- Open Access
Production of infectious human immunodeficiency virus type 1 does not require depletion of APOBEC3G from virus-producing cells
© Kao et al; licensee BioMed Central Ltd. 2004
- Received: 08 July 2004
- Accepted: 17 September 2004
- Published: 17 September 2004
The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.
In this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.
Our results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions.
- Long Terminal Repeat
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- Yellow Arrow Head
- Human APOBEC3G
- APOBEC3G Expression
Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T cell lines is dependent on the expression of a functional Vif protein. In the absence of Vif, virus replication is restricted by a host factor that was recently identified as CEM15 (now referred to as APOBEC3G) , a host cytidine deaminase targeting DNA substrates in vitro  but whose role in normal cells is unknown. In the absence of Vif, APOBEC3G is efficiently incorporated into virus particles [3–9] where it causes extensive cytidine to uracil changes on the viral minus-strand cDNA during reverse transcription [5, 10–12]. The conversion of cytidine to deoxyuridine on the minus-strand cDNA either results in guanine to adenine changes on the viral plus-strand cDNA to yield highly mutated viral genomes or triggers the degradation of the deaminated minus strand cDNA through the action of a DNA repair mechanism that involves removal of the uracil base by uracil DNA glycosylase and subsequent endonucleolytic cleavage at the abasic sites by apyrimidinic endonuclease (reviewed in [13, 14]). While both mechanisms are detrimental to virus replication, the reported inability of vif-defective viruses grown in restrictive cells to reverse transcribe the viral genome into full-length cDNA is more consistent with the latter mechanism involving the degradation of deaminated viral cDNA [15–19].
Vif is a 23-kDa basic protein that is expressed late during infection in a Rev-dependent manner . Immunocytochemical analyses revealed a largely cytoplasmic localization of Vif [21–23]. However, Vif is efficiently incorporated into HIV particles during productive infection through an interaction with viral genomic RNA and associates with viral nucleoprotein complexes [22, 24–26]. In the presence of Vif, the steady-state levels of cell-associated APOBEC3G – as judged by immunoblot analysis – are reduced by 3–10 fold [3–8, 27, 28]. This Vif-dependent reduction in APOBEC3G levels has been attributed to proteasome-dependent degradation of the protein and requires a direct interaction of Vif with APOBEC3G [3, 6–8].
Like Vif, APOBEC3G is a cytoplasmic protein. In fact, co-immunoprecipitation analyses demonstrated an interaction of Vif and APOBEC3G in transiently transfected cells [3, 5, 6, 27, 29–32]. The formation of stable Vif:APOBEC3G complexes seemed to be at odds with the reported proteasome-dependent degradation of APOBEC3G in Vif-expressing cells [3, 6–9, 27, 28]. Indeed, the identification of Vif:APOBEC3G complexes in mixtures of cell extracts that had been individually transfected to express either Vif or APOBEC3G suggested that the stable interaction of Vif and APOBEC3G during co-immunoprecipitation may occur after cell lysis . Thus, the co-immunoprecipitation of Vif and APOBEC3G from cell extracts is not necessarily an indication of the existence of stable intracellular complexes. Quite to the contrary, Marin et al reported a profound effect of Vif on the expression of APOBEC3G on a single cell level. They found that expression of Vif in transiently transfected COS7 cultures resulted in an almost complete segregation of cells expressing either APOBEC3G or Vif . Interestingly, this segregation of Vif and APOBEC3G into separate cells was seen only for wild type Vif. In fact, only 10% of cells expressing wild type Vif were double-positive while 95% of cells expressing an inactive Vif variant also contained APOBEC3G .
The current study aims at characterizing in more detail the effects of Vif on the expression of human APOBEC3G on a single cell level. The study was initiated because of the apparent discrepancy between the drastic effects of Vif on APOBEC3G reported by Marin et al and our own finding of only moderate effects of Vif on APOBEC3G expression in transiently transfected cells. In our study, Vif was expressed from a subviral construct in a Tat- and Rev-dependent manner while APOBEC3G was expressed either in a Tat-dependent manner from an HIV-1-LTR-based vector or independently from a CMV-promoter-based expression vector. The Tat-dependent APOBEC3G expression vector was used to restrict APOBEC3G expression to cells also expressing Tat (and thus Vif).
Confocal microscopic analysis of HeLa cells transiently transfected with Vif and APOBEC3G expression vectors revealed significant variations in the number of double-positive cells in identical samples ranging from as low as 40% to as high as 80% of transfected cells depending on fixation method and antibodies employed. Importantly, the appearance of cells expressing only Vif or APOBEC3G was observed both with wild type Vif and a biologically inactive variant and thus cannot be explained by Vif-induced degradation of APOBEC3G. Finally, despite the efficient co-expression of Vif and APOBEC3G, viruses produced in these cultures were fully infectious. We therefore conclude that the Vif-induced exclusion of APOBEC3G from virus-producing cells reported by Marin et al  does not apply to our system and because of that is not a universal property of all Vif proteins. This implies that elimination of APOBEC3G is not an obligate requirement for the production of infectious viruses from APOBEC3G-expressing cells.
Expression of Vif in the context of a proviral vector only moderately reduces cellular APOBEC3G levels
Co-expression of APOBEC3G and Vif in HeLa cells
Tat-dependent expression of APOBEC3G reduces the fraction of single-positive cells
In the experiment shown in figure 2, APOBEC3G was expressed under the control of a CMV promoter while Vif was expressed from the HIV-LTR promoter under the regulatory control of Tat and Rev. Because of the independent expression of Vif and APOBEC3G it cannot be ruled out that the large number of single-positive cells in that experiment – while statistically improbable – was caused by the selective transfection of cells with either the Vif or the APOBEC3G expression vector. To check this possibility, we expressed APOBEC3G from the HIV-1 long terminal repeat (LTR) promoter driven vector pHIV-APO3G . Because of its dependence on Tat, APOBEC3G expression from pHIV-APO3G is restricted to cells also expressing Vif. Indeed, transfection of pHIV-APO3G into cells in the absence of pNL-A1 or any other Tat expression vector did not reveal any APOBEC3G expression as judged by immunofluorescence analysis or immunoblotting attesting to the strict Tat-dependence of this APOBEC3G expression vector (data not shown).
In addition of measuring the context-dependent expression of APOBEC3G, we also wanted to determine the influence of the fixation procedure on the efficiency of Vif:APOBEC3G co-staining. It is well known that the choice of fixative can affect the ability of a given antibody to recognize a specific epitope on its target protein. Frequently, epitopes are masked because of the folding properties of a protein in vivo or because of pre-existing protein-protein interactions that may compete for antibody binding. To test this possibility we compared a formaldehyde fixation procedure employed previously  with the methanol fixation procedure employed in our own studies .
Co-expression of Vif and APOBEC3G: Protein degradation or epitope masking?
Exclusion of APOBEC3G from cells expressing biologically inactive Vif protein
Rescue of viral infectivity and Vif-induced reduction of cellular APOBEC3G levels are not directly linked
APOBEC3G is able to deaminate cytidine residues on the HIV minus-strand cDNA and cause hypermutation of the viral genome. Nevertheless, HIV-1 is able to efficiently replicate in APOBEC3G expressing cells thanks to the activity of the accessory protein Vif. One of the prerequisites for the antiviral activity of APOBEC3G is that it is packaged into the virions where it selectively targets the viral minus-strand cDNA [5, 10–12, 40, 41] and there is convincing evidence that HIV-1 Vif plays an important role in inhibiting the encapsidation of APOBEC3G. The question of how Vif accomplishes this remains under investigation. A number of groups have reported on the rapid Vif-induced degradation of APOBEC3G by cellular proteasomes [3, 6–9, 27, 28]. Consistent with this, treatment of cells with proteasome inhibitors was found to increase APOBEC3G expression levels despite the presence of Vif [6, 7, 9, 28]. This is contrasted by other reports that noted only a moderate effect of Vif on cellular APOBEC3G levels [4, 5]. In fact, our own studies with proteasome inhibitors did not yield a significant increase in APOBEC3G levels in the presence of Vif (manuscript in preparation). Nevertheless, the currently prevailing opinion is that Vif inhibits the encapsidation of APOBEC3G by inducing its rapid degradation in virus-producing cells.
While the results from our own study argue against a depletion of APOBEC3G in Vif-expressing cells – thus implying that Vif can rescue viral infectivity despite the presence of APOBEC3G in virus-producing cells – it is important to point out that our data do not rule out the possibility that Vif – under different experimental conditions – can indeed mediate the proteasome dependent degradation of APOBEC3G. In fact, expression of Vif from a codon-optimized vector consistently had a more pronounced effect on APOBEC3G steady-state levels than Vif expressed from pNL-A1 even though the Vif expression levels from the codon-optimized construct were consistently several-fold lower than those from pNL-A1 (manuscript in preparation). Experiments are ongoing to study the differential effects of Vif expressed from pNL-A1 and Vif expressed from a codon-optimized vector on APOBEC3G stability. However, these results could suggest that the effect of Vif on APOBEC3G steady-state levels may be influenced by the context in which Vif is expressed. At any rate, despite our inability to observe Vif-dependent cellular depletion of APOBEC3G, we were invariably able to recover fully infectious HIV under conditions were the intracellular levels of APOBEC3G were only moderately affected. We therefore conclude that (i) Vif has the ability to rescue viral infectivity even in the presence of APOBEC3G and (ii) that intracellular depletion of APOBEC3G and rescue of viral infectivity may be functionally separable activities of Vif.
For now, the reason for the differences in the sensitivity of APOBEC3G to Vif noted by us versus other research groups remains unexplained. APOBEC3G can form oligomeric structures and is able to interact with Vif. It is therefore possible that such complexes undergo conformational changes that can mask epitopes thus limiting the access of antibodies used in the experiments. Thus, the discrepancy between our findings of the coexpression of Vif and APOBEC3G in the majority of cells and the virtual exclusion of APOBEC3G from Vif-expressing cells reported by Marin et al.  may be attributed at least in part to differences in the experimental protocols. It is unlikely that the observed discrepancies are strain-specific variations. To this end we have compared the activities of two HIV-1 Vif isolates, HXB2 and NL4-3, which differ by 18 amino acids (9.4%), and found them to be equally active against APOBEC3G (manuscript in preparation). It is unclear why cotransfection of pHIV-APO3G with pNL-A1 produces a fraction of cells that are single-positive for Vif or for APOBEC3G. Since APOBEC3G expression from the pHIV-APO3G vector is strictly Tat-dependent, the results cannot be explained by differential transfection of cells with individual plasmids. Also, this phenomenon is clearly not a consequence of Vif function, since similar results were observed in the presence of a biologically inactive Vif variant (Fig. 6) or when APOBEC3G was co-expressed with HIV-1 Gag in the absence of Vif (data not shown).
The inability of Vif expressed from pNL-A1 to deplete APOBEC3G is consistent with our previous inability to observe APOBEC3G degradation in kinetic studies . More recent in-depth kinetic analyses of APOBEC3G employing multiple epitope tags and various antibodies confirm these initial findings and suggest that – instead of inducing APOBEC3G degradation – Vif induces conformational changes in APOBEC3G that affect the ability of antibodies to interact with the protein (manuscript in preparation). Experiments are ongoing to study the nature of the APOBEC3G/Vif complexes and to further decipher the mechanism(s) by which Vif inhibits the encapsidation of APOBEC3G under conditions of no or low intracellular degradation.
Expression of Vif and APOBEC3G in our experimental setup does not lead to the elimination of APOBEC3G from Vif expressing cells. In fact, more than 80% of successfully transfected cells efficiently co-expressed both proteins. Similar results were observed when a biologically inactive Vif variant was co-expressed with APOBEC3G suggesting that the absence of APOBEC3G in some of the Vif-positive cells is not due to Vif-mediated APOBEC3G degradation but reflects a general characteristic of the transient expression system. Moreover, APOBEC3G expression levels were very similar for Vif-positive and Vif-negative cells as judged from the immunostaining consistent with the only modest reduction in APOBEC3G steady-state levels observed in our immunoblot analyses. Nevertheless, viruses produced under such conditions were fully infectious in the presence but not in the absence of Vif attesting to the biological activity of all the proteins involved and demonstrating that Vif was expressed at saturating levels. We conclude that production of infectious viruses from APOBEC3G expressing cells is dependent on Vif but does not necessitate APOBEC3G exclusion from virus-producing cells.
The full-length molecular clone pNL4-3  was used for the production of wild type infectious virus. For transient expression of Vif, the subgenomic expression vector pNL-A1  was employed. This plasmid expresses all HIV-1 proteins except for gag and pol products. A vif-defective variant of pNL-A1, pNL-A1vif(-) was constructed by deletion of an NdeI/PflMI fragment . Plasmid pNL-A1/VifΔ23–43 expresses a Vif variant carrying a 21 amino acid deletion (residues 23 to 43) in its vif gene as reported elsewhere . This Vif variant is inactive and does not target APOBEC3G . Plasmids pcDNA-APO3G and pHIV-APO3G are vectors for the expression of human APOBEC3G under the control of the CMV immediate early promoter or the HIV promoter, respectively, and were constructed as described elsewhere .
Serum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. A monoclonal antibody to Vif (MAb #319) was used for all immunoblot analyses and some of the immunohistochemical analyses as indicated in the text and was obtained from Michael Malim through the NIH AIDS Research and Reference Reagent Program [23, 35, 36, 36, 37] For all other immunocytochemical analyses our Vif-specific polyclonal antibody (Vif93) was employed. APOBEC3G, carrying a C-terminal Myc epitope tag was identified either with the Myc-specific 9E10 monoclonal antibody or a polyclonal antibodies to the Myc epitope tag (both antibodies were obtained from Sigma-Aldrich, St. Louis). Alternatively, APOBEC3G was identified using a polyclonal rabbit serum against recombinant APOBEC3G . Tubulin was identified using a monoclonal antibody to α-tubulin (Sigma-Aldrich, St. Louis).
Tissue culture and transfections
HeLa cells were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 LTR . These cells were obtained through the NIH AIDS Research and Reference Reagent Program and were maintained in complete RPMI 1640 medium supplemented with 10% FBS and hygromycin B (300 μg/ml).
For transfection of HeLa cells, cells were grown in 25 cm2 flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS™ (Invitrogen Corp, Carlsbad CA) following the manufacturer's recommendations. A total of 5–6 μg of plasmid DNA per 25 cm2 flask was used. Cells were harvested 24 hr post-transfection. Transfection efficiency in our analyses was generally 30% to 40%.
Preparation of virus stocks
Virus stocks were prepared by transfecting HeLa cells with appropriate plasmid DNAs. Virus-containing supernatants were harvested 24 hr after transfection. Cellular debris was removed by centrifugation (3 min, 3000 × g) and clarified supernatants were filtered (0.45 μm) to remove residual cellular contaminants. For determination of viral infectivity, unconcentrated filtered viral supernatants were used for the infection of indicator cells. For immunoblot analysis of viral proteins, virus particles (7 ml) were concentrated by ultracentrifugation through 4 ml of 20% sucrose in PBS as described before .
For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as follows: Cells were washed once with PBS, suspended in PBS (400 μl/107 cells), and mixed with an equal volume of sample buffer (4% sodium dodecyl sulfate, 125 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue). Proteins were solubilized by boiling for 10 to 15 min at 95°C with occasional vortexing of the samples to shear chromosomal DNA. Residual insoluble material was removed by centrifugation (2 min, 15000 rpm in Eppendorf Minifuge). Viral proteins were obtained by boiling concentrated viral pellets in a 1:1 mixture of PBS and sample buffer. Cell lysates and viral extracts were subjected to SDS-PAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway NJ) and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).
To determine viral infectivity, virus stocks were normalized for equal reverse transcriptase activity and used to infect LuSIV cells (5 × 105) in a 24-well plate total volume 1.2 to 1.4 ml. Cells were incubated for 24 hours at 37°C. Cells were then harvested and lysed in 150 μl of Promega 1x reporter lysis buffer (Promega Corp., Madison WI). To determine the luciferase activity in the lysates, 50 μl of each lysate were combined with luciferase substrate (Promega Corp., Madison WI) by automatic injection and light emission was measured for 10 seconds at room temperature in a luminometer (Optocomp II, MGM Instruments, Hamden CT).
For the analysis of transfected HeLa cells, cells were scraped off the flasks 3 hr after transfection and reseeded into 12 well plates containing 0.13 mm cover slips. Cells were grown for 15 to 24 hrs at 37°C in DMEM containing 10% FBS. Cells were then fixed at -20°C in precooled methanol (-20°C) for 10 min followed by two washes in PBS or fixed in FA buffer (5% formaldehyde + 2% sucrose in PBS) for 20 min at room temperature followed by two washes in PBS. Coverslips were stored in PBS at 4°C until use. FA-fixed samples were permeabilized for 30 min at room temperature in permeabilization buffer (1% Triton X-100, 10% sucrose in PBS) prior to incubation with antibodies. For antibody staining, cover slips were incubated in a humid chamber at 37°C for 30 min with primary antibodies at appropriate dilutions in 1% BSA in PBS. Cover slips were washed once in PBS (5 min, room temp) and incubated with Texas-Red- or Cy2-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 30 min at 37°C in a humid chamber. Cover slips were then washed twice with PBS and mounted onto microscope slides with glycerol gelatin (Sigma-Aldrich, St. Louis) containing 0.1M N-propyl gallate (Sigma-Aldrich, St. Louis) to prevent photo bleaching and were stored at 4°C in the dark until analyzed by confocal microscopy.
For confocal microscopy, a Zeiss LSM410 inverted laser scanning microscope was employed. The microscope was equipped with a krypton/argon mixed-gas laser and was operated by the Microcosm Renaissance 410 (v2.3.4) software package. Images were acquired with a Plan-Apochromat 63x/1.4 oil immersion objective (Carl Zeiss, Thornwood). Additional optical magnification (up to 5-fold) was achieved using the zoom feature of the image acquisition software. For two-color analysis, objects were excited using 488/568 nm laser lines. Green and red emissions were recorded through appropriate filters (515–540 nm band pass filter for Cy2 and 590 nm long pass filter for Texas-Red) and stored in separate (red and green) image channels. At the same time, bright field images (Nomarski optics) were collected and stored in a third (blue) channel. Image quality was enhanced during data acquisition using the Renaissance 410 line average feature (8 or 16x).
We thank Michael Malim for the Vif monoclonal antibody (MAb #319) and Jason Roos and Janice Clements for the LuSIV indicator cell line. Both reagents were obtained through the NIH Research and Reference Reagent Program. Part of this work was supported by a Grant from the NIH Intramural AIDS Targeted Antiviral Program to K.S.
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