Wild-Type-Like Viral Replication Potential of Huma
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病菌学杂志 2005年第13期
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China
ABSTRACT
Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.
INTRODUCTION
Lipid rafts are highly specialized membrane microdomains present in both the plasma and endosomal membranes of eukaryotic cells (23, 55). These dynamic microdomains, which are organized in the lateral dimension of the plasma membrane, are characterized by detergent insolubility, light density, and enrichment of cholesterol and glycosphingolipids (for reviews, see references 3 and 55). Lipid rafts play important roles in various biological processes such as cell surface signal transduction, T-cell activation, and intracellular trafficking (17, 55, 56, 60). Many membrane-enveloped viruses also assemble and selectively bud from lipid rafts on the surfaces of infected cells; the envelope (Env) glycoproteins of some of these viruses are known to be associated with lipid rafts (2, 34, 62, 69). Acylation of viral Env proteins, particularly palmitoylation, may be important for the targeting of these Env proteins to lipid rafts (for reviews, see references 48 and 53). Lipid rafts also serve as important entry and assembly/budding sites for human immunodeficiency virus type 1 (HIV-1) (30, 33, 41, 46, 64). Removal of cellular cholesterol with ?-methyl cyclodextrin was shown to reduce HIV-1 budding and infectivity and Env-mediated syncytium formation (29, 33, 44, 61). Also, depletion of cholesterol in the viral envelope may impair viral internalization (16) and permeabilize the virion envelope (15).
In retroviruses, palmitoylation may play some roles in viral replication. For instance, palmitoylation-deficient Env mutants of murine leukemia virus (MLV) are mostly soluble when extracted using ice-cold Triton X-100, and they stay at the bottom of the sucrose gradient (28). In addition, treatment of cells with ?-methyl cyclodextrin abolishes the ability of the MLV Env to associate with lipid rafts (28). Those studies indicated that palmitoylation of the MLV Env is critical for lipid raft association. Nevertheless, mutant Env proteins of MLV are still able to mediate the syncytium-forming ability, suggesting that palmitoylation or raft association is not required for MLV-mediated fusion activity (28). In contrast, palmitoylation of the transmembrane (TM) protein of Rous sarcoma virus was shown to be required for protein stability and virus infectivity (42). Nevertheless, acylated Env glycoproteins of this virus are not sequestered into lipid raft domains (43). The TM proteins of HIV-1 and simian immunodeficiency virus are also posttranslationally modified by the addition of palmitate to the Cys residues located in the cytoplasmic domains of these viruses through a thioester bond (68). Palmitoylation of the T-cell (T)-tropic HXB2 strain of HIV-1 occurs on two Cys residues, Cys-764 and Cys-837, located in the cytoplasmic tail of gp41 (68). Although HIV-1 Env is associated with lipid rafts (41), whether palmitoylation of Env plays a crucial role in Env localization in lipid rafts and viral infectivity has been a longstanding question.
Rousso et al. found that substitutions of the two cytoplasmic Cys residues in the Env of the HXB-2D clone with Ser residues significantly decreased one-cycle viral infectivity by reducing incorporation of the C764S C837S double-mutant Env into the virus (50). Also, most of the double-mutant Envs could be extracted into the soluble fraction with Triton X-100 at 4°C, whereas both the C764S and C837S mutants, just like the wild-type (WT) Env, were still distributed in the membrane pellets (50). Their results indicated that palmitoylation of the HIV-1 Env is critical for viral infectivity through the targeting of Env to lipid rafts. Interestingly, the two palmitoylated Cys residues located in the HXB2 strain are not highly conserved among HIV-1 clade B and different HIV-1 clades (Fig. 1A) (22). The NL4-3 virus contains only one Cys residue at position 764, whereas the 837 position in this virus is a Tyr residue (Fig. 1A). Other isolates in the B clade, such as MN and JRCSF, do not contain Cys residues at these two positions (Fig. 1A). Moreover, some representative isolates of other clades neither encode these palmitoylated Cys residues nor contain any other Cys residues in the cytoplasmic domains of their TM proteins (Fig. 1A) (22). This suggests the interesting proposal that palmitoylation of the HIV-1 Env is not required for viral replication.
Bhattacharya et al. recently reported that replacement of both Cys-764 and Tyr-837 of the NL4-3 env gene by a Ser or Ala residue still mediated about 10% and 40%, respectively, of the single-cycle infectivity mediated by the WT Env (5). The degree of infectivity of these two mutants paralleled the amount of mutant Env incorporated into the virus. Interestingly, these two mutants were excluded from lipid rafts. These two mutant proteins also mediated cell-cell membrane fusion with an efficiency comparable to that of the WT Env. Bhattacharya et al. also found that substitutions of both Cys-764 and Tyr-837 residues with bulky hydrophobic side chains retained the raft association property of the Env, and these mutant proteins mediated over 80% of the one-cycle infectivity of the WT Env and were effectively incorporated into the virus, implying that these mutations may compensate for the lack of a palmitate group in the cytoplasmic tail of the NL4-3 Env. They therefore concluded that the cytoplasmic cysteines are required for Env targeting to lipid rafts but are not essential for Env incorporation into the virus or for viral infectivity.
Because of the disparate conclusions as to the effects of cytoplasmic tail palmitoylation on viral infection drawn by these two groups, we reexamined the role of cytoplasmic cysteines of the HIV-1 Env in virus replication. To truly monitor replication kinetics of mutant viruses carrying a deacylated Env, we examined the effects of deacylation of both Cys residues of the T-tropic HXB2 strain Env by Ser and Ala substitutions on viral replication, using a continuous cell culture system as well as a single-cycle viral replication assay. The properties of cell surface expression, lipid raft localization, and incorporation into the virus of mutant Env proteins were also studied. We further examined whether the effects of deacylation on viral replication are related to the T or macrophage (M) tropism of the viruses examined, which, respectively, utilize chemokine receptors CXCR4 and CCR5 as the entry coreceptors (and thus are referred to as the X4 and R5 viruses, respectively). In contrast to the earlier results (5, 50), we failed to detect any altered phenotypes of the deacylated Env mutants compared to those of the WT virus in any of the analyses performed. Our results strongly indicate that palmitoylation of Env does not play a critical role in the HIV-1 life cycle.
MATERIALS AND METHODS
Cells, hybridoma, and antibodies. 293T, HeLa, and HeLa-T4 cells were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (FBS). CEM-SS, PM1, and H938 cells were cultured in RPMI 1640 supplemented with 10% FBS. Hybridomas 902, Chessie 8, and 183 (clone H12-5C), which produce mouse monoclonal antibodies (MAbs) reactive with HIV-1 gp120, gp41, and p24, respectively, and sheep anti-gp120 were as previously described (9). The SIM2 hybridoma secreted a MAb that specifically recognizes human CD4. A MAb directed against the glycoprotein G of vesicular stomatitis virus (VSV) was purchased from Sigma (St. Louis, MO).
Construction of plasmids. For construction of Cys-to-Ser and Cys-to-Ala mutants of the Env protein of the T-tropic HXB2RU3 provirus (Fig. 1B), oligonucleotide-directed, site-specific mutagenesis using PCR overlap extension was performed as previously described (25). Oligonucleotides 8423f and 8933r were used as the 5' and 3' external primers, respectively. The paired internal primers used in the PCR encoding each Cys mutant were as follows: 764S, 5'-CTGCGGAGCCTGAGCCTCTTCAGCTACCACCGC-3' (sense) and 5'-GTAGCTGAAGAGGCTCAGGCTCCGCAGATCGTC-3' (antisense); 837S, 5'-GTACAAGGAGCTAGTAGAGCTATTCGCCACATA-3' (sense) and 5'-GCGAATAGCTCTACTAGCTCCTTGTACTACTTC-3' (antisense); 764A 5'-CTGCGGAGCCTGGCCCTCTTCAGCTACCACCGC-3' (sense) and 5-GTAGCTGAAGAGGGCCAGGCTCCGCAGATCGTC-3' (antisense); and 837A, 5'-GTACAAGGAGCTGCTAGAGCTATTCGCCACATA-3' (sense) and 5'-GCGAATAGCTCTAGCAGCTCCTTGTACTACTTC-3' (antisense). To construct the 764S 837S and 764A 837A double mutants, the 764S and 764A PCR products were used as templates, and the paired sense and antisense oligonucleotides for generating the 837S and 837A mutants were used as internal primers, respectively. All PCR amplifications were performed using Vent DNA polymerase (New England BioLabs, Beverly, MA) according to a previously described amplification program (26). The amplified mutated BamHI-XhoI fragments were cloned into the pHXB2RU3 provirus to generate various mutant proviruses. To construct HXB2-based mutant env expression plasmids, the BamHI-XhoI fragments isolated from Cys-to-Ser and Cys-to-Ala mutant pHXB2RU3 proviruses were cloned into corresponding sites in pSVE7puro, an HIV-1 long terminal repeat (LTR)-directed env expression plasmid. To generate ConB provirus-based Cys-to-Ala mutants, BamHI-XhoI fragments isolated from pHXB2RU3 mutant proviruses were used to replace the homologous sequences in the ConB provirus. The KpnI-XhoI fragments isolated from mutant ConB proviruses were cloned in the corresponding sites in pSVE7puro to generate ConB-based mutant env plasmids. All mutant clones were confirmed by DNA autosequencing.
Plasmid DNA transfection. 293T cells grown in 100-mm petri dishes were transfected with 10 μg each of the WT or mutant proviruses by a standard calcium phosphate coprecipitation method. To prepare VSV glycoprotein G-transcomplemented viral stocks, 293T cells were cotransfected with 7.5 μg of the human cytomegalovirus virus (HCMV) promoter-directed VSV G protein expression plasmid, pHCMV-VSV G, together with 7.5 μg each of the WT or mutant proviruses. To prepare HIV-1 Env pseudotypes, 293T cells were cotransfected with 7.5 μg each of pHXBBglCAT with each of the WT or mutant pSVE7puro plasmids. Cotransfection with pHXBBglCAT and an env-defective pSVE7puro(KS) was used as the negative control. For protein expression studies, HeLa cells grown in 100-mm petri dishes were transfected with 8 μg each of WT or mutant proviruses by the Lipofectamine transfection method (Life Technologies, Rockville, MD). For the [35S]methionine and [3H]palmitate labeling studies, HeLa cells grown in 6-cm petri dishes were transfected with 4 μg of WT or mutant proviruses or with 3.5 μg of WT or mutant pSVE7puro plasmids along with 1.5 μg of the Tat expression plasmid pIIIextat.
Viral infection, RT, and CAT assays. Two days after 293T transfection, cell-free culture supernatants were assayed for reverse transcriptase (RT) activity as previously described (11). Cell-free viruses containing 2 x 104 cpm of RT activity were used to challenge 106 CEM-SS or PM1 cells. Postinfection RT activity of viruses obtained from supernatants of infected cultures was then monitored. For VSV G-transcomplemented HIV-1 infection, 106 CEM-SS cells were challenged with VSV G-transcomplemented viruses containing 2 x 106 cpm of RT activity. For the env trans-complementation assay, Env-pseudotyped HXBBglCAT reporter viruses containing 2 x 105 cpm of RT activity were used to challenge subconfluent HeLa-T4 cells grown in 60-mm dishes, or 106 CEM-SS or PM1 cells, and chloramphenicol acetyltransferase (CAT) activity was assessed as previously described (10).
Isolation of human PBMCs and viral infection. Peripheral blood mononuclear cells (PBMCs) were isolated from normal donors by using a standard Ficoll/Hypaque separation method. Cells were then stimulated with interleukin-2 and phytohemagglutinin A. After stimulation, 106 cells were infected with WT or mutant viruses containing 106 cpm of RT activity, and postinfection virus production was then measured.
Western blot analysis. Two days after proviral transfection or 3 days after VSV G-transcomplemented HIV-1 infection, lysates of cells and virions were prepared as previously described (12). Equal volumes of cell and virion lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblotting analysis using MAbs 902, Chessie 8, and 183 to detect gp120, gp41, and capsid protein p24, respectively. Sheep anti-gp120 was used to detect gp160 and gp120 of the ConB virus. A MAb against VSV G and a MAb secreted from hybridoma SIM2 were used to react with VSV G and CD4, respectively. Horseradish peroxidase-conjugated anti-mouse or anti-sheep immunoglobulin G was used as the second antibody. The immune complexes were visualized with a chemiluminescence detection kit.
Sucrose gradient equilibrium ultracentrifugation and quantification of lipid raft-associated Env proteins. Flotation centrifugation to assess Env localization to lipid rafts was performed according to a previously described procedure (41) with modifications. Phosphate-buffered saline (PBS)-washed transfected 293T cells that expressed Env were extracted on ice with 0.5 ml of 1% Triton X-100 prepared in NTE buffer (25 mM Tris-HCl containing 0.15 M NaCl and 5 mM EDTA; pH 7.5) supplemented with phenylmethylsulfonyl fluoride and the protease inhibitor cocktail for 30 min. Cell lysates were centrifuged at 8,000 x g for 10 min at 4°C. The supernatants were adjusted to a final concentration of 42.5% sucrose prepared in NTE by adding an equal volume of 85% sucrose prepared in NTE. The mixed solutions were then loaded into SW41 tubes, and the extracts were overlaid with 6 ml of 30% sucrose prepared in NTE followed by 5 ml of 5% sucrose prepared in NTE. Tubes were centrifuged at 100,000 x g for 18 h at 4°C. The gradients were fractionated from the bottom of the gradient, and 1 ml was collected per fraction. Aliquots of samples from each fraction were precipitated with trichloroacetic acid and then subjected to SDS-PAGE followed by Western blotting. To quantitate lipid raft-associated Env proteins, soluble fractions 1 to 3 and lipid raft fractions 7 and 8 were individually pooled, and concentrations of total proteins were determined using a Bio-Rad (Hercules, CA) protein assay kit. Aliquots containing equal amounts of proteins from soluble and raft fractions were precipitated with trichloroacetic acid and then subjected to Western blotting using MAb Chessie 8. The ECL blot was scanned using a Microtek (Carson, CA) ScanMarker 8700 and quantitated using the MetaMorph software (Universal Imaging, Downing Town, PA).
Metabolic labeling, cell surface biotinylation, palmitoylation, and immunoprecipitation. Transfected HeLa cells grown in 6-cm petri dishes were metabolically labeled with [35S]methionine overnight, and labeled cells were surface biotinylated with 0.25 mg/ml of membrane-impermeable sulfo-N-hydroxysuccinimide-biotin. The levels of total and cell surface Env expressions were then assessed as previously described (9, 25). For palmitoylation studies, a set of transfected dishes was labeled with [35S]methionine for 5 h. Another set of transfected cells was washed twice with PBS and then incubated with 1 ml Dulbecco's modified Eagle's medium containing 5% dialyzed FBS and 5 mM sodium pyruvate at 37°C for 30 min. [3H]palmitic acid (30 to 60 Ci/mmol; New England Nuclear, Boston, MA) was dried to completeness to remove the ethanol and then dissolved in dimethyl sulfoxide before use. One millicurie of [3H]palmitate dissolved in 2 μl of dimethyl sulfoxide was diluted with PBS to 100 μl and added to each dish, and cell cultures were labeled for 5 h. Cell lysates were immunoprecipitated with anti-HIV preadsorbed onto protein A-Sepharose, and the immune complexes were separated by SDS-PAGE under nonreducing conditions.
Flow cytometry analyses. To assess Env expression on the cell surfaces, 293T cells were transfected with WT or mutant pSVE7puro plasmids together with pIIIextat. Two days after transfection, cells were divided into two portions. One portion of cells was used to determine total Env expression, and another portion of cells was processed for Env cell surface expression by fluorescence-activated cell sorter (FACS) analysis according to a procedure described previously (9).
RESULTS
Infectivity of gp41 cytoplasmic tail Cys-to-Ser mutants of a T-tropic HXB2 virus. To understand the role of palmitoylation of the Env cytoplasmic domain in viral replication, the two Cys residues located at residues 764 and 837 in the Env of the HIV-1 HXB2 strain were replaced individually or together by a Ser residue by using oligonucleotide-directed, site-specific mutagenesis with PCR overlap extension based on the pHXB2RU3 provirus (59). This molecular clone also carries functional vpr, vpu, and nef genes, which are not present in the parental HXB2 construct. These resultant mutants were referred to as the 764S, 837S, and 764S.837S mutants, respectively (Fig. 1B). Replication of these mutant viruses along with the WT virus was assessed using human CD4+ CEM-SS T cells. The 764S.837S double mutant virus, just like the WT and 764S and 837S mutant viruses, was still capable of productively replicating in CEM-SS cells (Fig. 2A). When the replication of these mutant viruses was examined in another CD4+ T-cell line, PM1, which is derived from Hut78 and expresses both CXCR4 and CCR5 coreceptors (32), all three of these Cys-to-Ser mutants productively replicated at a level similar to that of the WT virus (Fig. 2B).
Expression of viral proteins encoded by HXB2-based Cys-to-Ser mutants. To examine viral protein expression of these three Cys-to-Ser mutants in CD4+ T cells, which are natural target cells for HIV-1 infection, a high-level transient HIV-1 expression system based on pseudotyping with the VSV G protein (4, 31) was employed. This method has been shown to effectively express viral proteins in suspended T cells (9, 25, 40), which are usually transfected less efficiently than is required for biochemical analyses when other commonly used transfection methods are used. Equal amounts of cell-free VSV G-transcomplemented viruses obtained from each cotransfection and after normalization by RT activity were used to challenge CEM-SS cells. Equal portions of cell and virion lysates were analyzed by Western blotting using a cocktail containing the Env- and CA-specific MAbs. The 764S.837S double mutant produced levels of intracellular Gag and Env proteins comparable to those produced by the WT and the other two Ser mutants (Fig. 3A, lanes 1 to 4). Also, Gag assembly/budding of and Env incorporation into this double mutant virus were normal compared to those observed in the WT and the other two mutant viruses (Fig. 3A, lanes 5 to 8). When 293T cells were directly transfected with each of the WT and Cys-to-Ser mutant proviruses, these mutations did not affect Env synthesis, precursor processing, or Env incorporation into the viruses (Fig. 3B).
To determine whether these mutant Env proteins were expressed on the cell surface, HeLa cells transfected with the WT or each of the mutant proviruses were labeled with [35S]methionine overnight, and cell surface biotinylation using membrane-impermeable sulfo-N-hydroxysuccinimide-biotin was performed. This method has been widely used to examine cell surface expression of HIV Env proteins (9, 40, 51). All these mutant proviruses showed levels of the intracellular gp160 precursor and gp120, as well as cell surface gp120, that were comparable to those produced by the WT provirus upon transfection (Fig. 3C). As previously reported (9), no gp120 from WT transfection could be detected on the cell surface when cells were treated with brefeldin A, which prevents transport of glycoproteins out of the ER, prior to biotinylation (data not shown). Also, the cell surface expression of neither HIV-1 Gag Pr55 nor p24 from WT proviral transfection could be detected under this surface biotinylation condition (data not shown).
To confirm that these mutant Env proteins are as efficiently expressed on cell surface as the WT Env, these three mutations were introduced to the HIV-1 LTR promoter-driven env expression pSVE7puro plasmid. FACS analysis of 293T cells transfected with each of the WT and mutant pSVE7puro plasmids along with pIIIextat was performed. The two single mutants as well as the WT constructs produced comparable levels of Env in the cells and on the cell surface upon transfection (Fig. 3D). Although the double mutant produced a higher level of Env on the cell surface (Fig. 3D, bottom panels), this might reflect the fact that this mutant also produced a larger amount of Env in the cells than did the WT construct in this particular transfection study (Fig. 3D, top panels).
Cys-to-Ser mutant Env protein-mediated viral entry into CD4+ host cells. To determine whether these mutant Env proteins expressed on the cell surface are capable of mediating viral entry, an env trans-complementation assay which assesses single-cycle viral infectivity was performed. The double mutant, as well as the 764S and 837S mutants, effectively mediated one-cycle viral infectivity in HeLa-T4 cells (Fig. 3E and F).
Lipid raft association of the Cys-to-Ser mutants. To determine whether these Cys-to-Ser mutant Env proteins are localized in lipid rafts, sucrose gradient ultracentrifugation, which was used to separate raft-associated proteins from soluble cytosolic proteins (41), was employed. HeLa cells separately expressing CD4 and VSV G proteins were extracted with 1% Triton X-100 on ice, and extracted cell lysates were analyzed by sucrose gradient ultracentrifugation. VSV G and CD4 were predominantly distributed in the soluble bottom fractions and lipid raft fractions 7 and 8, respectively (Fig. 4A). This result was consistent with the membrane localization properties of these two membrane proteins (8, 45). When HeLa cells transfected with the WT pHXB2RU3 provirus were analyzed, the uncleaved gp160 precursor and gp41 were localized in the lipid raft fractions 7 and 8 (Fig. 4B). p17 was also localized in lipid rafts, whereas CA p24 was localized predominantly in the cytosolic fractions (Fig. 4B). This result was consistent with previous observations that the HIV-1 Env and p17 matrix proteins are located on lipid raft membranes (41, 50). When these HXB2-based Cys-to-Ser mutants were analyzed in parallel, all of the mutant Env proteins were localized in the lipid rafts (Fig. 4C). Next, 293T cells were cotransfected with each of the WT or mutant pSVE7puro plasmids and pIIIextat, and cells were extracted with cold Triton X-100 and analyzed by sucrose gradient ultracentrifugation. All three mutant Env proteins were localized in the lipid rafts (Fig. 4D). To quantify the expression of WT and mutant Env proteins in the soluble and raft fractions, the method previously used to quantify lipid raft-associated Nef, CD4, CXCR4, and CCR5 (36, 47, 67, 70) was employed. Soluble fractions 1 to 3 and raft fractions 7 and 8 were separately combined, and equal amounts of proteins from each pool were subjected to Western blotting using MAb Chessie 8. The percentages of mutant proteins distributed in the raft fractions versus the soluble fractions were comparable to those for the WT Env (Fig. 4E).
Characterization of HXB2-based Cys-to-Ala mutant viruses. To confirm that mutations at palmitoylated cysteine do not affect HIV-1 replication, Cys-764, Cys-837, or both residues were replaced by an Ala residue (Fig. 1B). These mutants were termed 764A, 837A, and 764A.837A, respectively. As shown for the Cys-to-Ser mutants, these three Ala-substituted mutants still productively replicated in CEM-SS (Fig. 5A) and PM1 (Fig. 5B) cells. In HeLa cells, mutations at either Cys-764, Cys-837, or both Cys residues did not apparently affect synthesis or precursor processing of Env and Gag (Fig. 5C, lanes 1 to 5), nor did they alter viral Gag budding or Env incorporation into the virus (Fig. 5C, lanes 6 to 10). Also, none of these mutations affected the synthesis or proteolytic cleavage of Gag and Env, Gag assembly/budding, or assembly of Env into the virus in 293T cells (Fig. 5D).
These mutant Env proteins were expressed on the cell surface at levels similar to those observed for the WT Env when cell surface biotinylation of transfected HeLa cells (Fig. 6A) or FACS analyses of transfected 293T cells (Fig. 6B) was performed. These Cys-to-Ala mutant Env proteins also mediated single-cycle viral entry into CEM-SS cells as effectively as did the WT Env (Fig. 6C). Sucrose gradient equilibrium centrifugation of cold Triton X-100-extracted lysates obtained from 293T cells transfected with each of the WT and mutant proviruses showed that these three mutant Env proteins as well as the WT Env were localized in lipid rafts (Fig. 6D). When equal amounts of proteins of the soluble and lipid raft fractions obtained from 293T cells cotransfected with each of the WT or mutant pSVE7puro plasmids along with pIIIextat were analyzed, the percentages of WT and mutant proteins localized in the lipid raft fractions versus the soluble fractions were comparable (Fig. 6E). These studies indicated that removal of the cysteine palmitoylation signals located in the cytoplasmic domain of the HXB2 Env did not alter the property of raft membrane localization of the Env.
Characterization of Cys-to-Ala mutants derived from an M-tropic ConB virus. To determine whether Env palmitoylation may function differentially in the life cycles of T- and M-tropic viruses, Cys-to-Ala mutations were introduced into an M-tropic ConB provirus (59), which contains the consensus V3 sequences of HIV-1 subtype B in the backbone of the molecular clone pHXB2RU3 and utilizes CCR5, but not CXCR4, as an entry coreceptor (65). The 764A.837A double mutant as well as the 764A and 837A mutants replicated with kinetics similar to that of the WT virus in PM1 cells (Fig. 7A). When expressed in HeLa cells, synthesis and precursor processing of the Env and Gag proteins of these M-tropic Cys-to-Ala mutants were normal compared to those observed with the WT ConB virus (Fig. 7B, lanes 1 to 5). These mutations did not affect virus assembly/budding into the culture medium or Env incorporation into the virus compared to the WT ConB virus (Fig. 7B, lanes 6 to 10). Furthermore, these mutations did not show an apparent effect on Env incorporation into the virus in 293T cells (data not shown). Similar levels of Env proteins were expressed on the surface of cells expressing either the WT or any of the three Ala substitution mutants when cell surface biotinylation of ConB provirus-transfected HeLa cells was performed (data not shown). Consistent with virus replication in PM1 cells, these Cys-to-Ala mutants also mediated entry of the Env-deficient reporter virus into PM1 cells as effectively as did the WT Env (data not shown). These Cys-to-Ala mutants and the WT ConB Env were all localized in lipid rafts (Fig. 7C).
Replication of mutant viruses in PBMCs. We then determined whether these Cys-to-Ala mutants could replicate in human primary CD4+ T cells. All single and double Cys-to-Ala mutants of the HXB2 and ConB viruses replicated as effectively as the WT virus in human PBMCs (Fig. 8A and B, respectively). These results indicated that no discernible replication potentials could be observed with these mutants even in human primary CD4+ T cells.
Effect of the Env/p24 ratio of the virus on its infectivity. To examine whether the Env/p24 ratios of the "real" viruses used in continuous cell culture assay may differ from those of the Env pseudotypes used in the one-cycle infectivity assay, 293T cells were transfected with WT pHXB2RU3 or with pHXBBglCAT and WT pSVE7puro, and cell and virion lysates were analyzed by Western blotting. The Env/p24 ratio of the WT Env pseudotype was much higher than that of the WT real virus (Fig. 9A). Since the one-cycle infectivity assay did not differentiate infectivities of WT and mutant Env pseudotypes, we then assayed the one-cycle-like infectivity of WT and mutant viruses produced from full-length proviral transfection. All of the Cys-to-Ala mutant viruses showed levels of infectivity similar to that of the WT virus when determined using H938 cells (Fig. 9B), a cat gene-carrying reporter cell line allowing measurement of viral infectivity by the ability of Tat to transactivate HIV-1 LTR-linked cat gene expression in this cell line.
Palmitoylation state of the Cys-to-Ala mutants. To confirm that the double Cys-to-Ala mutant is indeed deficient in palmitoylation, metabolic labeling with [35S]methionine and [3H]palmitic acid was performed on ConB viruses. A mutation at residue 837, but not at residue 764, significantly reduced incorporation of [3H]palmitate into gp160 and gp41 compared to the WT Env (Fig. 10). Likewise, palmitoylation of the double mutant was also greatly reduced (Fig. 10, lane 10). These results indicated that palmitoylation at residue 837, but not at residue 764, contributed the most to gp41 cytoplasmic tail palmitoylation under the conditions we used.
DISCUSSION
Rousso et al. previously characterized HIV-1 Env mutants in which Cys-764 and Cys-837 located in the cytoplasmic tail of the HXB-2D Env were individually or together replaced by Ser residues, and they concluded that palmitoylation of Env is critical for Env targeting to lipid rafts, Env incorporation into the virus, and viral infectivity (50). However, it was not clear why gp160, but not gp120, was used to assess Env incorporation. The uncleaved gp160 precursor is fusion defective, and gp160 incorporation into virions does not necessarily correlate with viral infectivity. In addition, release of gp160 into the culture medium may occur via its association with microvesicles (63), which are shed from cultured cells. Also, the cell fractionation method they used is not an ideal approach to assessing lipid raft localization, since Triton X-100 insolubility may result from association of target proteins with the cytoskeleton or other insoluble protein complexes. On the other hand, Bhattacharya et al. characterized the effects of replacing Cys-764 and Tyr-837 of the NL4-3 Env with various amino acid residues and concluded that although cytoplasmic cysteines are required for the association of Env with light detergent-resistant membranes, these Cys residues are not essential for Env incorporation or viral infectivity (5). In both of these studies, the single-round infectivity assay was employed to study the functions of mutant Env proteins. However, whether the 764S.837S double mutant replicates at all in CD4+ T cells was not elucidated. The reason for this concern is that the single-cycle infectivity assay measures virus-cell transmission of a defective virus pseudotyped with the Env, whereas replication of HIV-1 in CD4+ T cells also encounters cell-cell transmission. Because of the different results obtained by these two groups and because of the unanswered issues raised above, we reexamined the involvement of cytoplasmic palmitoylated cysteines of HIV-1 in the virus life cycle.
In contrast to the findings of the other two groups (5, 50), we found that in the context of the T-tropic HXB2 virus, substitutions of Ser and Ala for both Cys-764 and Cys-837 did not affect viral infectivity, as judged by continuous cultures and single-cycle infectivity assays in various host target cells, including HeLa-T4, CEM-SS, PM1, and human primary CD4+ T cells (Fig. 2, 3E, 3F, 5A, 5B, 6C and 8A). In addition, removal of the putative palmitoylation signals in the cytoplasmic tail of an M-tropic HIV-1 clone, ConB, also exerted no significant effect on viral replication (Fig. 7A and 8B and data not shown). Ser substitutions for either Cys-764 or Cys-837, or both, of the HXB2 strain Env were previously shown not to affect Env transport to the cell surface (68). Although the 764S.837S double mutant showed a higher level of surface expression than did the WT Env (Fig. 3D, bottom panels), this could simply have been due to a higher efficiency of transfection of this mutant in this particular experiment and not necessarily to higher surface expression of this mutant, since this mutant also produced a higher level of total Env in cells than did the WT Env (Fig. 3D, top panels). In support of this notion, surface biotinylation did not differentiate the WT and this double mutant (Fig. 3C), and the T- and M-tropic Cys-to-Ala double mutants showed no increase in surface expression compared to the WT and other single mutants (Fig. 6A and 6B and data not shown). Also, removal of these two cytoplasmic cysteines exhibited no significant effect on Env incorporation into the virus in CEM-SS (Fig. 3A), HeLa (Fig. 5C and 7B), or 293T (Fig. 3B and 5D and data not shown) cells.
On the other hand, abrogation of the palmitoylation signals in the cytoplasmic tail did not affect the raft association property of the T- and M-tropic Env proteins in HeLa (Fig. 4C) or 293T (Fig. 4D, 6D, and 7C) cells. In these lipid raft flotation analyses, only a fraction, ranging between 10% and 25%, of the total WT and mutant proteins was localized in the lipid rafts. When the expression of WT and mutant proteins in the soluble and raft fractions was quantified by normalization of protein amounts used in Western blotting, most of the WT as well as the Cys-to-Ser and Cys-to-Ala mutants was found to be located in the raft fractions, and the percentages of mutant proteins distributed in the raft fractions versus soluble fractions were comparable to those of the WT Env (Fig. 4E and 6E, respectively). These results indicate that the two cytoplasmic Cys residues are not critical for Env association with lipid rafts. Since the majority of proteins in the cells were not associated with lipid rafts, normalization for total proteins in the pooled raft and soluble fractions would result in much lower levels of Env being localized in the soluble fractions than in the raft fractions. Therefore, although this assay provides a means to compare the raft association properties of the WT and mutants, this approach provides no information on the absolute levels of gp41 present in the lipid rafts.
In agreement with previous studies (19, 35), we also noted that gp160 can be detected in viral particles. At a late stage of Env synthesis, the gp160 precursor is proteolytically cleaved by furin-like cellular proteases or other subtilisin-like proteases, most probably in the trans-Golgi network, to yield noncovalently associated gp120-gp41 heterodimers. Therefore, gp160 precursor cleavage is dependent on the cell types examined. Also, production of a native gp120-gp41 complex has been hampered by the limited efficiency of Env cleavage (18, 38, 39). These unprocessed gp160 molecules, likely forming homotrimers or heterotrimers with processed gp120-gp41, are also transported to the cell surface (40, 51), where they interact with Gag proteins and are incorporated into the virions.
Rousso et al. (50) employed an env-deficient, luciferase-containing NL4-3R–E–Luc provirus, which carries the functional vif and vpu genes but not the vpr or nef genes, along with the pCDNA3-based env gene derived from the HXB-2D strain for Env incorporation and env trans-complementation assays. Bhattacharya et al. (5) also used an NL4-3-derived env-deficient construct that encodes all viral proteins, except Env, and an HIV-1 LTR-driven NL4-3 env plasmid in their studies. Among these four accessory proteins, both Nef and Vpu encode activities that contribute to downregulation of the viral receptor CD4 from the cell surface (for a review, see reference 6). Nef increases viral infectivity (14, 37, 54), and downregulation of CD4 contributes to enhanced viral infectivity (1, 24, 49). Nef-enhanced viral infectivity occurs, at least in part, by an increase in Env incorporation into the virus, likely via Nef-enhanced steady-state levels of cell membrane-associated Env proteins (52). One of the well-characterized functions of Vpu is its ability to induce degradation of CD4 molecules trapped in intracellular complexes with Env, which allows the release of Env for transport to the cell surface (66). Vpu also mediates the efficient release of viral particles from virus-infected cells (21, 57). Vpu positively enhances viral infectivity by downmodulating the cell surface CD4 receptor (27), presumably removing the activity of CD4 that inhibits the virion release-promoting activity of Vpu (7, 58). Although the version of the infectious HXB2 molecular clone used in our study carries functional vif, vpr, vpu, and nef genes (59), these accessory genes might not be the primary factor responsible for the differential effects of the cytoplasmic tail palmitoylation observed. This was reasoned out by the observation that the pHXBBglCAT provirus used in our single-cycle infectivity assay carries none of the functional vpr, vpu, and nef genes.
It was reported that the entire cytoplasmic tail of the Env protein can be deleted without imposing a significant effect on Env incorporation into the virus in 293T cells (40). In the study by Bhattacharya et al. (5), the effect of mutations at positions 764 and 837 in the cytoplasmic tail on Env incorporation depended on the residue to be mutated and on what type of amino acid substitution was used. It is therefore likely that in 293T cells, the cytoplasmic tail per se does not positively contribute to Env incorporation; however, mutations in the cytoplasmic cysteines may induce alterations in Env folding and/or intracellular trafficking, which ultimately affect the effectiveness of Env incorporation into the virus.
Although the Env/p24 ratio of the Env-pseudotyped virus used in our one-cycle infectivity assay was greater than that of the "real" virus prepared from full-length, proviral transfection (Fig. 9A), the possibility that overexpression of mutant Env in pseudotypes may compensate for the impaired infectivity, if any, of the double mutant is unlikely. First, Bhattacharya et al. (5) also used LTR-controlled env expression plasmids for pseudotype preparation. Nevertheless, the viral entry of their Ser and Ala substitution double mutants was severely reduced, which is accounted for by insufficient Env incorporation of these two mutants. Second, if the possibility holds, it would be expected that the infectivity of the "real" WT and mutant viruses, which had lower Env/p24 ratios than the Env pseudotypes, would have been differentiated in the one-cycle infectivity assay. However, the WT and all mutant viruses showed similar levels of one-cycle-like infectivity in the LTR-lined cat gene-carrying H938 reporter cell line (Fig. 9B). Therefore, the WT-like infectivity of the mutant Env pseudotypes we examined was clearly due to the WT-like Env incorporation phenotype of the mutants.
A contiguous long sequence located in the cytoplasmic tail, beginning with the first palmitoylation site at Cys-764 and ending at the C terminus, is believed to be embedded in membranes (20). We previously showed that the C-terminal two-thirds of the cytoplasmic domain confers multimerization and plays a role in membrane association (13, 26). It is likely that minor amino acid differences between the env clones used in the studies by Rousso et al. (50), Bhattacharya et al. (5), and us may be responsible for the differential effects of the deacylated Env proteins on lipid raft association, Env incorporation, and viral infectivity observed by the three groups. The cytoplasmic tail of Env has specific interactions with cellular membranes, cellular proteins, and the matrix (MA) protein. It is also likely that subtle differences in the modes of interactions between Env and membranes, Env and cellular proteins, and Env and Gag, aroused by amino acid differences in the env and gag gene products, may contribute to the differential Env phenotypes of the deacylated Env mutants observed among different laboratories.
In the present study, we were unable to ascribe any apparent effects on replication of T- and M-tropic viruses to deacylation of the HIV-1 Env. Consistent with the notion that many isolates representative of different HIV-1 clades and the infectious molecular clones such as BORI, JRCSF, and DH123 do not contain the putative Cys palmitoylation sites in the cytoplasmic domains of their TM proteins, our results strongly support the notion that palmitoylation of the gp41 cytoplasmic tail does not play a critical role in the HIV-1 life cycle.
ACKNOWLEDGMENTS
The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: SIM2 hybridoma from James Hildreth, HeLa-T4 from Richard Axel, and H938 from Barbara K. Felber and Geroge N. Pavlakis. We are grateful to Jane C. Burns (School of Medicine, University of California, San Diego) for providing pHCMV-VSV G and to Ton-Hou Lee (Harvard School of Public Health, Boston, MA) for providing the pHXB2RU3 and pConB proviruses. We are also grateful to Sheau-Fen Lee and In-Ru Chen for technical help.
This work was supported by grants (NSC 92-2320-B-001-047 and NSC93-2320-B-001-016) from the National Science Council and Academia Sinica, Taipei, Taiwan, Republic of China.
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ABSTRACT
Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.
INTRODUCTION
Lipid rafts are highly specialized membrane microdomains present in both the plasma and endosomal membranes of eukaryotic cells (23, 55). These dynamic microdomains, which are organized in the lateral dimension of the plasma membrane, are characterized by detergent insolubility, light density, and enrichment of cholesterol and glycosphingolipids (for reviews, see references 3 and 55). Lipid rafts play important roles in various biological processes such as cell surface signal transduction, T-cell activation, and intracellular trafficking (17, 55, 56, 60). Many membrane-enveloped viruses also assemble and selectively bud from lipid rafts on the surfaces of infected cells; the envelope (Env) glycoproteins of some of these viruses are known to be associated with lipid rafts (2, 34, 62, 69). Acylation of viral Env proteins, particularly palmitoylation, may be important for the targeting of these Env proteins to lipid rafts (for reviews, see references 48 and 53). Lipid rafts also serve as important entry and assembly/budding sites for human immunodeficiency virus type 1 (HIV-1) (30, 33, 41, 46, 64). Removal of cellular cholesterol with ?-methyl cyclodextrin was shown to reduce HIV-1 budding and infectivity and Env-mediated syncytium formation (29, 33, 44, 61). Also, depletion of cholesterol in the viral envelope may impair viral internalization (16) and permeabilize the virion envelope (15).
In retroviruses, palmitoylation may play some roles in viral replication. For instance, palmitoylation-deficient Env mutants of murine leukemia virus (MLV) are mostly soluble when extracted using ice-cold Triton X-100, and they stay at the bottom of the sucrose gradient (28). In addition, treatment of cells with ?-methyl cyclodextrin abolishes the ability of the MLV Env to associate with lipid rafts (28). Those studies indicated that palmitoylation of the MLV Env is critical for lipid raft association. Nevertheless, mutant Env proteins of MLV are still able to mediate the syncytium-forming ability, suggesting that palmitoylation or raft association is not required for MLV-mediated fusion activity (28). In contrast, palmitoylation of the transmembrane (TM) protein of Rous sarcoma virus was shown to be required for protein stability and virus infectivity (42). Nevertheless, acylated Env glycoproteins of this virus are not sequestered into lipid raft domains (43). The TM proteins of HIV-1 and simian immunodeficiency virus are also posttranslationally modified by the addition of palmitate to the Cys residues located in the cytoplasmic domains of these viruses through a thioester bond (68). Palmitoylation of the T-cell (T)-tropic HXB2 strain of HIV-1 occurs on two Cys residues, Cys-764 and Cys-837, located in the cytoplasmic tail of gp41 (68). Although HIV-1 Env is associated with lipid rafts (41), whether palmitoylation of Env plays a crucial role in Env localization in lipid rafts and viral infectivity has been a longstanding question.
Rousso et al. found that substitutions of the two cytoplasmic Cys residues in the Env of the HXB-2D clone with Ser residues significantly decreased one-cycle viral infectivity by reducing incorporation of the C764S C837S double-mutant Env into the virus (50). Also, most of the double-mutant Envs could be extracted into the soluble fraction with Triton X-100 at 4°C, whereas both the C764S and C837S mutants, just like the wild-type (WT) Env, were still distributed in the membrane pellets (50). Their results indicated that palmitoylation of the HIV-1 Env is critical for viral infectivity through the targeting of Env to lipid rafts. Interestingly, the two palmitoylated Cys residues located in the HXB2 strain are not highly conserved among HIV-1 clade B and different HIV-1 clades (Fig. 1A) (22). The NL4-3 virus contains only one Cys residue at position 764, whereas the 837 position in this virus is a Tyr residue (Fig. 1A). Other isolates in the B clade, such as MN and JRCSF, do not contain Cys residues at these two positions (Fig. 1A). Moreover, some representative isolates of other clades neither encode these palmitoylated Cys residues nor contain any other Cys residues in the cytoplasmic domains of their TM proteins (Fig. 1A) (22). This suggests the interesting proposal that palmitoylation of the HIV-1 Env is not required for viral replication.
Bhattacharya et al. recently reported that replacement of both Cys-764 and Tyr-837 of the NL4-3 env gene by a Ser or Ala residue still mediated about 10% and 40%, respectively, of the single-cycle infectivity mediated by the WT Env (5). The degree of infectivity of these two mutants paralleled the amount of mutant Env incorporated into the virus. Interestingly, these two mutants were excluded from lipid rafts. These two mutant proteins also mediated cell-cell membrane fusion with an efficiency comparable to that of the WT Env. Bhattacharya et al. also found that substitutions of both Cys-764 and Tyr-837 residues with bulky hydrophobic side chains retained the raft association property of the Env, and these mutant proteins mediated over 80% of the one-cycle infectivity of the WT Env and were effectively incorporated into the virus, implying that these mutations may compensate for the lack of a palmitate group in the cytoplasmic tail of the NL4-3 Env. They therefore concluded that the cytoplasmic cysteines are required for Env targeting to lipid rafts but are not essential for Env incorporation into the virus or for viral infectivity.
Because of the disparate conclusions as to the effects of cytoplasmic tail palmitoylation on viral infection drawn by these two groups, we reexamined the role of cytoplasmic cysteines of the HIV-1 Env in virus replication. To truly monitor replication kinetics of mutant viruses carrying a deacylated Env, we examined the effects of deacylation of both Cys residues of the T-tropic HXB2 strain Env by Ser and Ala substitutions on viral replication, using a continuous cell culture system as well as a single-cycle viral replication assay. The properties of cell surface expression, lipid raft localization, and incorporation into the virus of mutant Env proteins were also studied. We further examined whether the effects of deacylation on viral replication are related to the T or macrophage (M) tropism of the viruses examined, which, respectively, utilize chemokine receptors CXCR4 and CCR5 as the entry coreceptors (and thus are referred to as the X4 and R5 viruses, respectively). In contrast to the earlier results (5, 50), we failed to detect any altered phenotypes of the deacylated Env mutants compared to those of the WT virus in any of the analyses performed. Our results strongly indicate that palmitoylation of Env does not play a critical role in the HIV-1 life cycle.
MATERIALS AND METHODS
Cells, hybridoma, and antibodies. 293T, HeLa, and HeLa-T4 cells were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (FBS). CEM-SS, PM1, and H938 cells were cultured in RPMI 1640 supplemented with 10% FBS. Hybridomas 902, Chessie 8, and 183 (clone H12-5C), which produce mouse monoclonal antibodies (MAbs) reactive with HIV-1 gp120, gp41, and p24, respectively, and sheep anti-gp120 were as previously described (9). The SIM2 hybridoma secreted a MAb that specifically recognizes human CD4. A MAb directed against the glycoprotein G of vesicular stomatitis virus (VSV) was purchased from Sigma (St. Louis, MO).
Construction of plasmids. For construction of Cys-to-Ser and Cys-to-Ala mutants of the Env protein of the T-tropic HXB2RU3 provirus (Fig. 1B), oligonucleotide-directed, site-specific mutagenesis using PCR overlap extension was performed as previously described (25). Oligonucleotides 8423f and 8933r were used as the 5' and 3' external primers, respectively. The paired internal primers used in the PCR encoding each Cys mutant were as follows: 764S, 5'-CTGCGGAGCCTGAGCCTCTTCAGCTACCACCGC-3' (sense) and 5'-GTAGCTGAAGAGGCTCAGGCTCCGCAGATCGTC-3' (antisense); 837S, 5'-GTACAAGGAGCTAGTAGAGCTATTCGCCACATA-3' (sense) and 5'-GCGAATAGCTCTACTAGCTCCTTGTACTACTTC-3' (antisense); 764A 5'-CTGCGGAGCCTGGCCCTCTTCAGCTACCACCGC-3' (sense) and 5-GTAGCTGAAGAGGGCCAGGCTCCGCAGATCGTC-3' (antisense); and 837A, 5'-GTACAAGGAGCTGCTAGAGCTATTCGCCACATA-3' (sense) and 5'-GCGAATAGCTCTAGCAGCTCCTTGTACTACTTC-3' (antisense). To construct the 764S 837S and 764A 837A double mutants, the 764S and 764A PCR products were used as templates, and the paired sense and antisense oligonucleotides for generating the 837S and 837A mutants were used as internal primers, respectively. All PCR amplifications were performed using Vent DNA polymerase (New England BioLabs, Beverly, MA) according to a previously described amplification program (26). The amplified mutated BamHI-XhoI fragments were cloned into the pHXB2RU3 provirus to generate various mutant proviruses. To construct HXB2-based mutant env expression plasmids, the BamHI-XhoI fragments isolated from Cys-to-Ser and Cys-to-Ala mutant pHXB2RU3 proviruses were cloned into corresponding sites in pSVE7puro, an HIV-1 long terminal repeat (LTR)-directed env expression plasmid. To generate ConB provirus-based Cys-to-Ala mutants, BamHI-XhoI fragments isolated from pHXB2RU3 mutant proviruses were used to replace the homologous sequences in the ConB provirus. The KpnI-XhoI fragments isolated from mutant ConB proviruses were cloned in the corresponding sites in pSVE7puro to generate ConB-based mutant env plasmids. All mutant clones were confirmed by DNA autosequencing.
Plasmid DNA transfection. 293T cells grown in 100-mm petri dishes were transfected with 10 μg each of the WT or mutant proviruses by a standard calcium phosphate coprecipitation method. To prepare VSV glycoprotein G-transcomplemented viral stocks, 293T cells were cotransfected with 7.5 μg of the human cytomegalovirus virus (HCMV) promoter-directed VSV G protein expression plasmid, pHCMV-VSV G, together with 7.5 μg each of the WT or mutant proviruses. To prepare HIV-1 Env pseudotypes, 293T cells were cotransfected with 7.5 μg each of pHXBBglCAT with each of the WT or mutant pSVE7puro plasmids. Cotransfection with pHXBBglCAT and an env-defective pSVE7puro(KS) was used as the negative control. For protein expression studies, HeLa cells grown in 100-mm petri dishes were transfected with 8 μg each of WT or mutant proviruses by the Lipofectamine transfection method (Life Technologies, Rockville, MD). For the [35S]methionine and [3H]palmitate labeling studies, HeLa cells grown in 6-cm petri dishes were transfected with 4 μg of WT or mutant proviruses or with 3.5 μg of WT or mutant pSVE7puro plasmids along with 1.5 μg of the Tat expression plasmid pIIIextat.
Viral infection, RT, and CAT assays. Two days after 293T transfection, cell-free culture supernatants were assayed for reverse transcriptase (RT) activity as previously described (11). Cell-free viruses containing 2 x 104 cpm of RT activity were used to challenge 106 CEM-SS or PM1 cells. Postinfection RT activity of viruses obtained from supernatants of infected cultures was then monitored. For VSV G-transcomplemented HIV-1 infection, 106 CEM-SS cells were challenged with VSV G-transcomplemented viruses containing 2 x 106 cpm of RT activity. For the env trans-complementation assay, Env-pseudotyped HXBBglCAT reporter viruses containing 2 x 105 cpm of RT activity were used to challenge subconfluent HeLa-T4 cells grown in 60-mm dishes, or 106 CEM-SS or PM1 cells, and chloramphenicol acetyltransferase (CAT) activity was assessed as previously described (10).
Isolation of human PBMCs and viral infection. Peripheral blood mononuclear cells (PBMCs) were isolated from normal donors by using a standard Ficoll/Hypaque separation method. Cells were then stimulated with interleukin-2 and phytohemagglutinin A. After stimulation, 106 cells were infected with WT or mutant viruses containing 106 cpm of RT activity, and postinfection virus production was then measured.
Western blot analysis. Two days after proviral transfection or 3 days after VSV G-transcomplemented HIV-1 infection, lysates of cells and virions were prepared as previously described (12). Equal volumes of cell and virion lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblotting analysis using MAbs 902, Chessie 8, and 183 to detect gp120, gp41, and capsid protein p24, respectively. Sheep anti-gp120 was used to detect gp160 and gp120 of the ConB virus. A MAb against VSV G and a MAb secreted from hybridoma SIM2 were used to react with VSV G and CD4, respectively. Horseradish peroxidase-conjugated anti-mouse or anti-sheep immunoglobulin G was used as the second antibody. The immune complexes were visualized with a chemiluminescence detection kit.
Sucrose gradient equilibrium ultracentrifugation and quantification of lipid raft-associated Env proteins. Flotation centrifugation to assess Env localization to lipid rafts was performed according to a previously described procedure (41) with modifications. Phosphate-buffered saline (PBS)-washed transfected 293T cells that expressed Env were extracted on ice with 0.5 ml of 1% Triton X-100 prepared in NTE buffer (25 mM Tris-HCl containing 0.15 M NaCl and 5 mM EDTA; pH 7.5) supplemented with phenylmethylsulfonyl fluoride and the protease inhibitor cocktail for 30 min. Cell lysates were centrifuged at 8,000 x g for 10 min at 4°C. The supernatants were adjusted to a final concentration of 42.5% sucrose prepared in NTE by adding an equal volume of 85% sucrose prepared in NTE. The mixed solutions were then loaded into SW41 tubes, and the extracts were overlaid with 6 ml of 30% sucrose prepared in NTE followed by 5 ml of 5% sucrose prepared in NTE. Tubes were centrifuged at 100,000 x g for 18 h at 4°C. The gradients were fractionated from the bottom of the gradient, and 1 ml was collected per fraction. Aliquots of samples from each fraction were precipitated with trichloroacetic acid and then subjected to SDS-PAGE followed by Western blotting. To quantitate lipid raft-associated Env proteins, soluble fractions 1 to 3 and lipid raft fractions 7 and 8 were individually pooled, and concentrations of total proteins were determined using a Bio-Rad (Hercules, CA) protein assay kit. Aliquots containing equal amounts of proteins from soluble and raft fractions were precipitated with trichloroacetic acid and then subjected to Western blotting using MAb Chessie 8. The ECL blot was scanned using a Microtek (Carson, CA) ScanMarker 8700 and quantitated using the MetaMorph software (Universal Imaging, Downing Town, PA).
Metabolic labeling, cell surface biotinylation, palmitoylation, and immunoprecipitation. Transfected HeLa cells grown in 6-cm petri dishes were metabolically labeled with [35S]methionine overnight, and labeled cells were surface biotinylated with 0.25 mg/ml of membrane-impermeable sulfo-N-hydroxysuccinimide-biotin. The levels of total and cell surface Env expressions were then assessed as previously described (9, 25). For palmitoylation studies, a set of transfected dishes was labeled with [35S]methionine for 5 h. Another set of transfected cells was washed twice with PBS and then incubated with 1 ml Dulbecco's modified Eagle's medium containing 5% dialyzed FBS and 5 mM sodium pyruvate at 37°C for 30 min. [3H]palmitic acid (30 to 60 Ci/mmol; New England Nuclear, Boston, MA) was dried to completeness to remove the ethanol and then dissolved in dimethyl sulfoxide before use. One millicurie of [3H]palmitate dissolved in 2 μl of dimethyl sulfoxide was diluted with PBS to 100 μl and added to each dish, and cell cultures were labeled for 5 h. Cell lysates were immunoprecipitated with anti-HIV preadsorbed onto protein A-Sepharose, and the immune complexes were separated by SDS-PAGE under nonreducing conditions.
Flow cytometry analyses. To assess Env expression on the cell surfaces, 293T cells were transfected with WT or mutant pSVE7puro plasmids together with pIIIextat. Two days after transfection, cells were divided into two portions. One portion of cells was used to determine total Env expression, and another portion of cells was processed for Env cell surface expression by fluorescence-activated cell sorter (FACS) analysis according to a procedure described previously (9).
RESULTS
Infectivity of gp41 cytoplasmic tail Cys-to-Ser mutants of a T-tropic HXB2 virus. To understand the role of palmitoylation of the Env cytoplasmic domain in viral replication, the two Cys residues located at residues 764 and 837 in the Env of the HIV-1 HXB2 strain were replaced individually or together by a Ser residue by using oligonucleotide-directed, site-specific mutagenesis with PCR overlap extension based on the pHXB2RU3 provirus (59). This molecular clone also carries functional vpr, vpu, and nef genes, which are not present in the parental HXB2 construct. These resultant mutants were referred to as the 764S, 837S, and 764S.837S mutants, respectively (Fig. 1B). Replication of these mutant viruses along with the WT virus was assessed using human CD4+ CEM-SS T cells. The 764S.837S double mutant virus, just like the WT and 764S and 837S mutant viruses, was still capable of productively replicating in CEM-SS cells (Fig. 2A). When the replication of these mutant viruses was examined in another CD4+ T-cell line, PM1, which is derived from Hut78 and expresses both CXCR4 and CCR5 coreceptors (32), all three of these Cys-to-Ser mutants productively replicated at a level similar to that of the WT virus (Fig. 2B).
Expression of viral proteins encoded by HXB2-based Cys-to-Ser mutants. To examine viral protein expression of these three Cys-to-Ser mutants in CD4+ T cells, which are natural target cells for HIV-1 infection, a high-level transient HIV-1 expression system based on pseudotyping with the VSV G protein (4, 31) was employed. This method has been shown to effectively express viral proteins in suspended T cells (9, 25, 40), which are usually transfected less efficiently than is required for biochemical analyses when other commonly used transfection methods are used. Equal amounts of cell-free VSV G-transcomplemented viruses obtained from each cotransfection and after normalization by RT activity were used to challenge CEM-SS cells. Equal portions of cell and virion lysates were analyzed by Western blotting using a cocktail containing the Env- and CA-specific MAbs. The 764S.837S double mutant produced levels of intracellular Gag and Env proteins comparable to those produced by the WT and the other two Ser mutants (Fig. 3A, lanes 1 to 4). Also, Gag assembly/budding of and Env incorporation into this double mutant virus were normal compared to those observed in the WT and the other two mutant viruses (Fig. 3A, lanes 5 to 8). When 293T cells were directly transfected with each of the WT and Cys-to-Ser mutant proviruses, these mutations did not affect Env synthesis, precursor processing, or Env incorporation into the viruses (Fig. 3B).
To determine whether these mutant Env proteins were expressed on the cell surface, HeLa cells transfected with the WT or each of the mutant proviruses were labeled with [35S]methionine overnight, and cell surface biotinylation using membrane-impermeable sulfo-N-hydroxysuccinimide-biotin was performed. This method has been widely used to examine cell surface expression of HIV Env proteins (9, 40, 51). All these mutant proviruses showed levels of the intracellular gp160 precursor and gp120, as well as cell surface gp120, that were comparable to those produced by the WT provirus upon transfection (Fig. 3C). As previously reported (9), no gp120 from WT transfection could be detected on the cell surface when cells were treated with brefeldin A, which prevents transport of glycoproteins out of the ER, prior to biotinylation (data not shown). Also, the cell surface expression of neither HIV-1 Gag Pr55 nor p24 from WT proviral transfection could be detected under this surface biotinylation condition (data not shown).
To confirm that these mutant Env proteins are as efficiently expressed on cell surface as the WT Env, these three mutations were introduced to the HIV-1 LTR promoter-driven env expression pSVE7puro plasmid. FACS analysis of 293T cells transfected with each of the WT and mutant pSVE7puro plasmids along with pIIIextat was performed. The two single mutants as well as the WT constructs produced comparable levels of Env in the cells and on the cell surface upon transfection (Fig. 3D). Although the double mutant produced a higher level of Env on the cell surface (Fig. 3D, bottom panels), this might reflect the fact that this mutant also produced a larger amount of Env in the cells than did the WT construct in this particular transfection study (Fig. 3D, top panels).
Cys-to-Ser mutant Env protein-mediated viral entry into CD4+ host cells. To determine whether these mutant Env proteins expressed on the cell surface are capable of mediating viral entry, an env trans-complementation assay which assesses single-cycle viral infectivity was performed. The double mutant, as well as the 764S and 837S mutants, effectively mediated one-cycle viral infectivity in HeLa-T4 cells (Fig. 3E and F).
Lipid raft association of the Cys-to-Ser mutants. To determine whether these Cys-to-Ser mutant Env proteins are localized in lipid rafts, sucrose gradient ultracentrifugation, which was used to separate raft-associated proteins from soluble cytosolic proteins (41), was employed. HeLa cells separately expressing CD4 and VSV G proteins were extracted with 1% Triton X-100 on ice, and extracted cell lysates were analyzed by sucrose gradient ultracentrifugation. VSV G and CD4 were predominantly distributed in the soluble bottom fractions and lipid raft fractions 7 and 8, respectively (Fig. 4A). This result was consistent with the membrane localization properties of these two membrane proteins (8, 45). When HeLa cells transfected with the WT pHXB2RU3 provirus were analyzed, the uncleaved gp160 precursor and gp41 were localized in the lipid raft fractions 7 and 8 (Fig. 4B). p17 was also localized in lipid rafts, whereas CA p24 was localized predominantly in the cytosolic fractions (Fig. 4B). This result was consistent with previous observations that the HIV-1 Env and p17 matrix proteins are located on lipid raft membranes (41, 50). When these HXB2-based Cys-to-Ser mutants were analyzed in parallel, all of the mutant Env proteins were localized in the lipid rafts (Fig. 4C). Next, 293T cells were cotransfected with each of the WT or mutant pSVE7puro plasmids and pIIIextat, and cells were extracted with cold Triton X-100 and analyzed by sucrose gradient ultracentrifugation. All three mutant Env proteins were localized in the lipid rafts (Fig. 4D). To quantify the expression of WT and mutant Env proteins in the soluble and raft fractions, the method previously used to quantify lipid raft-associated Nef, CD4, CXCR4, and CCR5 (36, 47, 67, 70) was employed. Soluble fractions 1 to 3 and raft fractions 7 and 8 were separately combined, and equal amounts of proteins from each pool were subjected to Western blotting using MAb Chessie 8. The percentages of mutant proteins distributed in the raft fractions versus the soluble fractions were comparable to those for the WT Env (Fig. 4E).
Characterization of HXB2-based Cys-to-Ala mutant viruses. To confirm that mutations at palmitoylated cysteine do not affect HIV-1 replication, Cys-764, Cys-837, or both residues were replaced by an Ala residue (Fig. 1B). These mutants were termed 764A, 837A, and 764A.837A, respectively. As shown for the Cys-to-Ser mutants, these three Ala-substituted mutants still productively replicated in CEM-SS (Fig. 5A) and PM1 (Fig. 5B) cells. In HeLa cells, mutations at either Cys-764, Cys-837, or both Cys residues did not apparently affect synthesis or precursor processing of Env and Gag (Fig. 5C, lanes 1 to 5), nor did they alter viral Gag budding or Env incorporation into the virus (Fig. 5C, lanes 6 to 10). Also, none of these mutations affected the synthesis or proteolytic cleavage of Gag and Env, Gag assembly/budding, or assembly of Env into the virus in 293T cells (Fig. 5D).
These mutant Env proteins were expressed on the cell surface at levels similar to those observed for the WT Env when cell surface biotinylation of transfected HeLa cells (Fig. 6A) or FACS analyses of transfected 293T cells (Fig. 6B) was performed. These Cys-to-Ala mutant Env proteins also mediated single-cycle viral entry into CEM-SS cells as effectively as did the WT Env (Fig. 6C). Sucrose gradient equilibrium centrifugation of cold Triton X-100-extracted lysates obtained from 293T cells transfected with each of the WT and mutant proviruses showed that these three mutant Env proteins as well as the WT Env were localized in lipid rafts (Fig. 6D). When equal amounts of proteins of the soluble and lipid raft fractions obtained from 293T cells cotransfected with each of the WT or mutant pSVE7puro plasmids along with pIIIextat were analyzed, the percentages of WT and mutant proteins localized in the lipid raft fractions versus the soluble fractions were comparable (Fig. 6E). These studies indicated that removal of the cysteine palmitoylation signals located in the cytoplasmic domain of the HXB2 Env did not alter the property of raft membrane localization of the Env.
Characterization of Cys-to-Ala mutants derived from an M-tropic ConB virus. To determine whether Env palmitoylation may function differentially in the life cycles of T- and M-tropic viruses, Cys-to-Ala mutations were introduced into an M-tropic ConB provirus (59), which contains the consensus V3 sequences of HIV-1 subtype B in the backbone of the molecular clone pHXB2RU3 and utilizes CCR5, but not CXCR4, as an entry coreceptor (65). The 764A.837A double mutant as well as the 764A and 837A mutants replicated with kinetics similar to that of the WT virus in PM1 cells (Fig. 7A). When expressed in HeLa cells, synthesis and precursor processing of the Env and Gag proteins of these M-tropic Cys-to-Ala mutants were normal compared to those observed with the WT ConB virus (Fig. 7B, lanes 1 to 5). These mutations did not affect virus assembly/budding into the culture medium or Env incorporation into the virus compared to the WT ConB virus (Fig. 7B, lanes 6 to 10). Furthermore, these mutations did not show an apparent effect on Env incorporation into the virus in 293T cells (data not shown). Similar levels of Env proteins were expressed on the surface of cells expressing either the WT or any of the three Ala substitution mutants when cell surface biotinylation of ConB provirus-transfected HeLa cells was performed (data not shown). Consistent with virus replication in PM1 cells, these Cys-to-Ala mutants also mediated entry of the Env-deficient reporter virus into PM1 cells as effectively as did the WT Env (data not shown). These Cys-to-Ala mutants and the WT ConB Env were all localized in lipid rafts (Fig. 7C).
Replication of mutant viruses in PBMCs. We then determined whether these Cys-to-Ala mutants could replicate in human primary CD4+ T cells. All single and double Cys-to-Ala mutants of the HXB2 and ConB viruses replicated as effectively as the WT virus in human PBMCs (Fig. 8A and B, respectively). These results indicated that no discernible replication potentials could be observed with these mutants even in human primary CD4+ T cells.
Effect of the Env/p24 ratio of the virus on its infectivity. To examine whether the Env/p24 ratios of the "real" viruses used in continuous cell culture assay may differ from those of the Env pseudotypes used in the one-cycle infectivity assay, 293T cells were transfected with WT pHXB2RU3 or with pHXBBglCAT and WT pSVE7puro, and cell and virion lysates were analyzed by Western blotting. The Env/p24 ratio of the WT Env pseudotype was much higher than that of the WT real virus (Fig. 9A). Since the one-cycle infectivity assay did not differentiate infectivities of WT and mutant Env pseudotypes, we then assayed the one-cycle-like infectivity of WT and mutant viruses produced from full-length proviral transfection. All of the Cys-to-Ala mutant viruses showed levels of infectivity similar to that of the WT virus when determined using H938 cells (Fig. 9B), a cat gene-carrying reporter cell line allowing measurement of viral infectivity by the ability of Tat to transactivate HIV-1 LTR-linked cat gene expression in this cell line.
Palmitoylation state of the Cys-to-Ala mutants. To confirm that the double Cys-to-Ala mutant is indeed deficient in palmitoylation, metabolic labeling with [35S]methionine and [3H]palmitic acid was performed on ConB viruses. A mutation at residue 837, but not at residue 764, significantly reduced incorporation of [3H]palmitate into gp160 and gp41 compared to the WT Env (Fig. 10). Likewise, palmitoylation of the double mutant was also greatly reduced (Fig. 10, lane 10). These results indicated that palmitoylation at residue 837, but not at residue 764, contributed the most to gp41 cytoplasmic tail palmitoylation under the conditions we used.
DISCUSSION
Rousso et al. previously characterized HIV-1 Env mutants in which Cys-764 and Cys-837 located in the cytoplasmic tail of the HXB-2D Env were individually or together replaced by Ser residues, and they concluded that palmitoylation of Env is critical for Env targeting to lipid rafts, Env incorporation into the virus, and viral infectivity (50). However, it was not clear why gp160, but not gp120, was used to assess Env incorporation. The uncleaved gp160 precursor is fusion defective, and gp160 incorporation into virions does not necessarily correlate with viral infectivity. In addition, release of gp160 into the culture medium may occur via its association with microvesicles (63), which are shed from cultured cells. Also, the cell fractionation method they used is not an ideal approach to assessing lipid raft localization, since Triton X-100 insolubility may result from association of target proteins with the cytoskeleton or other insoluble protein complexes. On the other hand, Bhattacharya et al. characterized the effects of replacing Cys-764 and Tyr-837 of the NL4-3 Env with various amino acid residues and concluded that although cytoplasmic cysteines are required for the association of Env with light detergent-resistant membranes, these Cys residues are not essential for Env incorporation or viral infectivity (5). In both of these studies, the single-round infectivity assay was employed to study the functions of mutant Env proteins. However, whether the 764S.837S double mutant replicates at all in CD4+ T cells was not elucidated. The reason for this concern is that the single-cycle infectivity assay measures virus-cell transmission of a defective virus pseudotyped with the Env, whereas replication of HIV-1 in CD4+ T cells also encounters cell-cell transmission. Because of the different results obtained by these two groups and because of the unanswered issues raised above, we reexamined the involvement of cytoplasmic palmitoylated cysteines of HIV-1 in the virus life cycle.
In contrast to the findings of the other two groups (5, 50), we found that in the context of the T-tropic HXB2 virus, substitutions of Ser and Ala for both Cys-764 and Cys-837 did not affect viral infectivity, as judged by continuous cultures and single-cycle infectivity assays in various host target cells, including HeLa-T4, CEM-SS, PM1, and human primary CD4+ T cells (Fig. 2, 3E, 3F, 5A, 5B, 6C and 8A). In addition, removal of the putative palmitoylation signals in the cytoplasmic tail of an M-tropic HIV-1 clone, ConB, also exerted no significant effect on viral replication (Fig. 7A and 8B and data not shown). Ser substitutions for either Cys-764 or Cys-837, or both, of the HXB2 strain Env were previously shown not to affect Env transport to the cell surface (68). Although the 764S.837S double mutant showed a higher level of surface expression than did the WT Env (Fig. 3D, bottom panels), this could simply have been due to a higher efficiency of transfection of this mutant in this particular experiment and not necessarily to higher surface expression of this mutant, since this mutant also produced a higher level of total Env in cells than did the WT Env (Fig. 3D, top panels). In support of this notion, surface biotinylation did not differentiate the WT and this double mutant (Fig. 3C), and the T- and M-tropic Cys-to-Ala double mutants showed no increase in surface expression compared to the WT and other single mutants (Fig. 6A and 6B and data not shown). Also, removal of these two cytoplasmic cysteines exhibited no significant effect on Env incorporation into the virus in CEM-SS (Fig. 3A), HeLa (Fig. 5C and 7B), or 293T (Fig. 3B and 5D and data not shown) cells.
On the other hand, abrogation of the palmitoylation signals in the cytoplasmic tail did not affect the raft association property of the T- and M-tropic Env proteins in HeLa (Fig. 4C) or 293T (Fig. 4D, 6D, and 7C) cells. In these lipid raft flotation analyses, only a fraction, ranging between 10% and 25%, of the total WT and mutant proteins was localized in the lipid rafts. When the expression of WT and mutant proteins in the soluble and raft fractions was quantified by normalization of protein amounts used in Western blotting, most of the WT as well as the Cys-to-Ser and Cys-to-Ala mutants was found to be located in the raft fractions, and the percentages of mutant proteins distributed in the raft fractions versus soluble fractions were comparable to those of the WT Env (Fig. 4E and 6E, respectively). These results indicate that the two cytoplasmic Cys residues are not critical for Env association with lipid rafts. Since the majority of proteins in the cells were not associated with lipid rafts, normalization for total proteins in the pooled raft and soluble fractions would result in much lower levels of Env being localized in the soluble fractions than in the raft fractions. Therefore, although this assay provides a means to compare the raft association properties of the WT and mutants, this approach provides no information on the absolute levels of gp41 present in the lipid rafts.
In agreement with previous studies (19, 35), we also noted that gp160 can be detected in viral particles. At a late stage of Env synthesis, the gp160 precursor is proteolytically cleaved by furin-like cellular proteases or other subtilisin-like proteases, most probably in the trans-Golgi network, to yield noncovalently associated gp120-gp41 heterodimers. Therefore, gp160 precursor cleavage is dependent on the cell types examined. Also, production of a native gp120-gp41 complex has been hampered by the limited efficiency of Env cleavage (18, 38, 39). These unprocessed gp160 molecules, likely forming homotrimers or heterotrimers with processed gp120-gp41, are also transported to the cell surface (40, 51), where they interact with Gag proteins and are incorporated into the virions.
Rousso et al. (50) employed an env-deficient, luciferase-containing NL4-3R–E–Luc provirus, which carries the functional vif and vpu genes but not the vpr or nef genes, along with the pCDNA3-based env gene derived from the HXB-2D strain for Env incorporation and env trans-complementation assays. Bhattacharya et al. (5) also used an NL4-3-derived env-deficient construct that encodes all viral proteins, except Env, and an HIV-1 LTR-driven NL4-3 env plasmid in their studies. Among these four accessory proteins, both Nef and Vpu encode activities that contribute to downregulation of the viral receptor CD4 from the cell surface (for a review, see reference 6). Nef increases viral infectivity (14, 37, 54), and downregulation of CD4 contributes to enhanced viral infectivity (1, 24, 49). Nef-enhanced viral infectivity occurs, at least in part, by an increase in Env incorporation into the virus, likely via Nef-enhanced steady-state levels of cell membrane-associated Env proteins (52). One of the well-characterized functions of Vpu is its ability to induce degradation of CD4 molecules trapped in intracellular complexes with Env, which allows the release of Env for transport to the cell surface (66). Vpu also mediates the efficient release of viral particles from virus-infected cells (21, 57). Vpu positively enhances viral infectivity by downmodulating the cell surface CD4 receptor (27), presumably removing the activity of CD4 that inhibits the virion release-promoting activity of Vpu (7, 58). Although the version of the infectious HXB2 molecular clone used in our study carries functional vif, vpr, vpu, and nef genes (59), these accessory genes might not be the primary factor responsible for the differential effects of the cytoplasmic tail palmitoylation observed. This was reasoned out by the observation that the pHXBBglCAT provirus used in our single-cycle infectivity assay carries none of the functional vpr, vpu, and nef genes.
It was reported that the entire cytoplasmic tail of the Env protein can be deleted without imposing a significant effect on Env incorporation into the virus in 293T cells (40). In the study by Bhattacharya et al. (5), the effect of mutations at positions 764 and 837 in the cytoplasmic tail on Env incorporation depended on the residue to be mutated and on what type of amino acid substitution was used. It is therefore likely that in 293T cells, the cytoplasmic tail per se does not positively contribute to Env incorporation; however, mutations in the cytoplasmic cysteines may induce alterations in Env folding and/or intracellular trafficking, which ultimately affect the effectiveness of Env incorporation into the virus.
Although the Env/p24 ratio of the Env-pseudotyped virus used in our one-cycle infectivity assay was greater than that of the "real" virus prepared from full-length, proviral transfection (Fig. 9A), the possibility that overexpression of mutant Env in pseudotypes may compensate for the impaired infectivity, if any, of the double mutant is unlikely. First, Bhattacharya et al. (5) also used LTR-controlled env expression plasmids for pseudotype preparation. Nevertheless, the viral entry of their Ser and Ala substitution double mutants was severely reduced, which is accounted for by insufficient Env incorporation of these two mutants. Second, if the possibility holds, it would be expected that the infectivity of the "real" WT and mutant viruses, which had lower Env/p24 ratios than the Env pseudotypes, would have been differentiated in the one-cycle infectivity assay. However, the WT and all mutant viruses showed similar levels of one-cycle-like infectivity in the LTR-lined cat gene-carrying H938 reporter cell line (Fig. 9B). Therefore, the WT-like infectivity of the mutant Env pseudotypes we examined was clearly due to the WT-like Env incorporation phenotype of the mutants.
A contiguous long sequence located in the cytoplasmic tail, beginning with the first palmitoylation site at Cys-764 and ending at the C terminus, is believed to be embedded in membranes (20). We previously showed that the C-terminal two-thirds of the cytoplasmic domain confers multimerization and plays a role in membrane association (13, 26). It is likely that minor amino acid differences between the env clones used in the studies by Rousso et al. (50), Bhattacharya et al. (5), and us may be responsible for the differential effects of the deacylated Env proteins on lipid raft association, Env incorporation, and viral infectivity observed by the three groups. The cytoplasmic tail of Env has specific interactions with cellular membranes, cellular proteins, and the matrix (MA) protein. It is also likely that subtle differences in the modes of interactions between Env and membranes, Env and cellular proteins, and Env and Gag, aroused by amino acid differences in the env and gag gene products, may contribute to the differential Env phenotypes of the deacylated Env mutants observed among different laboratories.
In the present study, we were unable to ascribe any apparent effects on replication of T- and M-tropic viruses to deacylation of the HIV-1 Env. Consistent with the notion that many isolates representative of different HIV-1 clades and the infectious molecular clones such as BORI, JRCSF, and DH123 do not contain the putative Cys palmitoylation sites in the cytoplasmic domains of their TM proteins, our results strongly support the notion that palmitoylation of the gp41 cytoplasmic tail does not play a critical role in the HIV-1 life cycle.
ACKNOWLEDGMENTS
The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: SIM2 hybridoma from James Hildreth, HeLa-T4 from Richard Axel, and H938 from Barbara K. Felber and Geroge N. Pavlakis. We are grateful to Jane C. Burns (School of Medicine, University of California, San Diego) for providing pHCMV-VSV G and to Ton-Hou Lee (Harvard School of Public Health, Boston, MA) for providing the pHXB2RU3 and pConB proviruses. We are also grateful to Sheau-Fen Lee and In-Ru Chen for technical help.
This work was supported by grants (NSC 92-2320-B-001-047 and NSC93-2320-B-001-016) from the National Science Council and Academia Sinica, Taipei, Taiwan, Republic of China.
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