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Journal of Virology, February 2005, p. 2484-2492, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2484-2492.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Phosphorylation and Proteolytic Cleavage of Gag Proteins in Budded Simian Immunodeficiency Virus

Sarah M. Rue,1,2 Jason W. Roos,1,2 Patrick M. Tarwater,3 Janice E. Clements,1,2 and Sheila A. Barber1*

Department of Comparative Medicine,1 Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland,2 School of Public Health, University of Texas Health Science Center at Houston, El Paso, Texas3

Received 19 March 2004/ Accepted 5 October 2004


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ABSTRACT
 
The lentiviral Gag polyprotein (Pr55Gag) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55Gag is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55Gag and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55Gag, several CA-containing cleavage intermediates of Pr55Gag, and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55Gag can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55Gag and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55Gag and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.


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INTRODUCTION
 
The gag gene of lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) encodes a polyprotein, Pr55Gag, which is composed of six distinct domains (from N terminus to C terminus): matrix (MA), capsid (CA), p2, nucleocapsid (NC), p1, and p6 (22, 24, 37, 39). In approximately 5% of gag translation events, a ribosomal frameshift causes the production of the Pr160Gag-Pol polyprotein, which contains the viral enzymes protease (PR), reverse transcriptase, and integrase at the C terminus of the Pr55Gag polyprotein (27). Because Pr55Gag and Pr160Gag-Pol are incorporated into the virion at approximately the same levels at which they are translated, the virion is composed mostly of Pr55Gag. At an undetermined point in the late stages of the lentivirus life cycle, PR cleaves itself out of the Pr160Gag-Pol polyprotein, homodimerizes, and separates the constituent domains of Pr55Gag, eventually releasing each of these domains as free proteins that have independent functions (for a review, see reference 11). Because the rate of cleavage (also referred to as proteolytic processing) by PR is different at each of the cleavage sites in Pr55Gag, several cleavage intermediates are detectable both in virions and in virus-producing cells (10, 38, 39).

The proteolytic processing of Pr55Gag enables the virion to undergo the process of maturation, a structural rearrangement in which MA stays primarily associated with the virion envelope while CA forms a cone-shaped core around NC and the viral RNA (16; for reviews, see references 15 and 17). Virion maturation and the prerequisite Pr55Gag cleavage are absolutely required for virion infectivity (32, 36, 44). It is generally agreed that virion maturation occurs outside the cell in lentiviruses, since budding virions have an immature morphology and mature virions are visible only outside the cell in electron micrographs of most virus-producing cells (macrophages are one exception) (17, 25, 33, 42). The timing of proteolytic cleavage of the Pr55Gag polyprotein is less clear, however, as Pr55Gag, its cleavage intermediates, and its final cleavage products (free MA, CA, etc.) are all present both in budded virions and in virus-producing cells (39). In HIV-1, it has been shown that Pr55Gag cleavage begins at the plasma membrane of the cell in the budding virion (30, 31), but a consensus has not been reached on whether cleavage is completed inside the budding virion before it pinches off from the plasma membrane or inside the budded virion that has been released from the cell. Furthermore, very little is known about proteolysis of Pr55Gag in SIV.

The processes of Pr55Gag cleavage and virion maturation are believed to be highly regulated (13, 14, 45, 46, 53), although the mechanisms of this regulation are not entirely clear. Some groups have proposed that phosphorylation of retroviral Gag proteins enhances their cleavage by PR (41, 55). HIV-1 studies have demonstrated that the free (completely cleaved) CA protein is posttranslationally modified by phosphorylation (7, 9, 10, 38, 39), and some groups have observed phosphorylated Pr55Gag (6, 10).

We became interested in the relationship between lentiviral CA phosphorylation and proteolytic processing of Pr55Gag when we detected phosphorylated forms of the free CA protein and several polyproteins that contain CA as a domain (Pr55Gag and some of its cleavage intermediates) in SIV virions but not in virus-producing cells (our unpublished observations). Here, we investigate this relationship in detail. By analyzing 35S- or 32P-labeled virus produced from cells transfected with a protease-inactive mutant of SIVmac239, we demonstrate that phosphorylation of SIV Pr55Gag can occur and does not require the presence of a functional viral protease. By cleaving Pr55Gag in 35S- and 32P-labeled wild-type and protease mutant virus with exogenous protease, we show that the CA domain of Pr55Gag can be phosphorylated in SIV virions. Furthermore, we provide evidence that the CA domain of Pr55Gag and/or its cleavage intermediates is phosphorylated to a greater extent than free, completely cleaved CA protein. By assaying changes in Gag protein levels over time in virus incubated in vitro, we demonstrate that SIV PR can cleave Gag proteins in budded virions. Using a similar assay, we show that the phosphorylation of Pr55Gag and CA-containing cleavage intermediates, but not the phosphorylation of free CA protein, increases over time in the virion, which indicates that phosphorylated free CA is likely derived from cleavage of phosphorylated CA domains by PR. Taken together, these results strongly suggest that, in budded SIV virions, phosphorylation of CA domains occurs before they are completely cleaved by PR.


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MATERIALS AND METHODS
 
Plasmid construction. The previously characterized clones of the 5' (p239SpSp5') and 3' (p239SpE3') halves of the SIVmac239 genome (AIDS Research and Reference Reagent Program) (34, 47) were used to generate the full-length D(25)APR SIVmac239 mutant exactly as described previously (49). The primers used to create this viral mutant made a (GAT->GCT) nucleotide substitution in codon 25 of the protease-encoding portion of pol. The full-length clone D(25)APR SIVmac239 pBS– was verified by complete sequencing of the viral coding region.

Cell culture. Human embryonic kidney 293T cells (American Type Culture Collection, Manassas, Va.) were maintained as described previously (3), except that 0.5 mg of gentamicin/ml was used instead of penicillin-streptomycin.

Metabolic labeling and immunoprecipitation of Gag proteins. 293T cells at ~40% confluence in 10-cm-diameter dishes or 150-cm2 flasks were transfected with a green fluorescent protein (GFP) control plasmid (pEGFP-N1; BD Biosciences Clontech, Palo Alto, Calif.) or infectious viral DNA with Lipofectamine and Plus reagents (Invitrogen Corporation, Carlsbad, Calif.) according to the manufacturer's recommendations. The next day, cells were metabolically labeled with [35S]Met/Cys alone or [35S]Met/Cys and [32P]orthophosphate in parallel. For 35S labeling, culture medium was replaced with methionine- and cystine-free Dulbecco's modified Eagle medium (DMEM) (Invitrogen) supplemented with 2% fetal bovine serum (Atlanta Biologicals, Norcross, Ga.), 2 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, and 0.5 mg of gentamicin/ml (mcfDMEM), and cells were serum starved for 30 min at 37°C. The medium was then replaced with fresh mcfDMEM containing ~0.1 mCi of Tran[35S]-Label ([35S]Met/Cys; ICN Pharmaceuticals, Inc., Costa Mesa, Calif.)/ml, and cells were incubated for 1 to 2 h at 37°C. For 32P labeling, culture medium was replaced with phosphate-free DMEM (Invitrogen) supplemented with 5% dialyzed fetal bovine serum (Invitrogen), 2 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, and 0.5 mg of gentamicin/ml, and cells were incubated for 30 min at 37°C. The medium was replaced with fresh phosphate-free DMEM containing [32P]orthophosphate (Perkin-Elmer Life and Analytical Sciences, Inc., Boston, Mass.) at ~1.0 mCi/ml, and cells were incubated for 2 h at 37°C. In experiments in which cells were labeled in parallel with 35S and 32P, [35S]Met/Cys or [32P]orthophosphate was added to parallel cultures simultaneously, and both cultures were labeled for 2 h.

Cell supernatants containing radiolabeled virus were pelleted briefly and filtered through a 0.45-µm-pore-size syringe filter (Millipore, Bedford, Mass.) to remove cell debris. Radiolabeled virus was purified by centrifugation at 133,000 x g for 2 h at 4°C through a cushion of 20% sucrose in TNE buffer (20 mM Tris [pH 8.0], 150 mM NaCl, and 2 mM EDTA). Virus pellets were lysed in modified radioimmunoprecipitation assay (RIPA) buffer (phosphate-buffered saline with 1% [vol/vol] Igepal [NP-40], 0.5% [wt/vol] sodium deoxycholate, 0.1% [wt/vol] sodium dodecyl sulfate [SDS], 1 mM sodium orthovanadate, and protease inhibitor cocktail III [Calbiochem, San Diego, Calif.]). Radiolabeled cells were washed once with ice-cold phosphate-buffered saline and lysed in RIPA buffer, and cell lysates were clarified by sonication and centrifugation.

For immunoprecipitation reactions, cell (500 µg of protein) and virus lysates prepared as described above were incubated for at least 1 h at 4°C with 3 µg of immunoglobulin G (IgG)-purified rabbit SIV CA polyclonal antiserum (HRP, Denver, Pa.). Protein A agarose beads (Sigma-Aldrich, St. Louis, Mo.) were then added, and lysates were incubated overnight at 4°C. Immunoprecipitates were washed with RIPA buffer and separated on 12.5% Tris-HCl Criterion precast gels (Bio-Rad, Hercules, Calif.) that were fixed and subjected to phosphorimager analysis with a Typhoon 9210 phosphorimager and Molecular Dynamics ImageQuant version 5.2 software (Amersham Pharmacia, Piscataway, N.J.). In experiments in which Gag was immunoprecipitated from 500 µg, 1 mg, and 1.5 mg of cell lysate protein (Fig. 1B), 3, 6, and 9 µg of IgG-purified SIV CA antiserum were used for immunoprecipitation, respectively, and volumes of the immunoprecipitation reactions were scaled up accordingly.



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  		FIG. 1. Analysis of SIV Gag phosphorylation in virions and virus-producing cells. (A) Virus lysates derived from 293T cells transfected with a GFP control vector or wild-type (WT) SIVmac239 proviral DNA and labeled with [35S]Met/Cys (35S) or [32P]orthophosphate (32P) were immunoprecipitated with IgG-purified SIV capsid (CA) polyclonal antiserum, resolved by SDS-PAGE, and visualized by phosphorimager analysis. (B) Cell lysate protein from the transfection in panel A (500 µg [1x], 1 mg [2x], or 1.5 mg [3x]) was immunoprecipitated with IgG-purified SIV CA antiserum as described in Materials and Methods and resolved by SDS-PAGE.

HIV-2 protease cleavage assay. 293T cells were transfected and metabolically labeled as described above, with the exception that cells were labeled in parallel with 0.04 mCi of [35S]Met/Cys/ml and 0.8 mCi of [32P]orthophosphate/ml for 2 h. Labeled virus-containing cell supernatants were pelleted briefly and filtered, and virus was purified by centrifugation as described above. Purified virus was then lysed, divided in half, and incubated with either HIV-2 protease (PR) (AIDS Research and Reference Reagent Program) (48) or PR suspension buffer (100 mM NaPO4 [pH 8.0] and 50 mM NaCl) in cleavage buffer (10 mM Tris-HCl [pH 7.0], 130 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride) for 15 h at 37°C. Cleavage products were directly resolved on a 15% Tris-HCl Criterion precast gel (Bio-Rad), which was fixed and subjected to phosphorimager analysis and band densitometry. Ratios of 32P to 35S (32P/35S ratios) for Gag proteins (see Fig. 3 and 5) were determined by dividing the band intensity of the 32P-labeled protein by the band intensity of the 35S-labeled protein corrected for methionine and cysteine content. The 32P/35S ratio is a measure of the extent of protein phosphorylation and is unaffected by the actual amount of protein present in each sample, because the amount of protein phosphorylation (32P band intensity) is standardized according to the amount of protein that is present in the sample (the 35S band intensity).



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  		FIG. 3. Analysis of phosphorylation of the domain form of SIV CA. Virus lysates derived from 35S- or 32P-labeled 293T cells transfected with a GFP control vector, D(25)APR SIVmac239 proviral DNA, or wild-type (WT) SIVmac239 proviral DNA were divided in half and either treated with HIV-2 PR (+) or mock treated (–), and products were subjected to SDS-PAGE and phosphorimager analysis. Gel lanes were rearranged with Adobe Photoshop to accommodate the flow of the text. MA, free matrix protein.



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  		FIG. 5. Phosphorylation of Gag proteins over time in budded virus. 35S-labeled (A) or 32P-labeled (B) 293T-derived wild-type SIVmac239 virus-containing cell supernatants were assayed as described in the legend to Fig. 4. (C) Phosphorylation (n-fold) of Gag proteins over time relative to the 0-min time point (t = 0), calculated from band densitometry data from the gels in panels A and B as described in Materials and Methods. Top panel, Pr55Gag versus cleavage intermediates; bottom panel, free CA versus cleavage intermediates. Arrows indicate the precursor-product relationship between Pr55Gag and its cleavage intermediates (top panel) or the cleavage intermediates and free CA (bottom panel). The vertical dotted line indicates the window of time (0 min to 6 h) during which the most dramatic increases in Gag protein phosphorylation were detected.

Analysis of Gag protein cleavage and phosphorylation over time in budded virus. Virus derived from transfected 293T cells that had been labeled with ~0.2 mCi of [35S]Met/Cys/ml or ~0.6 mCi of [32P]orthophosphate/ml for 1 (35S only) or 2 (35S and 32P in parallel) h as described above was briefly pelleted, filtered, aliquoted, and incubated at 37°C for the indicated amounts of time. At each time point, an aliquot was immediately put on ice and then placed at –80°C to stop further proteolytic activity. After the time course was complete, all aliquots were thawed in an ice bath, and virus was purified by centrifugation through 20% sucrose in TNE buffer, lysed, and immunoprecipitated at 4°C as described above. Immunoprecipitates were resolved on 12.5% Tris-HCl Criterion gels (Bio-Rad) that were fixed and subjected to phosphorimager analysis and band densitometry. Data points for the graph of the fraction of initial protein remaining over time (see Fig. 4C) were calculated from the band densitometry data as the band intensity of the protein at time point x (t = x) divided by the band intensity of the protein at the 0-min time point (t = 0). Data points for graphs of the phosphorylation (n-fold) of proteins over time relative to t = 0 (see Fig. 5C) were calculated by dividing the 32P/35S ratio (see above) for the protein at t = x by the 32P/35S ratio for the protein at t = 0.



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  		FIG. 4. Proteolytic processing of Gag in budded SIV virions. Virus-containing cell supernatants from 293T cells transfected with wild-type (A) or D(25)APR (B) proviral DNA and labeled with 35S were aliquoted and incubated at 37°C for the indicated number of minutes. Upon completion of the time course, virus from each time point was purified, lysed, immunoprecipitated with SIV CA antiserum, resolved by SDS-PAGE, and visualized with a phosphorimager. (C) Fraction of initial protein remaining over time calculated for Gag proteins as described in Materials and Methods from band densitometry of the gels in panels A and B. Data for the first 6 h of the time course are shown, as cleavage of Gag proteins was not detected past the 6-h (360-min) time point. Results shown are representative of at least three independent experiments.


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RESULTS
 
Phosphorylated capsid-containing Gag proteins are detectable in SIV virions but not in cells. In our studies of the role of phosphorylation of SIV proteins in viral replication, we consistently observed that free CA protein, the SIV Pr55Gag precursor polyprotein, and several CA-containing cleavage intermediates were phosphorylated in budded virions (our unpublished observations; see below). To examine whether these Gag proteins are also phosphorylated in SIV-producing cells, 293T cells transfected with a GFP control vector or infectious wild-type SIVmac239 DNA were in vivo labeled with [35S]Met/Cys and [32P]orthophosphate in parallel. Cell and virus lysates were immunoprecipitated with SIV CA polyclonal antiserum (immunoprecipitation is necessary for visualization of Gag proteins in cell lysates), resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and subjected to phosphorimager analysis (Fig. 1). By convention, in this and other assays presented in this paper, Gag protein that is detected in virus lysates represents Gag present in virus that has budded from the cell, while Gag protein that is detected in cell lysates represents both Gag present in the cytoplasm of the cell and Gag present in virions that are budding (i.e., not yet released) from the plasma membrane at the time of cell harvest.

Several CA-containing proteins were detected in 35S-labeled wild-type SIV virus lysates: the Pr55Gag precursor polyprotein, free CA protein, and five processing intermediates that we have named according to their apparent molecular masses determined from this and other experiments (p50, p42, p41, p35, and p28 [CA-p2]) (Fig. 1A). The CA-p2 protein is the last processing intermediate in the cleavage of lentiviral CA, such that cleavage of the CA-p2 boundary by PR produces free CA (39, 45, 46, 52). For the sake of simplicity, from this point on we refer to CA that has been completely cleaved as "free" and CA that is present as a domain in Pr55Gag and/or the cleavage intermediates as "domain form(s) of CA" or "CA domains."

Pr55Gag, p50, p41, CA-p2, and a 27-kDa protein that was confirmed by mass spectroscopy to be SIV CA (data not shown) were all detectably phosphorylated in 32P-labeled virus (Fig. 1A), indicating that most but not all of the different cleavage forms of the CA protein are phosphorylated in the virion. To our knowledge, phosphorylated Pr55Gag has been observed in only one other study in a system using full-length HIV or SIV virus: it was detected intracellularly in a T-lymphocyte line infected with HIV-1 (10). Furthermore, the detection of phosphorylated forms of cleavage intermediates of Pr55Gag in lentiviral virions has not been reported. It is important to note that because the SIVmac239 p2 protein does not contain any of the amino acids that are typical acceptors for phosphate modification (serine, threonine, tyrosine, or histidine) (21, 51), the CA domain, not the p2 domain, of the CA-p2 intermediate is phosphorylated in SIV virions.

In contrast, no phosphorylated Pr55Gag, cleavage intermediates, or free CA was detected in cell lysates, even in immunoprecipitates from 1.5 mg of cell lysate (three times the amount of lysate required for optimal detection of 35S-labeled Gag proteins) (Fig. 1B). The same CA-containing Gag proteins were detected as phosphorylated proteins in budded virus but not in virus-producing cells when either primary rhesus macrophages or CEMx174 cells were used in similar assays (data not shown), indicating that the SIV Gag protein phosphorylation profile is independent of cell type. The lack of detectable phosphorylation of free SIV CA inside virus-producing cells presents a potential difference between SIV and HIV-1, since at least one study has shown convincingly that free HIV-1 CA is phosphorylated both inside the cell and in virus particles (40). Collectively, these data indicate that phosphorylation of CA-containing Gag proteins occurs predominantly (if not exclusively) inside the virion subsequent to budding and is likely mediated by one or more virion-associated kinases.

Phosphorylation of Pr55Gag in a protease-inactive mutant of SIVmac239. In addition to CA, at least two other previously characterized phosphoproteins (MA and p6) (5, 12, 20, 39, 40) are also present as domains within lentiviral Pr55Gag. To determine whether the phosphorylated Pr55Gag polyprotein present in SIV virions was phosphorylated on CA or another domain, we initially wanted to cleave phosphorylated Pr55Gag in virus lysates with exogenous protease and to analyze which of the resultant Pr55Gag constituent proteins are phosphorylated. The limitation of using wild-type virus lysates in such an experiment is that they contain not only Pr55Gag but also several of its CA-containing cleavage intermediates (Fig. 1A). The origin of free CA produced by treating wild-type virus lysates with exogenous protease would therefore be uncertain. To circumvent this problem, we constructed a viral mutant that encoded an alanine substitution at the active site aspartate of PR, D(25)APR SIVmac239. Cells transfected with this mutant should produce virions that contain Pr55Gag but none of its processing intermediates, making it easier to examine the phosphorylation state of the CA domain of Pr55Gag. We observed no detectable PR activity, virion maturation, or virus replication in our D(25)APR SIVmac239 mutant (data not shown), a phenotype identical to that reported for HIV-1 proviral clones with active site substitutions in PR (18, 36).

To determine whether Pr55Gag was phosphorylated in D(25)APR SIVmac239 virus, 293T cells transfected with a GFP control vector, wild-type viral DNA, or D(25)APR viral DNA were labeled in parallel with [35S]Met/Cys and [32P]orthophosphate, and virus and cell lysates from these samples were immunoprecipitated with IgG-purified SIV CA antiserum and resolved by SDS-PAGE (Fig. 2). As in wild-type virus lysates, Pr55Gag in D(25)APR virus lysates was phosphorylated, indicating that phosphorylation of Pr55Gag can occur in the absence of an active viral protease (Fig. 2A). Consistent with our earlier results, no intracellular phosphorylated Gag proteins were detected for either wild-type or D(25)APR SIVmac239 (Fig. 2B).



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  		FIG. 2. Gag phosphorylation in a protease-inactive mutant of SIVmac239. Virus (A) and cell (B) lysates derived from 35S- or 32P-labeled 293T cells transfected with a GFP control vector, wild-type (WT) SIVmac239 proviral DNA, or D(25)APR SIVmac239 proviral DNA were immunoprecipitated with IgG-purified SIV CA antiserum, resolved by SDS-PAGE, and visualized with a phosphorimager.

The CA and MA domains of Pr55Gag are phosphorylated in SIV virions. To examine whether the CA domain of Pr55Gag is phosphorylated in budded virus, we labeled 293T cells transfected with a GFP control vector, D(25)APR proviral DNA, or wild-type proviral DNA in parallel with 35S or 32P. Virus was purified by centrifugation, lysed, divided in half, and either mock treated or treated with exogenous HIV-2 PR (purified SIV PR is not readily available). The products of these reactions were then directly resolved by SDS-PAGE (Fig. 3). Phosphorylated forms of both free MA and free CA were detected in D(25)APR virus lysates that had been treated with HIV-2 PR, indicating that the phosphorylated Pr55Gag observed in wild-type virus is likely phosphorylated on both the MA and CA domains. Because we had difficulty detecting 35S-labeled p6 in this and other assays (data not shown), we cannot determine whether the p6 domain of SIV Pr55Gag is phosphorylated as well.

Although, consistent with earlier results, phosphorylated free CA was detected in mock-treated wild-type virus lysates in this assay, there appeared to be more phosphorylated free CA in HIV-2 PR-treated wild-type virus lysates (Fig. 3). To quantitatively address this possibility, band densitometry was performed on the 35S- and 32P-labeled free CA protein in both mock-treated and HIV-2 PR-treated wild-type virus lysates. The band densitometry data were used to calculate the 32P/35S ratio for free CA in both mock-treated [(32P/35S)mock] and HIV-2 PR-treated [(32P/35S)HIV-2 PR] wild-type virus lysates as described in Materials and Methods. The free CA present in mock-treated lysates represents only CA that was completely cleaved by the innate (SIV) PR, while the free CA in HIV-2 PR-treated lysates represents both CA that was cleaved by the innate PR and CA that originated in the domain form in Pr55Gag and/or its cleavage intermediates but was released by treatment with exogenous (HIV-2) PR. Therefore, (32P/35S)mock represents the extent of phosphorylation of free CA, while (32P/35S)HIV-2 PR represents the extent of phosphorylation of both free CA and domain form CA. As such, if domain form CA is phosphorylated to the same extent as free CA, we would expect that (32P/35S)HIV-2 PR would equal (32P/35S)mock. However, if domain form CA is phosphorylated to a greater extent than free CA, we would expect that (32P/35S)HIV-2 PR would be greater than (32P/35S)mock.

Analysis of the band densitometry data for free CA in wild-type virus indicated that (32P/35S)HIV-2 PR was greater than (32P/35S)mock: in this experiment, (32P/35S)HIV-2 PR was 5.9, while (32P/35S)mock was 3.3. This trend was observed in three additional independent replicates of this experiment (data not shown). Therefore, the domain form of CA is phosphorylated to a greater extent than free (completely cleaved) CA protein in budded SIV virions. We cannot determine whether the phosphorylated free CA protein released by HIV-2 PR treatment of wild-type virus originated as a domain in Pr55Gag or in one of the CA-containing cleavage intermediates. However, it is very likely that at least some of this phosphorylated free CA originated as a domain in Pr55Gag, since phosphorylated free CA was released from Pr55Gag when D(25)APR SIVmac239 virus was treated with HIV-2 PR.

Pr55Gag processing in virions. We next identified the origin of phosphorylated free CA present in budded SIV virions. Phosphorylated forms of CA are detectable only in budded virus; thus, there are only two possible origins for phosphorylated free CA: either (i) phosphorylated domain forms of CA are cleaved within the virion to produce phosphorylated free CA or (ii) the domain forms of CA and free CA protein are independently phosphorylated by one or more virion-associated kinases.

As discussed above, no studies have directly addressed whether PR can cleave Pr55Gag and its intermediates in SIV virions. Therefore, to assess whether the first scenario is even possible, we assayed the proteolysis of CA-containing Gag proteins over time in virus-containing cell supernatants. 293T cells transfected with wild-type or D(25)APR SIVmac239 were labeled with [35S]Met/Cys, and virus-containing cell supernatants were briefly pelleted and filtered to remove cell debris. Filtered virus was then aliquoted and incubated at 37°C for different amounts of time. At each time point, an aliquot of virus was immediately frozen to stop proteolytic processing. When the time course was complete, all aliquots were thawed in an ice bath, virus was purified by centrifugation and lysed, and proteins were immunoprecipitated with IgG-purified SIV CA antiserum and resolved by SDS-PAGE (Fig. 4).

We observed that SIV Pr55Gag and all of the previously detected CA-containing cleavage intermediates (p50, p42, p41, p35, and CA-p2) are in fact cleaved over time in budded wild-type virus (Fig. 4A). Further, this cleavage is specific to the viral protease, since we observed very little change in Pr55Gag levels over time in protease-inactive D(25)APR SIVmac239 virus (Fig. 4B). Band densitometry was performed for each protein, and the fraction of initial protein remaining over time was calculated for Pr55Gag, free CA, and the cleavage intermediates (see Materials and Methods). All of the detectable cleavage of CA-containing Gag proteins in wild-type virus in this assay occurred between the 0-min and 6-h (360-min) time points (Fig. 4C).

Similar results were obtained for this time course experiment when [35S]Met/Cys-labeled, 293T-derived lysed wild-type virus was analyzed by direct electrophoresis rather than immunoprecipitation (data not shown), indicating that the results of this experiment are not biased by immunoprecipitation. Moreover, similar trends were also observed when these time course experiments were performed with virus derived from transfection of COS-1 cells (data not shown), indicating that the intravirion cleavage of CA-containing Gag proteins by PR is not specific to 293T-derived virus.

It is clear that cleavage of SIV Pr55Gag and its intermediates does not occur exclusively in budded virions. Both cleavage intermediates of Pr55Gag and completely cleaved proteins such as free CA are present in budded virus produced from 35S-labeled cells at the earliest time point at which it can be detected (these cleavage products are likely the result of proteolysis that occurs at the plasma membrane in the budding virion [our unpublished observations]). Furthermore, cleavage intermediates of Pr55Gag and free CA can be detected in lysates of 35S-labeled cells producing SIV (Fig. 1B) (these cleavage products [like those in budded virus] are detected only when an active viral protease is present, and they likely originate from cleavage of Pr55Gag by PR both in the cytoplasm and in virus that is budding from the plasma membrane of the cell [30, 31]). Nevertheless, the results of our time course assays clearly indicate that SIV PR can cleave Pr55Gag and its cleavage intermediates in virus that has budded from the cell.

Phosphorylation of CA-containing Gag proteins over time in budded virus. Since cleavage of Pr55Gag by PR can occur in budded virus, it is possible that phosphorylated free CA protein is derived from the proteolytic release of phosphorylated domain forms of CA from Pr55Gag and/or its cleavage intermediates. However, we have not yet eliminated the possibility that free CA and proteins that contain CA as a domain (Pr55Gag and the phosphorylated cleavage intermediates p50, p41, and CA-p2) are independently phosphorylated. To examine this possibility, we labeled 293T cells transfected with wild-type SIVmac239 with 35S (Fig. 5A) and 32P (Fig. 5B) in parallel and performed the same assay shown in Fig. 4 with the budded virus. Consistent with earlier results, Pr55Gag, free CA, and the CA-containing Pr55Gag cleavage intermediates p50, p41, and CA-p2 were detectably phosphorylated in budded virus (Fig. 5B). Band densitometry of these gels was performed, and the phosphorylation of each of the proteins relative to the 0-min time point (t = 0) was calculated for each time point (Fig. 5C) (see Materials and Methods for calculations). We repeatedly observed that phosphorylation of Pr55Gag and, to a greater extent, phosphorylation of the cleavage intermediates p50, p41, and CA-p2 increased over time in budded virus (Fig. 5C, top panel). Although active phosphorylation of Pr55Gag and the cleavage intermediates p50, p41, and CA-p2 was detected throughout the entire 48-h time course, the greatest increases in phosphorylation of these proteins occurred between the 0-min and 6-h time points. In contrast, free CA phosphorylation did not significantly increase relative to the phosphorylation of the cleavage intermediates (Fig. 5C, bottom panel). Thus, we conclude that free CA is not likely to be actively phosphorylated over time by a virion-associated kinase. Furthermore, we propose that the phosphorylated free CA detected in SIV virions is derived from intravirion cleavage of phosphorylated domain form CA by PR. One could argue that, instead, the phosphorylated free CA detected in virions is derived from independent phosphorylation events that occur before the 0-min time point of our assay for active phosphorylation of Gag (Fig. 5) and that intravirion cleavage of phosphorylated CA domains by PR does not contribute to the pool of phosphorylated free CA in SIV virions. However, Fig. 4 demonstrates that at least some CA domains are cleaved all the way to free CA within the virion: the total amount of free CA in virions increases over time due to PR activity. Thus, in order for intravirion cleavage of phosphorylated domain form CA to not contribute to the pool of phosphorylated free CA in virions, PR would have to cleave only nonphosphorylated CA domains all the way to free CA in the virion. If this were true, the phosphorylation of free CA over time (an indicator of changes in the CA 32P/35S ratio) would decrease: the numerator in the 32P/35S ratio (the free CA 32P band intensity) would remain constant, while the denominator (the free CA 35S band intensity) would increase over time. This is not the case: the phosphorylation of free CA over time does not change significantly (Fig. 5C). Therefore, we conclude that at least some of the phosphorylated free CA present in SIV virions must be derived from the intravirion cleavage of phosphorylated CA domains by PR. This result indicates that when CA domains are both phosphorylated by a virion-associated kinase and cleaved by PR in SIV virions, phosphorylation precedes the completion of cleavage.


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DISCUSSION
 
In this study, we provide definitive evidence that proteolytic processing of Pr55Gag and its cleavage intermediates can occur in budded SIV virions. Several viral proteins are known to be cleaved inside retroviral virions by PR, such as HIV-1 Vif (35), HIV-1 and HIV-2 Nef (4, 43, 50, 54), and, in several retroviruses, the cytoplasmic tail of the transmembrane portion of Env (2, 19, 23). However, up to this point, it has not been generally agreed that Gag proteins can be cleaved in budded virus, with the exception of the CA-p2 intermediate of Rous sarcoma virus Gag (1). In fact, there are conflicting reports in the literature as to whether Gag processing occurs in HIV-1 virions. Ten years ago, one group was unable to detect changes in Gag protein levels over time in HIV-1 virions using an assay quite similar to the one used in this study with the exception that changes in Gag protein levels were assayed by immunoblotting (29). As such, it was believed that, in HIV-1, processing must be completed either before the virion is released from the cell or in a very small window of time (less than 10 s) after the virion has been released (29). In contrast, in their recent studies of intravirion Vif cleavage, Khan and colleagues observed cleavage of Pr55Gag in HIV-1 virions over a period of several hours postbudding (35), similar to our findings in SIV. In our view, the apparently contradictory results in HIV-1 can be explained by differences in experimental design: 35S labeling of virus (performed as described in reference 35 and this study) is necessary to attain the level of sensitivity required to detect subtle changes in Gag protein levels over time. Similar assays using other retroviruses will ultimately be required to determine whether intravirion cleavage of Pr55Gag and its intermediates by PR is specific to HIV and SIV or conserved among retroviruses.

Two of our findings indicate that active phosphorylation of CA-containing Gag proteins occurs predominantly, if not exclusively, in budded SIV virions: (i) phosphorylated forms of Pr55Gag, three of its CA-containing cleavage intermediates (p50, p41, and CA-p2), and free CA are detectable in budded virions but not in virus-producing cells (Fig. 1) and (ii) Pr55Gag and the same three cleavage intermediates are actively phosphorylated over time in budded virus (Fig. 5). These findings also indicate that at least one cellular kinase capable of Gag phosphorylation is incorporated into SIV virions. CA is clearly a substrate for this virion-associated kinase: the observed phosphorylation of CA-p2 over time in budded virions (Fig. 5) must occur on the CA domain, and (although we cannot be certain) it is possible that the phosphorylation of Pr55Gag, p50, and p41 over time occurs on the CA domains of these proteins as well. However, as free CA is not actively phosphorylated over time in budded virions, the virion-associated kinase clearly prefers domain forms of CA to free CA as a substrate. Although the identity of the virion-associated kinase(s) responsible for phosphorylation of SIV CA remains to be determined, several kinases that are known or suspected to phosphorylate Gag proteins are incorporated into HIV virions: the catalytic subunit of cellular protein kinase A (C-PKA) (8), active extracellular signal-regulated kinase 2 (ERK-2) (7, 28), and a 53-kDa serine/threonine kinase that remains to be identified (7). Virion-associated kinase activity appears to be a common feature of enveloped viruses (reviewed in reference 26), making it tempting to speculate that virion-associated kinases play an important role in the life cycle of enveloped viruses.

If, as our data suggest, cleavage of phosphorylated domain form CA by PR is the source of phosphorylated free CA (Fig. 5), why is domain form CA more phosphorylated than free CA (Fig. 3)? In our view, there are two possible explanations: (i) there is a pool of hyperphosphorylated domain form CA that is not cleaved to free CA by SIV PR or (ii) the domain form of CA is phosphorylated by a virion-associated kinase and then dephosphorylated by a virion-associated phosphatase before the completion of CA cleavage. Identification of the kinase(s) and/or phosphatase(s) incorporated into SIV virions would greatly facilitate determination of which of these scenarios occurs in budded virus.

We have demonstrated that both phosphorylation and cleavage of CA-containing Gag proteins can occur in budded SIV virions. It should be noted that cleavage of Gag proteins was detected within only the first 6 h of our time course assays (Fig. 4) and that the most dramatic increases in Gag protein phosphorylation were detected within the same window of time (Fig. 5). These early time points may in fact be the most biologically relevant, as several factors may contribute to a decline in virion infectivity over time. Therefore, it is quite possible that the phosphorylation and proteolysis of Gag that we detect in our in vitro assays represent Gag modifications that are functionally important for virus replication in vivo.

Based on the studies presented in this paper, we propose the following model for the phosphorylation and cleavage of CA-containing Gag proteins in the late stages of the SIV life cycle. Gag proteins assemble at the plasma membrane of the cell, and PR initiates Gag cleavage inside the virion as it buds. One or more cellular kinases are incorporated into the virion during this assembly and budding process. Once the virion is released from the cell, the incorporated kinase begins to phosphorylate CA domains in the Gag polyproteins (Pr55Gag and several of its intermediates) that have not yet been cleaved by PR. PR then cleaves some of these phosphorylated CA domains all the way to free CA.

As discussed, the most dramatic increases in phosphorylation of CA-containing Gag proteins were detected in the same window of time during which all of the cleavage of these proteins by PR was detected (0 min to 6 h). This observation, coupled with studies of Gag proteins in other retroviruses which indicate that phosphorylation of Gag stimulates its cleavage by PR (41, 55), leads us to hypothesize that the phosphorylation and proteolysis of Gag proteins may be functionally linked in SIV. Since we also demonstrated that, in budded virions, the phosphorylation of CA domains in Pr55Gag and/or its cleavage intermediates precedes the cleavage of these domains by the viral protease (Fig. 5), it is possible that phosphorylation of CA domains regulates the timing of either Gag cleavage by PR or virion maturation in SIV. Efforts focused on identifying the site(s) of CA phosphorylation should clarify the function of this modification in the life cycle of SIV.


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ACKNOWLEDGMENTS
 
We thank Robert Cole for his contributions as Director of the AB Mass Spectrometry/Proteomics Facility at the Johns Hopkins University School of Medicine (www.hopkinsmedicine.org/msf/), which is supported by a National Center for Research Resources shared instrumentation grant (1S10-RR14702), the Johns Hopkins Fund for Medical Discovery, and the Institute for Cell Engineering. We also thank Jef Boeke, Carolyn Machamer, and James Hildreth for insightful discussions. The reagents p239SpSp5' and p239SpE3' from Ronald Desrosiers and HIV-2 protease from Bret Shirley and Michael Cappola, Boehringer Ingelheim Pharmaceuticals, Inc., were obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

This work was supported by grants to J.E.C. from the National Institutes of Health (NS35751, NS38008, MH70306, and NS47984).


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FOOTNOTES
 
* Corresponding author. Mailing address: 733 N. Broadway Rm. 831, Baltimore, MD 21205. Phone: (410) 955-9770. Fax: (410) 955-9823. E-mail: sabarber{at}jhmi.edu. Back


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Journal of Virology, February 2005, p. 2484-2492, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2484-2492.2005
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