- ability of HIV-1 virions produced in the presence of all the galovirus (CMV) promoter element (Fig. 1A). pLL3.7 was viral proteins except Vif to specifically package an HA packaged into virions by co-transfection into 293T cells epitope tagged form of hAPOBEC3G. To confirm this together with plasmids expressing the vesicular stomatitis hypothesis, we transfected 293T cells with pLL3.7 together virus G (VSV-G) protein and the HIV-1 gag and pol genes with psynGag/Pol, a Rev-independent, codon optimized as well as a plasmid expressing HIV-1 Rev. The 293T cells Gag/Pol expression plasmid (Cowan et al., 2002), and a were also co-transfected with vectors expressing human plasmid-expressing hAPOBEC3G-HA (Bogerd et al., APOBEC3G (hAPOBEC3G), mouse APOBEC3 (mAPO- 2004). As shown in lane 1 of Fig. 2B, virions released BEC3) and/or HIV-1 Vif (Bogerd et al., 2004). Released from this culture were indeed able to package hAPO- LL3.7 virions were then used to infect a second culture of BEC3G-HA effectively. This packaging was specific, as no 293T cells. hAPOBEC3G-HA was recovered from the supernatant As shown in Fig. 1B, the LL3.7 lentiviral vector was able media of 293T cells transfected with hAPOBEC3G-HA to efficiently transduce 293T cells with the EGFP gene. Co- and pLL3.7 in the absence of the psynGag/Pol expression expression of hAPOBEC3G or mAPOBEC3 resulted, plasmid (Fig. 2B, lane 5). Moreover, a control HA-tagged however, in an almost complete inhibition of EGFP trans- cytoplasmic protein of similar size, h-arrestin 2 (Oakley et duction (Fig. 1C and D). Additional co-expression of HIV-1 al., 2000), was not detectably packaged into released HIV-1 Vif fully restored the infectivity of LL3.7 virions produced virions (Fig. 2B, lane 6). in the presence of hAPOBEC3G (Fig. 1F) but did not To more accurately determine the requirements for relieve the inhibition caused by mAPOBEC3 (Fig. 1G), as hAPOBEC3G-HA packaging into HIV-1 virions, we also also previously demonstrated for wild-type HIV-1 virions generated non-infectious virions produced in the absence of (Mariani et al., 2003). We therefore conclude that APO- a packageable RNA genome (Fig. 2, lane 2), in the presence BEC3G is able to block the infectivity of HIV-1 virions of only the Gag polyprotein and the protease component of containing only the HIV-1 Gag and Pol proteins and very pol (Fig. 2, lane 3) and finally in the presence of only the limited segments from the viral RNA genome. These data Gag polyprotein (Fig. 2, lane 4). In all cases, hAPOBEC3G- therefore confirm and extend a previous report showing that HA continued to be efficiently packaged into the released hAPOBEC3G can inhibit the infectivity of HIV-1 virions HIV-1 virion particles (Fig. 2B, lanes 2 to 4). We therefore Rapid Communication 165

Fig. 2. The HIV-1 Gag polyprotein is sufficient for virion packaging of hAPOBEC3G. (A) Western analysis of the lysates of virion producer cells using HIV-1 capsid (upper panel) or HA epitope tag-specific mouse monoclonal antibodies (lower panel). (B) HIV-1 virions released by the cultures analyzed in panel A were collected and also subjected to Western blot. For both panels, 293T cells were transfected with: lane 1, 1 Ag of pLL3.7, 500 ng of psynGag/Pol and 500 ng of ph3G-HA; lane 2, 500 ng of psynGag/Pol and 500 ng ph3G-HA; lane 3, 500 ng of psynGag/Pro and 500 ng ph3G-HA; lane 4, 500 ng of psynGag and 500 ng ph3G-HA; lane 5, 1 Ag of pLL3.7 and 500 ng of ph3G-HA; lane 6, 1 Ag of pLL3.7, 500 ng of psynGag/Pol and 500 ng of phARR-2-HA. Levels of transfected DNA were held constant by supplementation to 2 Ag per transfection with a control plasmid. (C) Similar to panel A. (D) Similar to panel B. For panels C and D, 293T cells were transfected with: lane 1, 1 Ag of pLL3.7 and 500 ng of ph3G-HA; lane 2, 500 ng of psynGag and 500 ng of ph3G-HA; lane 3,

500 ng of psynGag/ZWT and 500 ng of ph3G-HA. conclude that the HIV-1 Gag polyprotein precursor is the 6). We therefore conclude that hAPOBEC3G specifically only viral protein required for virion incorporation of interacts with the NC/p6 domain of the Gag polyprotein hAPOBEC3G. precursor and that this interaction likely mediates pack- If HIV-1 Gag mediates hAPOBEC3G packaging, then aging of hAPOBEC3G into HIV-1 virion particles. We note Gag should contain one or more specific binding sites for that as this experiment used proteins recovered from hAPOBEC3G. To test this hypothesis, we used in vitro expressing cells (APOBEC3G-HA) or translated in a translation in the presence of 35S-methionine to synthesize reticulocyte lysate (NC/p6), we cannot be certain that the the full-length ~55 kDa Gag precursor, or portions of Gag observed interaction is direct. consisting of the ~17 kDa matrix (MA) protein, the ~24 It has previously been demonstrated that hAPOBEC3G kDa capsid (CA) protein or the ~16 kDa carboxy terminal can inhibit the infectivity of a wide range of retroviruses, region of Gag, consisting of the ~7 kDa NC protein linked including HIV-1 as well as other primate and non-primate to p6 (Fig. 3A). Recombinant hAPOBEC3G-HA was lentiviruses and the distinct oncoretrovirus murine leukemia prepared by overexpression in transfected 293T cells and virus (MLV) (Mangeat et al., 2003; Mariani et al., 2003). It was recovered from the lysate by incubation in the presence therefore seems probable that packaging of hAPOBEC3G of an HA affinity matrix (Fig. 3C). Mock-transfected 293T must involve one of the few protein sequence motifs that are cells served as a negative control. After loading with conserved across the lentivirus and oncoretrovirus families. hAPOBEC3G-HA, the HA affinity matrix was incubated APOBEC3G specifically binds to the carboxy-terminal ~16 with the 35S-labeled Gag proteins and bound proteins kDa of the ~55 kDa HIV-1 Gag polyprotein, which includes recovered by centrifugation. As shown in Fig. 3B, we noted the ~7 kDa NC protein as well as p6 (Fig. 3). However, p6 is specific binding of the full-length Gag protein to the not conserved in MLV, thus suggesting that NC may be key hAPOBEC3G-HA loaded affinity matrix but not to the for hAPOBEC3G incorporation into HIV-1 virions. control matrix (lanes 1 and 2). In addition, we also To directly test whether the HIV-1 NC protein mediates observed strong binding of the NC/p6 protein to the hAPOBEC3G incorporation into progeny virions, we took hAPOBEC3G-HA matrix (lanes 7 and 8) but no detectable advantage of the previous observation (Accola et al., 2000) binding of either the HIV-1 MA or CA protein (lanes 3 to that the NC domain of HIV-1 Gag, which is normally 166 Rapid Communication

3). Therefore, the NC protein indeed plays an essential role in mediating virion packaging of APOBEC3G, as predicted by the in vitro binding data presented in Fig. 3. A key function of the NC domain of Gag in the retroviral life cycle is the specific incorporation of the viral RNA genome into virion particles. While the NC domain of Gag is therefore critical for the specific interaction of the Gag precursor with the viral RNA genome, NC can also interact with RNA non-specifically (Dorfman et al., 1993; Gorelick et al., 1993). We have presented evidence demonstrating that the viral RNA genome is not required for hAPOBEC3G packaging into HIV-1 virion particles (Fig. 2)orfor hAPOBEC3G binding by the NC/p6 protein (Fig. 3). However, it remains possible that non-specific RNA binding by NC is important. To test this hypothesis, we subjected both the HA-affinity matrix-bound hAPOBEC3G protein and the in vitro translated NC/p6 protein to exhaustive digestion with RNase A. Interestingly, this digestion resulted in the loss of a slower migrating form of the NC/p6 fusion protein (see asterisk in Fig. 4A) and an increase in the intensity of the protein band migrating at the predicted approximately 16 kDa size (compare lanes 4 to 6 with lanes 1 to 3 in Fig. 4A). We therefore hypothesize that this slower migrating band represents a NC–RNA complex that remains stable even when subjected to electrophoresis through an SDS–poly- acrylamide gel. We note that less complete digestion with RNase A, as performed in Fig. 3, did not result in loss of Fig. 3. The hAPOBEC3 protein specifically interacts with the NC/p6 domain of the Gag polyprotein. (A) 35S-methionine-labeled forms of the this hypothetical complex (see lanes 7 and 8 in Fig. 3A) and full-length approximately 55 kDa Gag polyprotein (lanes 1 and 2), the indeed did not completely remove all RNA (data not approximately 24 kDa CA protein (lanes 3 and 4), the approximately 17 shown). kDa MA protein (lanes 5 and 6) and the approximately 16 kDa carboxy- The key observation shown in Fig. 4B is, however, the terminal domain of Gag, consisting of NC and p6 as well as the p2 and p1 finding that exhaustive RNase A digestion effectively spacers, were generated by in vitro translation. This gel shows the input- labeled proteins used in the binding reaction, as well as molecular weight blocks hAPOBEC3G binding by the NC/p6 protein. We markers. (B) This panel shows the 35S-labeled proteins retained by an HA therefore conclude that RNA plays an essential role in affinity matrix loaded with HA-APOBEC3G expressed in 293T cells (+). supporting the interaction of NC with hAPOBEC3G. While The negative control lanes (À) used a 293T cell lysate lacking HA- the RNA may simply be important for maintaining NC in a APOBEC3G. (C) Expression of HA-APOBEC3G in 293T cells transfected functional conformation, we note that hAPOBEC3G can with ph3G-HA or with the control pcDNA3 plasmid was visualized by Western analysis. These lysates were used to load the HA affinity matrix bind RNA directly (Yu et al., 2004), thus raising the utilized in panel B. possibility that RNA plays a direct role in mediating formation of a hAPOBEC3G–NC complex, perhaps by acting as a bridge. essential for virion production, can be functionally substituted with a 34 aa leucine zipper derived from the yeast GCN4 protein. Although this substitution effectively res- Discussion cues virion production, it does not rescue viral infectivity. We precisely substituted this GCN4 coding sequence, In this manuscript, we have sought to identify the basis termed ZWT (Accola et al., 2000), in place of sequences for the selective incorporation of hAPOBEC3G into virion encoding the 7 kDa NC protein, and a flanking 1 kDa spacer particles. We have demonstrated that Gag is the only viral peptide, leaving behind fully intact forms of the HIV-1 Gag protein required for hAPOBEC3G incorporation (Fig. 2), matrix, capsid, and p6 domains. As shown in Fig. 2C and D, that hAPOBEC3G specifically interacts with the NC/p6 the Gag-ZWT protein is effectively expressed and gives rise domain of HIV-1 Gag (Fig. 3), that NC is essential for to levels of virus-like particles that are similar to those seen incorporation of hAPOBEC3G into HIV-1 virus-like par- with HIV-1 Gag. However, these NC-deficient virions differ ticles (Fig. 2), and perhaps most surprisingly, that non- from the wild-type HIV-1 Gag particles in that they do not specific RNA plays a critical role in mediating hAPO- incorporate detectable levels of hAPOBEC3G (Fig. 2D, lane BEC3G binding by the HIV-1 NC protein in vitro (Fig. 4). Rapid Communication 167

specific RNA which in our hands was only removed by exhaustive digestion with RNase A (Fig. 4).

Materials and methods

Immunofluorescence analysis

293T cells were transfected with 1 Ag of the pLL3.7 lentiviral vector plasmid together with 250 ng of each of the packaging plasmids pVSV-G, pRSV-Rev and pMDL- gag/pol RRE (Rubinson et al., 2003). The cells were also co-transfected with 500 ng of ph3G-HA or pm3-HA, or 500 ng of pcDNA3.1 as a negative control. Finally, the cells were also co-transfected with 250 ng of pgVif or the pgDVif control plasmid (Bogerd et al., 2004). At 48 h after transfection, the virus containing supernatant media were harvested, passed through a 0.45-Am filter and used to infect 293T cells grown on coverslips. At 36 h post- infection, EGFP expression was visualized by fluorescence microscopy.

Western analyses

Fig. 4. hAPOBEC3G binding to HIV-1 NC/p6 is mediated by RNA. This 293T cells were transfected with a combination of HIV-1 binding assay was performed as described in Fig. 3, except that the RNA and/or Gag expression plasmids, together with a hAPOBEC3G-HA and NC/p6 proteins were either not treated with RNase plasmid expressing an HA-tagged form of APOBEC3G, as A or were subjected to exhaustive treatment with RNase A before binding. described in the legend of Fig. 2. At 44 h after transfection, 35 (A) These gels show the S-labeled input NC/p6 proteins used in the the producer cells and the supernatant media were harvested binding reaction. Note that exhaustive RNase A digestion results in the loss of a slower migrating form of the approximately 16 kDa NC/p6 protein for Western analysis. Released virions were collected by (asterisk) that we hypothesize is a complex of NC/p6 with RNA. (B) In pelleting through a sucrose cushion (Bogerd et al., 2004), vitro binding of the 35S-labeled NC/p6 protein to HA-APOBEC3G. Mock- resuspended and then normalized for minor differences in transfected 293T cells, or cells transfected with a plasmid expressing HA- p24 production as determined by ELISA. Both the cell and tagged h-arrestin 2 (harr2) provided negative control cell lysates. (C) virion lysates were prepared and analyzed by Western blot Western analysis of the 293T cell lysates used to load the HA affinity matrix used in panel B, using a mouse anti-HA monoclonal antibody. as previously described (Bogerd et al., 2004) using mouse monoclonal antibodies specific for HIV-1 capsid (Chesebro et al., 1992) or the HA epitope tag (Covance). All the plasmids used in these experiments have been previously Given that hAPOBEC3G is able to inhibit the replication of described (Cowan et al., 2002; Rubinson et al., 2003; a wide range of retroviruses (Mangeat et al., 2003; Mariani Bogerd et al., 2004) except for psynGag-ZWT, which is et al., 2003), it will be of interest to determine which identical to psynGag except that the NC domain and the conserved attributes of retroviral NC proteins are critical for adjacent p1 spacer have been precisely substituted with a specific binding to hAPOBEC3G. leucine zipper derived from residues 247 to 280 of the yeast While this manuscript was in preparation, two other GCN4 protein, as previously described (Accola et al., groups also reported experiments designed to analyze the 2000). requirements for APOBEC3G into HIV-1 virions. While Cen et al. (2004) also found that the NC domain of Gag was In vitro binding assays critical for APOBEC3G incorporation, they differed in that they did not observe any requirement for RNA. In contrast, 35S-methionine-labeled Gag proteins were generated by Svarovskaia et al. (2004) reported that APOBEC3G in vitro translation using a TNT coupled transcription/ incorporation into virions required RNA, but did not depend translation system. In parallel, 293T cells were transfected on the NC domain. Our results may reconcile these very with 1.5 Ag of ph3G-HA (Bogerd et al., 2004) or the different conclusions by demonstrating that, while NC is pcDNA3.1 control plasmid. Forty-eight hours after trans- indeed critical for APOBEC3G incorporation into virions fection, cultures were suspended in lysis buffer (50 mM (Fig. 2) and specific binding of APOBEC3G by Gag (Fig. TRIS, pH 7.4; 150 mM NaCl; 0.5% NP40), cleared by 3), this interaction is also dependent on the presence of non- centrifugation, and then added to 250 Al of HA affinity 168 Rapid Communication matrix (Covance). After binding for 1 h at 4 8C, equal T-cell-tropic isolates: definition of critical amino acids involved in cell aliquots of the bound fraction were either taken for Western tropism. J. Virol. 66, 6547–6554. Conticello, S.G., Harris, R.S., Neuberger, M.S., 2003. The Vif protein of blot analysis, using a monoclonal mouse antibody specific HIV triggers degradation of the human antiretroviral DNA deaminase for the HA epitope tag, or added to 20 Al of each in vitro APOBEC3G. Curr. Biol. 13, 2009–2013. translated Gag protein. 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