1. Gladstone Institute of Virology and Immunology,
2. Departments of Medicine, Microbiology and Immunology, University of California, San Francisco, California 94143, USA

Correspondence to: Warner C. Greene^1,2 Correspondence and requests for materials should be addressed to W.C.G. (Email: wgreene@gladstone.ucsf.edu).

APOBEC3G belongs to a family of tissue-restricted (deoxy)cytidine deaminases⁶ that edit RNA and mutates DNA^{6, 7}. In the case of HIV, the incorporation of APOBEC3G into virions leads to extensive mutation of nascent HIV DNA formed during reverse transcription in the next round of infection^{5, 8, 9, 10, 11}. The Vif protein of HIV circumvents this anti-HIV defence mechanism by enhancing the 26S proteasome-mediated degradation of APOBEC3G^{12, 13, 14} and decreasing its synthesis^{12, 15}. These events make APOBEC3G unavailable for incorporation into budding virions, and thus, its anti-retroviral action is forfeited.

One puzzling aspect of APOBEC3G biology is why cytoplasmic forms of this enzyme in target CD4⁺ T cells undergoing HIV infection fail to recapitulate the antiviral effects of intra-virion APOBEC3G. Perhaps cellular APOBEC3G is subject to some form of negative regulation. APOBEC1 (ref. 16) and activation-induced cytidine deaminase (AID)¹⁷ are two well-characterized members of this (deoxy)cytidine deaminase family. APOBEC1 is the central component of an RNA-editing complex and is regulated by its assembly with APOBEC1-complementing factor¹⁶. AID catalyses the deamination of dC residues, yielding dU on single-stranded DNA in vitro, but has no measurable deaminase activity unless pre-treated with RNase to remove small inhibitory RNAs bound to AID¹⁸. These findings prompted us to determine whether APOBEC3G assembles into HMM complexes and to assess the potential role of RNA binding in the regulation of APOBEC3G.

Proteins in lysates of H9 T cells harbouring endogenous APOBEC3G were size fractionated by gel filtration on a Superose 6HR 10/30 column using a fast performance liquid chromatography (FPLC) apparatus followed by SDS–polyacrylamide gel electrophoresis (PAGE) and anti-APOBEC3G immunoblotting of the individual fractions. The endogenous 46-kDa APOBEC3G enzyme resided principally in a HMM complex that is >700 kDa in mass (Fig. 1a, top). However, when lysates were treated with RNase A before fractionation, APOBEC3G was detected as a 46–100-kDa LMM form (Fig. 1a, bottom). This effect of RNase was blocked by the addition of a specific inhibitor of pancreatic-type RNase A (Supplementary Fig. 1). As shown by transfection of haemagglutinin-tagged APOBEC3G (HA-APOBEC3G) expression vector DNA¹², all of the putative cellular cofactors needed for the assembly of the HMM APOBEC3G complex appear to be expressed in 293T cells, which lack endogenous APOBEC3G expression (Fig. 1b).

Figure 1: APOBEC3G is negatively regulated by recruitment into an enzymatically inactive HMM complex.

a, b, Endogenous APOBEC3G in human H9 T cells (a) and exogenous HA-APOBEC3G expressed in 293T cells (b) reside principally in >700-kDa HMM complexes that can be converted to LMM forms after RNase A treatment. c, HMM (b, top, fractions 7–9) and LMM (b, bottom, fractions 15–17) forms of HA-APOBEC3G resolved by FPLC were immunoprecipitated (IP) with anti-HA (Covance), and HA-APOBEC3G protein content was assessed by anti-APOBEC3G antibody (lanes 1 and 2). The immunoprecipitates were tested in a deoxycytidine deaminase assay with or without RNase treatment. ssDNA, single-stranded DNA.

High resolution image and legend (72K)

Next, we examined the deoxycytidine deaminase activity of the two forms of APOBEC3G. In an in vitro assay, LMM APOBEC3G displayed enzymatic activity, but the HMM form did not (Fig. 1c, lanes 3 and 4). RNase treatment conferred activity on isolated HMM complexes but did not further increase the activity of the LMM form (Fig. 1c, lanes 5 and 6). Thus, recruitment of APOBEC3G into the HMM ribonucleoprotein complex negatively regulates its enzymatic activity within cells, and degradation of the RNA component leads to disassembly of the complex and acquisition of enzymatic activity.

To assess the effect of Vif on the HMM APOBEC3G complex, we coexpressed HIV-1 Vif, HA-APOBEC3G and His₆-ubiquitin in 293T cells. In the presence of the proteasome inhibitor MG132, Vif co-fractionated with HMM APOBEC3G, and a ladder of potentially polyubiquitinated forms of the APOBEC3G enzyme was detected in the presence but not absence of Vif (Fig. 2a, middle and top panels). In the absence of APOBEC3G, Vif fractionated in a lower-molecular-mass range (Fig. 2a, bottom). Physical assembly of Vif and APOBEC3G in the HMM complex was confirmed by the detection of Vif in anti-HA-APOBEC3G immunoprecipitates (Fig. 2b, lane 5). Additionally, polyubiquitinated forms of APOBEC3G in the HMM complex were detected when Vif and APOBEC3G were coexpressed (Fig. 2b, lane 9), but not when APOBEC3G was expressed in the absence of Vif (Fig. 2b, lane 7). We also demonstrated that Vif targets APOBEC3G for high-level polyubiquitination by recruitment of Cul5 and elongin B, which are components of an E3 ubiquitin ligase complex¹⁹, to the HMM complex (Supplementary Fig. 2).

Figure 2: Vif assembles with APOBEC3G in HMM complexes and promotes polyubiquitination of APOBEC3G.

a, Vif co-fractionates with the HMM APOBEC3G complex. 293T cells were transfected as indicated, cultured for 32 h, treated with proteasome inhibitor (MG132, 12.5 M) for 16 h, and subjected to FPLC analysis. Vif shifted to HMM fractions in the presence of HA-APOBEC3G, and a ladder of potentially polyubiquitinated forms of the APOBEC3G enzyme was detected (lane 6, upper middle panel). An asterisk indicates nonspecific bands detected by anti-HA antibody (12CA5, Roche). b, HMM fractions from 293T cells transfected with HA-APOBEC3G and His₆-ubiquitin (His-Ubi) in the absence (pNL-A1 Vif) (a, top panel, lane 6) or presence of Vif (pNL-A1) (a, middle panels, lane 6) serve as input materials for an immunoprecipitation (IP) assay with anti-HA, resolved by SDS–PAGE, and immunoblotted with anti-HA, anti-Vif or anti-His antibodies (Santa Cruz Biotechnology). Protein G-agarose beads in the absence of anti-HA were used for control immunoprecipitation. In the left panel, note the co-immunoprecipitation of Vif and APOBEC3G (lane 5). In the right panel, when coexpressed with Vif, APOBEC3G in the HMM complex was polyubiquitinated (lane 9).

High resolution image and legend (62K)

We next surveyed primary cells for APOBEC3G complexes. Notably, only LMM forms of APOBEC3G were detected in purified, unstimulated CD4⁺ T cells and monocytes from peripheral blood (Fig. 3). However, when the CD4⁺ T cells were activated with phytohaemagglutinin (PHA) and interleukin-2 (IL-2), RNase-sensitive HMM APOBEC3G complexes were detected (Fig. 3 and data not shown). When monocytes were induced to differentiate into macrophages by plastic adherence, HMM APOBEC3G complexes appeared (Fig. 3). These findings were of particular interest because activated CD4⁺ T cells and macrophages readily support HIV replication, whereas unstimulated peripheral blood T cells and freshly isolated monocytes do not. This phenomenon is due, at least in part, to an early post-entry block occurring at or soon after the reverse transcription step^{1, 2, 3, 4, 20}.

Figure 3: Inducibility of HMM APOBEC3G complex formation in primary CD4⁺ T cells with different stimuli, and correlation of the HMM complex with permissivity for HIV infection.

APOBEC3G complexes were identified by FPLC, SDS–PAGE and immunoblotting with anti-APOBEC3G antibody. Single-round infection experiments were performed with the VSV-G-pseudotyped NL4-3 HSA virus. Ranges of HIV infectivity were determined by normalizing the percentage of HSA-positive cells obtained with each treatment to the response in cells stimulated with PHA and IL-2 (assigned a value of 100%). HIV infectivity: >70% (high, + + + ); 30–70% (moderate, + + ); 5–30% (low, +); <5% (none, -). Scoring was based on the infectivity data obtained from cells isolated from four healthy donors. PMA, phorbol 12-myristate 13-acetate.

High resolution image and legend (53K)

To explore further the effects of CD4⁺ T-cell activation on HMM APOBEC3G complex formation and single-cycle HIV infectivity, we used vesicular stomatitis virus-G (VSV-G)-pseudotyped NL4-3 viruses expressing a heat-stable antigen (HSA, mouse CD24) reporter gene in lieu of Nef (Supplementary Fig. 3). Co-stimulation of CD4⁺ T cells with anti-CD3 and anti-CD28 antibodies strongly induced HMM APOBEC3G complex formation, and these cells were highly permissive for HIV infection (Fig. 3; see also Supplementary Fig. 3). Anti-CD3 antibodies modestly induced HMM complex assembly; anti-CD28 antibodies did not (Fig. 3). When cells were pre-treated with aphidicolin to block cell-cycle progression at the G1/S border or n-acetyl butyric acid (n-butyrate) to block progression from G1a to G1b (ref. 2) before anti-CD3 and anti-CD28 co-stimulation, markedly different results were obtained. Aphidicolin-treated cells displayed HMM APOBEC3G complexes and were permissive for HIV infection, whereas n-butyrate-treated cells contained only LMM forms of APOBEC3G and were not permissive (Fig. 3; see also Supplementary Fig. 3). These results suggest that progression of cells into G1b is necessary for HMM APOBEC3G complex formation and, as previously reported, HIV infection², but entry into S phase is not required for either event. Activation of CD4⁺ T cells with phorbol 12-myristate 13-acetate also induced HMM APOBEC3G complex formation (Fig. 3) and increased permissivity for HIV infection (Supplementary Fig. 3).

These composite findings raised the possibility that cellular LMM APOBEC3G functions as an effective early post-entry restriction factor for HIV in unstimulated peripheral blood CD4⁺ T cells and freshly isolated monocytes. To test this hypothesis, we introduced APOBEC3G-specific small interfering RNA (siRNA; APOBEC3G-si240) into unstimulated CD4⁺ T cells using an AMAXA nucleofector apparatus. In studies with fluorescein isothiocyanate (FITC)-conjugated siRNA, about 80% of the nucleofected cells acquired the siRNA (data not shown). siRNA targeting APOBEC3G decreased APOBEC3G protein expression by a maximum of 70% between 48 and 88 h after nucleofection (Fig. 4a, b). Despite equivalent cellular uptake, a mutant siRNA (APOBEC3G-si240 mm) differing by two ribonucleotides did not alter APOBEC3G expression (Fig. 4a). Thus, the expression of endogenous APOBEC3G was decreased by nearly 90% in the cells acquiring the APOBEC3G-specific siRNA. Importantly, nucleofection did not activate the CD4⁺ T cells as assessed by either anti-CD25 or anti-CD69 immunostaining (Fig. 4c).

Figure 4: APOBEC3G is a post-entry restriction factor for HIV in unstimulated CD4⁺ T cells.

a, APOBEC3G protein levels (normalized to tubulin) in lysates of PBL-derived CD4⁺ T cells nucleofected with APOBEC3G-specific (APOBEC3G-si240) or mutant siRNAs (APOBEC3G-si240 mm; specificity control). b, Time course of siRNA-mediated knockdown of APOBEC3G in PBL-derived CD4⁺ T cells. c, siRNA-mediated knockdown of APOBEC3G renders unstimulated CD4⁺ T cells permissive for HIV infection. PBL-derived CD4⁺ T cells nucleofected with buffer or siRNAs were cultured in the absence or presence of the reverse transcriptase inhibitor 3TC (10 M) and infected with NL4-3 HSA reporter virus 48 h after siRNA treatment. Untreated cells not receiving siRNA (unstimulated) served as a negative control, and cells stimulated with PHA and IL-2 as a positive control. Expression of the HSA reporter gene and the activation status of CD4⁺ T cells were analysed 48 h after infection. Similar results were obtained using cells from four different donors.

High resolution image and legend (95K)

To assess the susceptibility of unstimulated peripheral blood CD4⁺ T cells to HIV infection after siRNA treatment, we performed single-round infection experiments with the VSV-G-pseudotyped NL4-3 HSA reporter virus. As expected, unstimulated CD4⁺ T cells did not support reporter virus expression, but cells activated with PHA and IL-2 were readily infected (Fig. 4c). Of note, APOBEC3G-specific, but not mutant, siRNA markedly relieved the HIV replication block in these unstimulated CD4⁺ T cells (Fig. 4c). As expected, APOBEC3G siRNA-dependent expression of HSA required effective reverse transcription of the reporter virus because surface HSA expression did not occur in cells previously treated with 3TC, a reverse transcriptase inhibitor (Fig. 4c).

Next, studies were performed to pinpoint the replication block induced by LMM APOBEC3G in unstimulated peripheral blood CD4⁺ T cells. Studies of HIV virion binding and fusion in cells treated with the mutant versus APOBEC3G-specific siRNA revealed no differences (data not shown). These results indicate that the APOBEC3G siRNA relieves a block occurring after virion binding and fusion in the unstimulated CD4⁺ T cells. Taqman-based real-time polymerase chain reaction (PCR) was used to quantify the synthesis of HIV-1 complementary DNA in cells nucleofected with APOBEC3G-specific or control siRNA. To limit infection to a single round, cells were infected with VSV-G-pseudotyped, DNase I-treated NL4-3 HSA reporter virus as described above. Early and late reverse transcription products were quantified using specific primer and probe combinations (Supplementary Table 1). As shown in Fig. 5a, late reverse transcription products accumulated much more slowly in unstimulated CD4⁺ T cells than in activated T cells, requiring 2–3 days to reach peak levels. These findings are consistent with previously published results³. Cells pre-treated with APOBEC3G-specific, but not mutant, siRNA accumulated late reverse transcription products much more rapidly. These late reverse transcription products were readily detectable within 8 h after infection (Fig. 5a). This pattern of more rapid accumulation of reverse transcripts in cells receiving APOBEC3G siRNA was observed with cells isolated from multiple donors. Of note, the synthesis of early reverse transcription products was indistinguishable in cells treated with the mutant versus APOBEC3G-specific siRNA (data not shown). These results suggest the presence of a late reverse transcription defect in unstimulated CD4⁺ T cells that is alleviated upon siRNA-mediated knockdown of endogenous APOBEC3G expression. Delayed accumulation of late reverse transcription products has also been described in monocytes²⁰, strengthening the possibility that LMM APOBEC3G also mediates the replication delay in these cells.

Figure 5: LMM APOBEC3G impairs the accumulation of late HIV-1 reverse transcription products in unstimulated CD4⁺ T cells.

a, siRNA-mediated knockdown of APOBEC3G alleviates the reverse transcription delay present in unstimulated PBL-derived CD4⁺ T cells. Untreated CD4⁺ T cells or cells nucleofected with buffer or siRNAs or activated with PHA/IL-2 were infected with VSV-G-pseudotyped, DNase I-treated NL4-3 HSA reporter viruses. Total cellular DNA was isolated at the indicated times, and the presence of early (not shown) and late reverse transcription products was assessed by real-time PCR. Data represent the means s.d. derived from assays independently performed three times. b, Sequence analysis of the env region (6581–6842) of viral reverse transcripts synthesized in unstimulated PBL-derived CD4⁺ T cells of three donors. Nine of 110 (8%) independent env sequences displayed extensive dG dA hypermutations. Sequences are compared to the starting NL4-3 HSA plasmid, and only nucleotides differing from this sequence are shown.

High resolution image and legend (118K)

To determine whether the antiviral function of the LMM APOBEC3G was mediated through its deoxycytidine deaminase activity, we analysed the sequence of the HIV reverse transcripts in the env region appearing in unstimulated CD4⁺ T cells 48 h after infection. Of a total of 110 independent env sequences from the unstimulated cells derived from three different donors, 27 contained mutations but only 9 of the 110 displayed dG dA hypermutations (Fig. 5b). In contrast, of 109 independent env sequences isolated from cells treated with APOBEC3G-specific siRNA, no dG dA hypermutations were detected despite the presence of potential sites for deamination (data not shown). Although we have been unable to test the enzymatic activity of LMM APOBEC3G from unstimulated CD4⁺ T cells owing to the lack of suitable antibody for immunoprecipitation, the detection of dG dA hypermutations in reverse transcripts from these cells argues that the endogenous LMM APOBEC3G is enzymatically active.

The LMM form of APOBEC3G functions as a highly active post-entry restriction factor for HIV in unstimulated peripheral blood CD4⁺ T cells and probably in freshly isolated monocytes. Interestingly, unstimulated CD4⁺ T cells present in human lymphoid tissues are permissive for HIV infection²¹, and APOBEC3G is predominantly in the HMM form in these cells (J. F. Kreisberg et al., unpublished data). The cause of these differences is currently under investigation. The detection of dG dA hypermutations in only 8% of the late reverse transcription products suggests that the replication block induced by cellular LMM APOBEC3G involves a non-enzymatic mechanism. Indeed, APOBEC3G-mediated DNA editing may not always be necessary for its antiviral activity²². One strong possibility involves the RNA binding properties of APOBEC3G. Binding of LMM APOBEC3G to the viral RNA in the reverse transcription complex could decrease the efficiency of reverse transcriptase action, leading to a kinetic delay in the accumulation of late reverse transcription products. This model could also explain why subsequent mitogen activation of infected resting CD4⁺ T cells rescues productive viral infection^{1, 2, 3}. Under these conditions, APOBEC3G may be recruited into the HMM complex, thus relieving the late block in reverse transcription. Viral inhibition mediated through the RNA-binding properties of APOBEC3G has also been described in the setting of the hepatitis B virus²³. Why the accumulating reverse transcripts in unstimulated CD4⁺ T cells display such unexpectedly low levels of dG dA hypermutation is unclear. It is possible that the effectiveness of deoxycytidine deamination differs depending on whether the enzyme is originally recruited into virions or resides in the cytoplasm. Alternatively, our studies would underestimate the number of late reverse transcription products containing dG dA hypermutations if these deaminated minus-strand precursors are particularly sensitive to degradation by uracil N-glycosylase and apurinic-apyrimidinic endonuclease in unstimulated CD4⁺ T cells. Such an effect would be consistent with the accelerated decay of HIV reverse transcripts previously described^{1, 3}. APOBEC3F is a deoxycytidine deaminase that also exerts antiviral activity, albeit at levels lower than APOBEC3G^{24, 25, 26, 27}. APOBEC3G and APOBEC3F are coordinately expressed in a wide range of human tissues, including unstimulated T cells, and APOBEC3F-signature hypermutations are detectable in the viral cDNA sequences isolated from unstimulated CD4⁺ T cells (Fig. 5b). Thus, it will be important to explore whether APOBEC3F is also negatively regulated within a latent HMM complex and whether it similarly functions as a post-entry restriction factor for HIV in unstimulated CD4⁺ T cells and monocytes.

APOBEC3G thus joins a growing list of innate cellular factors (for example, Murr1 and TRIM5-^{28, 29}) that impair various early phases of the HIV life cycle. The overall function of APOBEC3G as a post-entry restriction factor, although mechanistically different, is reminiscent of the effects of rhesus TRIM5-, which impairs the growth of HIV in monkey cells²⁸. However, although human TRIM5- exerts essentially no anti-HIV activity, human APOBEC3G effectively restricts the early growth of HIV in human cells when present in its active LMM form. Our findings further highlight how APOBEC3G can exert both 'Vif-insensitive' and 'Vif-sensitive' antiviral functions during the early and late stages in the retroviral life cycle, respectively. Because production of the viral vif gene product depends on its expression from the integrated provirus and only negligible amounts of Vif are present in HIV virions³⁰, the early action of LMM APOBEC3G effectively impairs the growth of both wild-type and Vif forms of HIV. Our findings also raise an intriguing possibility: agents that promote the disassembly of the HMM APOBEC3G complex might confer resistance to HIV infection in cells normally highly permissive for HIV infection. However, it will be important to exclude an effect of such disassembled forms of APOBEC3G on host genomic DNA in these activated and often proliferating cellular hosts.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank K. Strebel for the gift of the pNL-A1 plasmid, X.-F. Yu for the gift of the Myc-Cul5 plasmid, D. Bohmann for the gift of the His₆-tagged ubiquitin expression vector DNA, N. R. Landau for the gift of NL4-3 HSA R-E- reporter provirus through the AIDS Research and Reference Reagent Program at NIH, J. Burns for the gift of expression plasmid pVSV-G, and M. Malim for providing the monoclonal anti-Vif (319) antibody. We thank A. O'Mahony and H. Kwon for assistance with the FPLC experiments; J. Neidleman for assistance preparing the primary cells; M. Cavrois, D. Fenard, A. Yonezawa, J. Bohuslav, L.-F. Chen, C. Martin and S. Williams for discussions; G. Howard and S. Ordway for editorial assistance; and S. Cammack, R. Givens and J. Carroll for assistance in preparation of the manuscript and the graphics. Different components of this work were supported by funding from the National Institutes of Health (Women's HIV Interdisciplinary Network and NIMH; W.C.G.) and the Universitywide AIDS Research Program (W.C.G.) and the American Foundation for AIDS Research (Y.-L.C.).

Competing interests statement

The authors declare no competing financial interests.

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