ATPS Disrupts Human Immunodeficiency Virus Type 1
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病菌学杂志 2005年第9期
Departments of Microbiology
Medicine, Columbia University, New York, New York
Department of Biochemistry, Biomedical Center, Uppsala, Sweden
ABSTRACT
Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPS and GTPS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPS. The effects of ATPS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.
INTRODUCTION
The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is required for assembly and release of enveloped virions from infected cells (reviewed in reference 31). Concurrent with the budding of nascent virions, the Gag polyprotein is cleaved by the viral protease to produce matrix (MA), which lines the virion envelope, nucleocapsid (NC), which coats the viral RNA, capsid (CA), which forms the virion core, and the carboxyl-terminal p6 protein, which is required for virion release from host cells. While the Gag polyprotein is essential for the formation and release of virion particles, gag-encoded proteins are also important for viral entry, disassembly, reverse transcription, and integration.
Since viruses are obligate intracellular parasites and the roles played by HIV-1 gag-encoded proteins are numerous, it has been hypothesized that host proteins are required for HIV-1 gag-encoded functions. Attempts to identify these putative factors have revealed many Gag-interacting proteins, a subset of which have been shown to be incorporated into HIV-1 virions (4, 20, 22, 30, 36, 37, 43, 48, 55-58, 66, 69, 73). However, conclusive evidence that these virion-incorporated proteins are required for early steps of the HIV-1 life cycle has, in most cases, remained elusive.
We previously identified the ATPase heat shock protein 70 (Hsp70) as a constituent of the HIV-1 virion and showed that the HIV-1 Gag polyprotein is sufficient for Hsp70 incorporation (37). Hsp70 protein family members facilitate assembly and disassembly of oligomeric protein complexes as well as their folding and intracellular transport (21, 38, 39). There is also ample evidence that Hsp70 plays an active role in the life cycle of a variety of DNA and RNA viruses. Hsp70 is found inside purified rabies virus virions (61), and it may be involved in the assembly of adenovirus, enterovirus, and polyomavirus capsid protein complexes (25, 46, 47).
Incorporation of Hsp70 into HIV-1 virions suggests that intravirion Hsp70 participates in early events of infection, such as virion uncoating, in a manner similar to uncoating of clathrin-coated cages (21) or targeting of the reverse transcription complex to the appropriate cellular compartment. Indeed, it has been shown that Hsp70 is required for nuclear import of adenovirus DNA (62) and that Hsp70 might target the HIV-1 viral preintegration complex to the nucleus in the absence of HIV-1 Vpr protein (3).
In this manuscript, we extend our previous findings by showing that Hsp70 remains associated with detergent-stripped virion cores and that nonlentiviruses such as murine leukemia virus (MLV) also incorporate Hsp70 family members. We took a chemical approach to inhibit the ATPase activity of intravirion Hsp70 because standard genetic approaches designed to block Hsp70 precluded virion assembly. Treatment of purified HIV-1 and MLV virions with the slowly hydrolyzable ATP analog ATPS disrupts virion core morphology, and this is accompanied by defects in reverse transcription and infectivity.
MATERIALS AND METHODS
Plasmids. pNL4-3 contains a complete infectious HIV-1 provirus, HIV-1NL4-3 (2). pNL4-3env(–) has an env deletion (59). pSIVMAC239 contains a complete infectious simian immunodeficiency virus (SIV) provirus (42). pCSGW is an HIV-1-derived vector that provides the cis-acting information required for RNA packaging into virions and for subsequent reverse transcription and integration (6). It expresses green fluorescent protein (GFP) from an internal cytomegalovirus promoter and bears a deletion in the enhancer of the 3' long terminal repeat to prevent transcriptional interference in the resulting provirus. R8.9 and pMD-G encode HIV-1 structural proteins and vesicular stomatitis virus (VSV) G protein, respectively (81, 82). pNCA contains an infectious copy of MLV proviral DNA (24). pNCAIRES-GFP was constructed by cloning the enhanced green fluorescent protein (EGFP, Clontech) downstream of MLV env in pNCA under the translational control of the encephalomyocarditis virus internal ribosome entry site (IRES). pCMV-?laM-Vpr encodes a ?-lactamase-Vpr fusion protein (19).
To generate pCMV-Hsp70, Hsp701A cDNA was amplified by PCR with Pfu DNA polymerase (Stratagene) with the forward primer 5'-ATGGCCAAAGCCGCGGCG-3' and the reverse primer 5'-CTAATCTACCTCCTCAATGGTGGGGCC-3' and was cloned into pcDNA 3.1(+) (Invitrogen). With pCMV-Hsp70 as the template, the K71E mutation was engineered with the primers above and the mutagenic primers 5'-GTTTGACGCGGAGCGGCTGATC-3' and 5'-GATCAGCCGCTCCGCGTCAAAC-3'; this plasmid was designated pCMV-Hsp70(K71E).
Antibodies and immunoblots. MLV virions were harvested from the supernatant of chronically infected Rat-2 cells or 293T cells transfected with pNCA, pelleted through a 25% sucrose cushion, and subjected to a protease protection assay with subtilisin (37, 56). Proteins associated with the purified virions were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and detected by Coomassie stain or by Western blot with rabbit polyclonal anti-Hsp70 antibody (Stressgen, Inc.) or rabbit polyclonal antibody against Hsc70 (Santa Cruz Biotech). To analyze cell lysates, infected Rat-2 cells or transfected 293T cells were lysed with radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride). Lysate was cleared by centrifugation at 16,000 x g for 15 min at 4°C and subjected to SDS-PAGE, and proteins were detected as described above for virions.
Isolation of HIV-1 cores. HIV-1 cores were prepared as described previously (75). HIV-1 virions were harvested from the supernatant of 293T cells transfected with provirus pNL4-3, and 30 ml of this supernatant was pelleted through a 25% sucrose cushion at 100,000 x g for 2 h. The viral pellet was resuspended in 100 μl of phosphate-buffered saline and mixed with an equal volume of 200 mM NaCl-100 mM morpholinepropanesulfonic acid (MOPS) (pH 7.0); virions were lysed for 2 min at room temperature by adding Triton X-100 to a final concentration of 0.5%. HIV-1 cores were recovered by centrifugation in a microcentrifuge at full speed (16,000 x g) for 8 min at 4°C. The pellets were washed twice with 100 mM NaCl-50 mM MOPS (pH 7.0), resuspended in SDS-PAGE loading buffer, and subjected to immunoblotting.
Dot blot analysis. Cores were isolated as described above and lysed with 0.1% Triton X-100 in phosphate-buffered saline, transferred to a nylon membrane with the Minifold I dot blot apparatus (Schleicher & Schuell), and probed with a 32P-end-labeled DNA oligonucleotide as described (23).
Drugs. ATP, ATPS, and GTPS were purchased as 100 mM stocks (Roche Biochemicals). ADP, AMP-PNP, and AMP-CPP were purchased from Sigma and prepared as 100 mM stock solutions according to the manufacturer's instructions. Lamivudine was extracted from an Epivir pill (GlaxoWellcome) with 3 ml of dimethyl sulfoxide; a 1:200 dilution of this stock was used to inhibit HIV-1 replication.
Cells and tissue culture. Human 293T embryonic kidney cells and Rat2 cells chronically infected with MLVNCA were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. GHOST cells (National Institutes of Health AIDS Research and Reference Reagent Program, catalog number3942) were maintained in DMEM supplemented with 10% fetal calf serum, 500 μg of G418 per ml, 100 μg of hygromycin per ml, and 1 μg of puromycin per ml; these HOS cell derivatives are transduced with the MV7neo-T4 retroviral vector and stably transfected with an HIV-2 long terminal repeat-human GFP construct. Coreceptor CXCR4 and CCR5 cDNAs were introduced via retroviral transduction. Jurkat T cells were maintained in RPMI supplemented with antibiotics and 10% fetal calf serum. Owl monkey kidney (OMK) cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum.
Virus, vector, and virus-like particle production. To produce HIV-1 and SIV virions, 293T cells were transfected by calcium phosphate precipitation with pNL4-3 or pSIVMac239, respectively. To produce HIV-1 vectors, 293T cells were cotransfected with pCSGW, R8.9, and pMDG, or cells were transfected with pNL4-3env(–) in combination with pMDG. To produce MLV GFP reporter virions, 293T cells were transfected with pMLVNCA/IRES-GFP in combination with pMDG. To produce HIV-1 virus-like particles (VLPs), 293T cells were transfected with R8.9 and pMDG. All virions, vectors, and VLPs were harvested from the transfected cell supernatants 2 days posttransfection, syringe filtered (0.45 μm pore size, Acrodisc), and stored at –80°C. MLVNCA virions were harvested from the supernatant of chronically infected Rat-2 cells, syringe filtered, and stored at –80°C.
Assay for effect of Hsp70 overexpression on HIV-1 virion production. 293T cells were cotransfected by calcium phosphate precipitation in six-well plates with pNL4-3 and pCMV-Hsp70 or pCMV-Hsp70(K71E); 48 h posttransfection, virion production was measured by monitoring reverse transcriptase activity in the culture supernatant.
Saturation of owl monkey cell restriction activity by HIV-1 VLPs. We seeded 7 x 104 OMK cells per well in 24-well plates the day before infection. The cells were coinfected by spinoculation at 1,200 x g for 72 min with VSV G-pseudotyped HIV-1 vector and either mock- or ATPS-treated HIV-1 VLPs; 48 h postinfection, the number of GFP-positive cells was determined by flow cytometry.
Virion incubation with nucleotides and assessment of infectivity. Virion or vector stocks were concentrated by sedimentation through a 25% sucrose cushion as previously described (9). Pellets were resuspended in 10 mM Tris-HCl (pH 7.5) and 100 mM NaCl. The buffer was then adjusted to 5 mM MgCl2 and 5 mM of the indicated nucleotide and then incubated at 37°C for 6 h.
To assess HIV-1 virion infectivity after incubation with nucleotide, 4 x 104 GHOST cells were seeded per well in 24-well plates the day before infection. The cells were challenged with nucleotide-treated virions for 16 h, after which the medium was replaced with fresh DMEM containing dextran sulfate to block syncytium formation and further rounds of infection (44); 48 h postinfection, cells were fixed with 3% formaldehyde, and the number of GFP-positive cells was determined by flow cytometry (FACScalibur, Becton Dickinson).
To assess the infectivity of VSV-G-pseudotyped MLV or HIV-1 vector, 105 293T cells were seeded per well in 24-well plates the day before infection, and the cells were exposed to nucleotide-treated MLV virions or HIV-1 vector. At 48 h postinfection, cells were fixed with 3% formaldehyde, and the number of GFP-positive cells was determined by flow cytometry.
HIV-1 entry assay. The HIV-1 virion-based fusion assay was performed as described previously (19) with the GeneBLAzer in vivo detection kit (Invitrogen). Jurkat T cells were incubated for 4 h with HIV-1 virions carrying the ?-lactamase-Vpr chimeric protein. Cells were washed once with Hanks' balanced salt solution and resuspended in serum-free RPMI containing 2 μM CCF2-AM dye, 1% probenecid, and 1 mM HEPES. After an overnight incubation at room temperature, cells were washed twice with Hanks' balanced salt solution and fixed with 1.2% paraformaldehyde. The change in CCF2 fluorescence emission after cleavage by the ?-lactamase-Vpr chimera was detected by flow cytometry with a FACS Aria (Becton Dickinson). CCF2 dye was excited at 407 nm with a krypton laser; green fluorescence from uncleaved CCF2 was detected at an emission peak of 520 nm and blue fluorescence from cleaved CCF2 was detected at 450 nm. Data is analyzed with FACS Diva software, and results are reported as ratio of blue to green fluorescence.
Monitoring HIV-1 cDNA synthesis by real-time PCR. Jurkat T cells (107) were infected with VSV G-pseudotyped HIV-1NL4-3/env (–) at a multiplicity of infection of 1. Cells were washed with phosphate-buffered saline 24 h postinfection and total cellular DNA was extracted with the blood And cell culture DNA midi kit (Qiagen). PCR products (40 cycles: 94°C, 15 s; 65°C, 30 s; 72°C, 30 s) were generated with an ABI Prism 7700 sequence detector (Applied Biosystems), as previously described (5). Products were detected with SYBR Green (Molecular Probes, 1:100,000) and quantitated by comparison with standard curves generated by serial dilution of plasmid template containing the relevant amplicon. Full-length linear viral cDNA was detected with a primer pair which distinguishes between plasmid DNA and viral DNA synthesized de novo; this was possible due to nucleotide mismatches between the two long terminal repeats of pNL4-3 (9). All other viral cDNA products were detected with previously published primer sets (41).
Natural endogenous reverse transcription. Natural endogenous reverse transcription was performed as described previously (78). HIV-1, SIV, and MLV virions were pelleted through a 25% sucrose cushion and incubated in natural endogenous reverse transcription buffer composed of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl2, 50 μCi of [-32P]dCTP (3,000 Ci/mmol, Perkin Elmer) and 0.1 mM deoxynucleoside triphosphates (1 mM for MLV). After 6 h of incubation at 37°C, reactions were stopped by adding 10 mM EDTA, 1% SDS, and 50 μg of proteinase K per ml, for 1 h at 60°C. Viral DNA was extracted by phenol-chloroform (1:1), precipitated with ethanol, and free [-32P]dCTP was removed by passing the sample through a MicroSpin S-200 HR column (Amersham Pharmacia). The labeled viral DNA was then electrophoresed on an alkaline-denatured agarose gel, dried, and visualized by autoradiography.
Low deoxynucleoside triphosphate extension assay. HIV-1 reverse transcriptase was prepared with the expression plasmid pRT6H/NB-PROT, as described previously (8). For each extension assay, 2 pmol of the M13 –47 primer (New England Biolabs) was 5'-end labeled with [-32P]ATP with T4 polynucleotide kinase. After incubating at 37°C for 1 h, unincorporated nucleotide was removed by passing the primer through a MicroSpin S-200 HR column and the labeled primer was annealed to 0.25 μg of M13mp18 DNA (New England Biolabs) by heating to 96°C and slowly cooling to room temperature; 0.5 μg of reverse transcriptase was added to the labeled template-primer complex in 25 mM Tris-HCl (pH 8.0)-75 mM KCl-8 mM MgCl2-2 mM dithiothreitol-100 μg of bovine serum albumin per ml-10 mM CHAPS. The reactions were supplemented with 0.5 μM each deoxynucleoside triphosphate as well as the indicated concentrations of ATP or ATPS and allowed to proceed for 30 min at 37°C. After phenol-chloroform extraction and isopropanol precipitation, the labeled DNA was electrophoresed on a 6% polyacrylamide gel and processed for autoradiography.
Transmission electron microscopy. After incubation with ATP or ATPS, purified HIV-1 virions were mixed with uninfected 293T cells to provide contrast for electron microscopy staining. Samples were fixed with freshly made 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, embedded in Epon, and poststained with 1% uranyl acetate. Sections were approximately 60 nm thick to accommodate the volume of the capsid structure parallel to the plane of section. Electron microscopy was performed as described (40), and a series of electron micrographs were evaluated, focusing on virion core structure.
RESULTS
Intravirion Hsp70 is associated with HIV-1 cores. Previously, we showed that Hsp70 is incorporated into primate lentiviral virions, where it is protected within the virion membrane from exogenously added protease (37). To determine if virion-associated Hsp70 copurifies with HIV-1 core preparations, concentrated virion stocks were treated with detergent and cores were pelleted, as previously described (75). The protein content of these cores was analyzed by immunoblotting with antibodies against viral proteins and Hsp70. Viral genomic RNA content was examined by hybridization with an oligonucleotide probe. As expected, matrix and TM(gp41), two proteins associated with the virion membrane, were lost during core preparation (Fig. 1). Capsid, nucleocapsid, reverse transcriptase, integrase, and viral genomic RNA remained associated with the cores (Fig. 1). Hsp70 was also stably associated with the cores, suggesting that it plays a role in early steps of the virus life cycle.
Genetic approaches to study the function of intravirion Hsp70. HIV-1 Gag is sufficient for Hsp70 incorporation into HIV-1 particles (37). To determine if virion-associated Hsp70 is required for infectivity, we first attempted to identify an assembly-competent gag mutant that failed to incorporate Hsp70 into particles. A panel of gag mutations that delete the matrix, p6, much of the nucleocapsid, and the amino terminus of the capsid through the major homology region all produced particles that incorporate Hsp70 (data not shown). Larger deletions failed to assemble particles, as previously reported (1), and thus could not be evaluated for Hsp70 incorporation. We then evaluated HIV-1 capsid mutations P38A, E45A, T54A, Q63A/Q67A, L136D, D152A, D163A, K170A, and K203A, which were selected for assembly competence but lack of infectivity (73). Virions bearing these capsid mutations had no defect in Hsp70 incorporation, as determined by immunoblotting following subtilisin treatment (data not shown). Thus, we were unable to identify gag mutations that produce Hsp70-deficient virions.
Next, we tried to knockdown Hsp70 expression in virion-producing cells with RNA interference. Hsp70 family members are encoded by 11 genes, five of which encode cytoplasmic proteins with the potential to be incorporated into HIV-1 virions. Our efforts to simultaneously silence all five genes were not successful (data not shown).
Finally, we attempted to produce virions bearing Hsp70 mutants (K71E, D199S, or D206S) with defective ATPase activity (53, 76) to determine if they block infectivity of the released virions. While overexpression of wild-type Hsp70 did not affect HIV-1 virion release, expression of the mutants in HIV-1-producing cells disrupted virion assembly in a dose-dependent manner (Fig. 1B and data not shown), and thus we were unable to study the role of intravirion Hsp70 with the mutants.
Treatment of HIV-1 virions with ATPS or GTPS decreases infectivity. Hsp70 binds and releases target proteins in an ATP-hydrolytic reaction cycle in which ATP binding induces dissociation of Hsp70 from bound polypeptides (38). Since retroviral virion envelopes are reported to be permeable to nucleotides (7, 79), we purified HIV-1 virions by pelleting through a sucrose cushion and incubating them with ATP or various analogues, hoping to disturb the interaction of Hsp70 with putative substrates in the virion core. Incubation with ATP or ADP for 6 h at 37°C had no effect on virion infectivity in a single-cycle assay (Fig. 2 and data not shown). Incubation under identical conditions with ATPS, an ATP analogue that is resistant to hydrolysis by Hsp70, reduced infectivity by 10-fold (Fig. 2). The reduced infectivity resulted from effects of ATPS on the virions and was not due to toxicity to the target cells because no reduction in infectivity was observed when ATPS was added directly to the cells (data not shown). GTPS, another slowly hydrolysable nucleotide analogue, was as effective as ATPS at inhibiting HIV-1 virion infectivity (data not shown).
The effect of ATPS on HIV-1 infectivity is independent of HIV-1 env and most accessory genes. To determine which viral proteins are required for ATPS inhibition, we examined its effects on HIV-1 vectors with inactivating mutations in env as well as the accessory genes vif, vpr, vpu, and nef. GFP transducing HIV-1 vector particles pseudotyped with VSV G were treated with ATPS and used to infect 293T cells. Flow cytometric analysis of GFP-positive cells 2 days postinfection revealed that ATPS inhibited HIV-1 vector infectivity as efficiently as it inhibited HIV-1 virion infectivity (Fig. 3). Hence, the effect of the drug is independent of env and the deleted accessory genes. Additionally, ATPS was effective at inhibiting virion infectivity even at high multiplicities of infection (Fig. 3).
Hsp70 family members are incorporated into MLV virions. When virions from the prototypical oncoretrovirus Moloney murine leukemia virus (MLV) were purified from chronically infected Rat2 cells, we failed to detect Hsp70 in association with the particles, perhaps because Hsp70 is expressed at low levels in these cells (Fig. 4) (37). However, Rat-2-derived MLV particles incorporate the constitutively expressed paralogue, Hsc70 (Fig. 4). Furthermore, when 293T cells are transfected with an MLV proviral plasmid, Hsp70 is incorporated into the resulting virions as efficiently as it is incorporated into 293T-derived HIV-1 virions (Fig. 4). Additionally, we found that Hsp70 is associated with highly purified, subtilisin-treated virions from visna virus, bovine immunodeficiency virus, equine infectious anemia virus, and human T-cell leukemia virus (data not shown), which were provided by David Ott and Julian Bess.
ATPS and GTPS inhibit MLV virion infectivity. Having determined that MLV particles package Hsp70 family members, the effect of ATPS and GTPS on MLV infectivity was examined. Purified MLV-GFP reporter virions pseudotyped with VSV G were treated with ATPS or GTPS and then used to infect 293T cells. GFP-positive cells were counted by flow cytometry and both ATPS and GTPS were found to inhibit MLV virion infectivity (Fig. 5). These results suggest that these two nucleotide triphosphate analogues inhibit a step of the retroviral life cycle that is common to both HIV-1 and MLV. In side-by-side comparisons, ATPS inhibition curves were performed for HIV-1 virions, VSV G-pseudotyped HIV-1 vectors, and VSV G-pseudotyped MLV virions. The 50% inhibitory concentration for HIV-1 virus and vector was 750 μM, and for MLV virus it was 250 μM.
ATPS does not inhibit HIV-1 virion membrane fusion with the plasma membrane of susceptible target cells. To determine how ATPS inhibits virion infectivity, we first determined whether ATPS treatment inhibits HIV-1 fusion with target cells. We used an enzymatic assay in which HIV-1 virions incorporate a ?-lactamase-Vpr chimeric protein and the target cells are loaded with CCF2-AM, a fluorescent, membrane-permeating ?-lactamase substrate (19). Virions were treated with ATPS and used to infect target Jurkat cells. Entry of the virions into the cells was monitored by measuring the amount of CCF2-AM cleaved by the ?-lactamase-Vpr protein. ATPS-treated virions entered target cells as efficiently as mock-treated virions (Table 1).
ATPS-treated HIV-1 virions fail to complete reverse transcription. Next we determined if ATPS inhibited viral cDNA synthesis. VSV G-pseudotyped HIV-1NL4-3/env (–) virions were treated with ATPS and used to infect Jurkat T cells at an multiplicity of infection of 1. Total cellular DNA was subjected to real-time PCR analysis with sets of oligonucleotide primers that monitor discrete steps in reverse transcription, as shown in Fig. 6. Compared to control virions, a progressive defect in reverse transcription was observed after infection with virions exposed to ATPS: minus-strand strong stop was reduced 2.5-fold, and full-length linear cDNA was reduced 30-fold (Fig. 6). The reverse transcription defect was of sufficient magnitude to fully account for the decrease in infectivity associated with ATPS treatment.
ATPS inhibits natural endogenous reverse transcription. When supplied with deoxynucleoside triphosphates and Mg2+, purified retroviral virions are capable of viral cDNA synthesis in the absence of detergent, a process that has been called natural endogenous reverse transcription (78). Since ATPS blocked HIV-1 cDNA synthesis acutely after infection in cells, we examined the effect of the drug on natural endogenous reverse transcription. HIV-1NL4-3, SIVMac239, and MLV virions were incubated with deoxynucleoside triphosphates, Mg2+, and -32P-labeled dCTP. Following natural endogenous reverse transcription, radiolabeled DNA was extracted and the cDNA products were analyzed by alkaline agarose gel electrophoresis. The presence of ATPS during natural endogenous reverse transcription significantly reduced the amount of product for all three retroviruses (Fig. 7). Thus, ATPS treatment of virions inhibits both intracellular and intravirion reverse transcription.
ATPS does not inhibit the processivity of HIV-1 reverse transcriptase. Inhibition of retroviral reverse transcription by ATPS suggested that the ATP analogue might act directly on reverse transcriptase itself. To test this possibility, virions were lysed with Triton X-100, and reverse transcriptase activity was measured with a homopolymeric poly(rA)-oligo(dT) template. Reverse transcriptase activity in this assay was not inhibited by ATPS even when it was added at a 500-fold excess over the dTTP in the assay (data not shown).
To take a more careful look at possible effects of ATPS on HIV-1 reverse transcriptase processivity, we performed a low deoxynucleoside triphosphate extension assay with recombinant HIV-1 reverse transcriptase in vitro (8). Even at ATPS concentrations high enough to inhibit virion infectivity (2 mM) the drug had minimal effects on reverse transcriptase processivity (Fig. 8). At a concentration of 5 mM, 2,500 times the concentration of deoxynucleoside triphosphates in the reaction, some interference with reverse transcriptase processivity was observed. However, this inhibition was not specific to ATPS, since at this high concentration even ATP inhibited reverse transcriptase processivity, even though it had no observable effect on virion infectivity.
ATPS disrupts the core morphology in HIV-1 virions. Aberrant core morphology is often associated with defects in reverse transcription and loss of infectivity (60, 67, 72). Since treatment of HIV-1 virions with ATPS inhibited reverse transcription and virion infectivity, we wanted to determine if this inhibition correlated with morphological changes in the virion core. Purified HIV-1 virions were treated with either ATP or ATPS, and then the virions were fixed and examined by transmission electron microscopy. Compared with ATP treatment, a significant reduction in the number of mature, conical core structures (Fig. 9A) was observed when cell-free HIV-1 virions were incubated with ATPS (Table 2). A significant increase in grossly aberrant core morphology was also observed with ATPS, including partially filled, irregular, and empty core structures (Fig. 9B to E). These data suggest that the defects in reverse transcription and infectivity that we observed with ATPS-treated virions are secondary to the morphological changes that this drug induces in the virion core.
Biochemical and functional assessment of HIV-1 cores from ATPS-treated virions. In light of the electron microscopy results, we examined the effect of ATPS on HIV-1 virion core yield and stability with a previously described method (29). Cores were purified by exposure to detergent with subsequent acceleration through a sucrose density gradient. Cores purified on the gradient were incubated at 37°C for up to 3 h. Significant increases in core yield and stability were found when virions were treated with ATPS (data not shown). However, the same effect was observed when virions were treated with ATP, our control compound that does not inhibit virion infectivity or disrupt core morphology.
HIV-1 infection of owl monkey kidney cells is restricted by TRIMCyp, a gene that encodes a fusion protein of TRIM5 and cyclophilin A (52, 63). This restriction can be saturated by HIV-1 virus-like particles (VLPs) bearing the viral protease (70). Recently, it was demonstrated that capsid mutations that disrupt core stability and integrity decrease the ability of VLPs to abrogate HIV-1 restriction in OMK cells (28). To determine if ATPS disrupts the integrity of VLP cores, OMK cells were infected with the HIV-1-GFP reporter vector and either untreated HIV-1 VLPs or VLPs treated with ATP or ATPS. Challenge of OMK cells with modest quantities of HIV-1-GFP resulted in few infected cells (Fig. 10, two top panels). As expected, coinfection with a large quantity of VLPs released HIV-1 from restriction (Fig. 10). ATP treatment had no effect on the ability of VLPs to saturate owl monkey TRIMCyp (data not shown), but ATPS-treated VLPs were completely defective at blocking HIV-1 restriction (Fig. 10). Similarly, ATPS treated N-tropic MLV VLPs were defective at abrogating MLV restriction in TE671 rhabdomyosarcoma cells (data not shown). These results confirm that retroviral core structure is altered by ATPS treatment.
DISCUSSION
Incorporation of Hsp70 family members into retroviral virions. Our results demonstrate that Hsp70 is incorporated not only into primate lentiviral virions, as previously shown (37), but probably into all retroviruses. This pattern of incorporation is quite different from what is seen with another host cell protein, cyclophilin A, which is only incorporated into HIV-1 virions (10). Hsp70 is also distinguished from cyclophilin A in that it remains stably associated with detergent-stripped HIV-1 virion cores. Cyclophilin A is recruited to virions via contacts with capsid surface residues that are unique to HIV-1 (30, 32). The contacts are quite minimal, and so cyclophilin A is easily stripped from cores (75). Though we have shown that gag is sufficient for virion incorporation of Hsp70 (37), our genetic approaches to generate Hsp70-deficient virions have been thwarted by effects on virion assembly. Hence, we do not know which viral factors mediate Hsp70's stable interaction with purified cores.
Effects of Hsp70 on virion assembly. Though conditions have been found in which purified Gag fragments multimerize into ordered structures in vitro (12-14, 33, 35, 77), the formation of higher-order structures by full Gag polyprotein seems to require factors in mammalian cell lysate that include ATP (11, 26, 45, 71, 74). These results suggest that cellular ATP-binding proteins play an essential role in HIV-1 virion assembly. Indeed, one such ATPase, HP68, associates with Gag assembly intermediates, and a fragment lacking the ATP binding motif inhibits virion release from cells (26, 80). Our finding of similar defects when ATPase-deficient Hsp70 mutants are expressed in virion producer cells (Fig. 1B) suggests that Hsp70 is also crucial for virion assembly. Additionally, ATPase-defective Hsp70 mutants interfere with vesicular trafficking between endosomal compartments and the cell surface (51). Since a significant portion of HIV-1 Gag produced in cells buds into the endosomal compartment, from which it shuttles to the plasma membrane for virion release (54, 64), the block to assembly that we observed may be due to the fact that mutant Hsp70 inhibits transport of virus-laden endosomes to the plasma membrane.
ATPS disrupts virion core morphology. ATPS inhibition of HIV-1 virion infectivity is independent of the mode of virion fusion with target cells and does not require viral accessory genes, since minimal HIV-1 vectors pseudotyped with VSV-G are as susceptible to ATPS as is full-length HIV-1. MLV virions are also inhibited by ATPS, indicating that the target of the drug is either a viral protein with a function shared by all retroviruses or a cellular factor that all retroviruses require for infectivity, perhaps Hsp70.
ATPS-treated virions fuse normally with susceptible target cells and presumably release the virion core into the host cell cytoplasm, yet the reverse transcription complex is somehow defective. ATPS does not act directly on reverse transcriptase, as determined by the in vitro processivity assay, but on another factor required for both intravirion and intracellular reverse transcription.
Avian sarcoma and leukosis virus uncoating and reverse transcription in a cell-free system require ATP hydrolysis and are inhibited by ATPS (50). In this system it is not known if ATP is required by a molecular chaperone that aids uncoating or if it is used for some other purpose, such as the phosphorylation of a viral or cellular protein. When HIV-1 virions were treated with ATPS, few conical cores were detected by electron microscopy (Fig. 9 and Table 2). In many instances it has been found that amino acid changes in HIV-1 capsid have no effect on viral assembly, but these virions fail to form proper cores and are noninfectious, with defects in reverse transcription (60, 67, 72). Thus, a properly formed HIV-1 conical core seems necessary for infectivity, and the irregular core structures that result from ATPS treatment of virions are likely to be causally related to the block in infectivity and reverse transcription.
We found no significant defect in the stability of cores from ATPS-treated virions, but noninfectious capsid mutants have been reported with hypo-, hyper-, or relatively normal core stability (29, 68). Saturation of retroviral restriction factors by VLPs requires that the Gag polyprotein be processed by the viral protease (65) and that the capsid be reasonably intact (28). Our finding that ATPS disrupts the ability of VLPs to saturate restriction factors suggests that capsid conformation is altered by this treatment.
How does ATPS inhibit HIV-1 infectivity? The morphological changes in HIV-1 cores reported here may result from effects of ATPS on the peptide binding properties of virion-associated Hsp70. While bound to ATP, Hsp70 rapidly associates with and dissociates from peptide substrates. Upon ATP hydrolysis, Hsp70 binds substrate tightly and is not released until ADP is replaced by ATP (38, 39). When bound to ATPS, the conformation of Hsp70 resembles that of Hsp70 bound to ADP (49), and it binds to peptides with an especially low dissociation rate, since ATPS cannot be hydrolyzed (34). Thus, the changes in core structure may result from the Hsp70-ATPS complex locking onto a virion core protein in a nonphysiological manner, leading to collapse of the core and defects in reverse transcription.
Alternatively, decreased infectivity from ATPS treatment might result from thiophosphorylation of a virion protein. ATPS is an excellent substrate for protein kinases. Since thiophosphorylated proteins are resistant to phosphatases, using ATPS as a kinase substrate would result in the irreversible transfer of the thiophosphate residue to proteins that are normally regulated by reversible phosphorylation (27). Thiophosphorylation of glycogen phosphorylase and myosin light chain leads to constitutive activation of these proteins, while thiophosphorylation of the epidermal growth factor receptor blocks its degradation (17, 18). At least three kinases, cyclic AMP-dependent protein kinase A, ERK-1, and ERK-2, are packaged into HIV-1 virions (15, 16). It is thus possible that irreversible thiophosphorylation of a virion component may be responsible for the defects in infectivity and the morphological changes in core structure.
ACKNOWLEDGMENTS
We thank Marianne Ljungkvist for valuable contribution to this study and Julian Bess, Heinrich Gottlinger, Warner Green, Ned Landau, David Ott, David Sayah, Elena Sokolskaja, Gilda Tachedjian, and Uta von Schwedler for reagents and technical advice.
This work was supported by National Institutes of Health grant RO1AI41857 to J.L. and core facilities of the Columbia-Rockefeller Center for AIDS Research.
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Medicine, Columbia University, New York, New York
Department of Biochemistry, Biomedical Center, Uppsala, Sweden
ABSTRACT
Heat shock protein 70 (Hsp70) is incorporated within the membrane of primate lentiviral virions. Here we demonstrate that Hsp70 is also incorporated into oncoretroviral virions and that it remains associated with membrane-stripped human immunodeficiency virus type 1 (HIV-1) virion cores. To determine if Hsp70 promotes virion infectivity, we attempted to generate Hsp70-deficient virions with gag deletion mutations, Hsp70 transdominant mutants, or RNA interference, but these efforts were confounded, largely because they disrupt virion assembly. Given that polypeptide substrates are bound and released by Hsp70 in an ATP-hydrolytic reaction cycle, we supposed that incubation of HIV-1 virions with ATP would perturb Hsp70 interaction with substrates in the virion and thereby decrease infectivity. Treatment with ATP or ADP had no observable effect, but ATPS and GTPS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPS-treated virions were competent for fusion with susceptible target cells, but viral cDNA synthesis was inhibited to an extent that correlated with the magnitude of decrease in infectivity. Intravirion reverse transcription by HIV-1, simian immunodeficiency virus, or murine leukemia virus was also inhibited by ATPS. The effects of ATPS on HIV-1 reverse transcription appeared to be indirect, resulting from disruption of virion core morphology that was evident by transmission electron microscopy. Consistent with effects on capsid conformation, ATPS-treated viruslike particles failed to saturate host antiviral restriction activity. Our observations support a model in which the catalytic activity of virion-associated Hsp70 is required to maintain structural integrity of the virion core.
INTRODUCTION
The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is required for assembly and release of enveloped virions from infected cells (reviewed in reference 31). Concurrent with the budding of nascent virions, the Gag polyprotein is cleaved by the viral protease to produce matrix (MA), which lines the virion envelope, nucleocapsid (NC), which coats the viral RNA, capsid (CA), which forms the virion core, and the carboxyl-terminal p6 protein, which is required for virion release from host cells. While the Gag polyprotein is essential for the formation and release of virion particles, gag-encoded proteins are also important for viral entry, disassembly, reverse transcription, and integration.
Since viruses are obligate intracellular parasites and the roles played by HIV-1 gag-encoded proteins are numerous, it has been hypothesized that host proteins are required for HIV-1 gag-encoded functions. Attempts to identify these putative factors have revealed many Gag-interacting proteins, a subset of which have been shown to be incorporated into HIV-1 virions (4, 20, 22, 30, 36, 37, 43, 48, 55-58, 66, 69, 73). However, conclusive evidence that these virion-incorporated proteins are required for early steps of the HIV-1 life cycle has, in most cases, remained elusive.
We previously identified the ATPase heat shock protein 70 (Hsp70) as a constituent of the HIV-1 virion and showed that the HIV-1 Gag polyprotein is sufficient for Hsp70 incorporation (37). Hsp70 protein family members facilitate assembly and disassembly of oligomeric protein complexes as well as their folding and intracellular transport (21, 38, 39). There is also ample evidence that Hsp70 plays an active role in the life cycle of a variety of DNA and RNA viruses. Hsp70 is found inside purified rabies virus virions (61), and it may be involved in the assembly of adenovirus, enterovirus, and polyomavirus capsid protein complexes (25, 46, 47).
Incorporation of Hsp70 into HIV-1 virions suggests that intravirion Hsp70 participates in early events of infection, such as virion uncoating, in a manner similar to uncoating of clathrin-coated cages (21) or targeting of the reverse transcription complex to the appropriate cellular compartment. Indeed, it has been shown that Hsp70 is required for nuclear import of adenovirus DNA (62) and that Hsp70 might target the HIV-1 viral preintegration complex to the nucleus in the absence of HIV-1 Vpr protein (3).
In this manuscript, we extend our previous findings by showing that Hsp70 remains associated with detergent-stripped virion cores and that nonlentiviruses such as murine leukemia virus (MLV) also incorporate Hsp70 family members. We took a chemical approach to inhibit the ATPase activity of intravirion Hsp70 because standard genetic approaches designed to block Hsp70 precluded virion assembly. Treatment of purified HIV-1 and MLV virions with the slowly hydrolyzable ATP analog ATPS disrupts virion core morphology, and this is accompanied by defects in reverse transcription and infectivity.
MATERIALS AND METHODS
Plasmids. pNL4-3 contains a complete infectious HIV-1 provirus, HIV-1NL4-3 (2). pNL4-3env(–) has an env deletion (59). pSIVMAC239 contains a complete infectious simian immunodeficiency virus (SIV) provirus (42). pCSGW is an HIV-1-derived vector that provides the cis-acting information required for RNA packaging into virions and for subsequent reverse transcription and integration (6). It expresses green fluorescent protein (GFP) from an internal cytomegalovirus promoter and bears a deletion in the enhancer of the 3' long terminal repeat to prevent transcriptional interference in the resulting provirus. R8.9 and pMD-G encode HIV-1 structural proteins and vesicular stomatitis virus (VSV) G protein, respectively (81, 82). pNCA contains an infectious copy of MLV proviral DNA (24). pNCAIRES-GFP was constructed by cloning the enhanced green fluorescent protein (EGFP, Clontech) downstream of MLV env in pNCA under the translational control of the encephalomyocarditis virus internal ribosome entry site (IRES). pCMV-?laM-Vpr encodes a ?-lactamase-Vpr fusion protein (19).
To generate pCMV-Hsp70, Hsp701A cDNA was amplified by PCR with Pfu DNA polymerase (Stratagene) with the forward primer 5'-ATGGCCAAAGCCGCGGCG-3' and the reverse primer 5'-CTAATCTACCTCCTCAATGGTGGGGCC-3' and was cloned into pcDNA 3.1(+) (Invitrogen). With pCMV-Hsp70 as the template, the K71E mutation was engineered with the primers above and the mutagenic primers 5'-GTTTGACGCGGAGCGGCTGATC-3' and 5'-GATCAGCCGCTCCGCGTCAAAC-3'; this plasmid was designated pCMV-Hsp70(K71E).
Antibodies and immunoblots. MLV virions were harvested from the supernatant of chronically infected Rat-2 cells or 293T cells transfected with pNCA, pelleted through a 25% sucrose cushion, and subjected to a protease protection assay with subtilisin (37, 56). Proteins associated with the purified virions were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and detected by Coomassie stain or by Western blot with rabbit polyclonal anti-Hsp70 antibody (Stressgen, Inc.) or rabbit polyclonal antibody against Hsc70 (Santa Cruz Biotech). To analyze cell lysates, infected Rat-2 cells or transfected 293T cells were lysed with radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride). Lysate was cleared by centrifugation at 16,000 x g for 15 min at 4°C and subjected to SDS-PAGE, and proteins were detected as described above for virions.
Isolation of HIV-1 cores. HIV-1 cores were prepared as described previously (75). HIV-1 virions were harvested from the supernatant of 293T cells transfected with provirus pNL4-3, and 30 ml of this supernatant was pelleted through a 25% sucrose cushion at 100,000 x g for 2 h. The viral pellet was resuspended in 100 μl of phosphate-buffered saline and mixed with an equal volume of 200 mM NaCl-100 mM morpholinepropanesulfonic acid (MOPS) (pH 7.0); virions were lysed for 2 min at room temperature by adding Triton X-100 to a final concentration of 0.5%. HIV-1 cores were recovered by centrifugation in a microcentrifuge at full speed (16,000 x g) for 8 min at 4°C. The pellets were washed twice with 100 mM NaCl-50 mM MOPS (pH 7.0), resuspended in SDS-PAGE loading buffer, and subjected to immunoblotting.
Dot blot analysis. Cores were isolated as described above and lysed with 0.1% Triton X-100 in phosphate-buffered saline, transferred to a nylon membrane with the Minifold I dot blot apparatus (Schleicher & Schuell), and probed with a 32P-end-labeled DNA oligonucleotide as described (23).
Drugs. ATP, ATPS, and GTPS were purchased as 100 mM stocks (Roche Biochemicals). ADP, AMP-PNP, and AMP-CPP were purchased from Sigma and prepared as 100 mM stock solutions according to the manufacturer's instructions. Lamivudine was extracted from an Epivir pill (GlaxoWellcome) with 3 ml of dimethyl sulfoxide; a 1:200 dilution of this stock was used to inhibit HIV-1 replication.
Cells and tissue culture. Human 293T embryonic kidney cells and Rat2 cells chronically infected with MLVNCA were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. GHOST cells (National Institutes of Health AIDS Research and Reference Reagent Program, catalog number3942) were maintained in DMEM supplemented with 10% fetal calf serum, 500 μg of G418 per ml, 100 μg of hygromycin per ml, and 1 μg of puromycin per ml; these HOS cell derivatives are transduced with the MV7neo-T4 retroviral vector and stably transfected with an HIV-2 long terminal repeat-human GFP construct. Coreceptor CXCR4 and CCR5 cDNAs were introduced via retroviral transduction. Jurkat T cells were maintained in RPMI supplemented with antibiotics and 10% fetal calf serum. Owl monkey kidney (OMK) cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum.
Virus, vector, and virus-like particle production. To produce HIV-1 and SIV virions, 293T cells were transfected by calcium phosphate precipitation with pNL4-3 or pSIVMac239, respectively. To produce HIV-1 vectors, 293T cells were cotransfected with pCSGW, R8.9, and pMDG, or cells were transfected with pNL4-3env(–) in combination with pMDG. To produce MLV GFP reporter virions, 293T cells were transfected with pMLVNCA/IRES-GFP in combination with pMDG. To produce HIV-1 virus-like particles (VLPs), 293T cells were transfected with R8.9 and pMDG. All virions, vectors, and VLPs were harvested from the transfected cell supernatants 2 days posttransfection, syringe filtered (0.45 μm pore size, Acrodisc), and stored at –80°C. MLVNCA virions were harvested from the supernatant of chronically infected Rat-2 cells, syringe filtered, and stored at –80°C.
Assay for effect of Hsp70 overexpression on HIV-1 virion production. 293T cells were cotransfected by calcium phosphate precipitation in six-well plates with pNL4-3 and pCMV-Hsp70 or pCMV-Hsp70(K71E); 48 h posttransfection, virion production was measured by monitoring reverse transcriptase activity in the culture supernatant.
Saturation of owl monkey cell restriction activity by HIV-1 VLPs. We seeded 7 x 104 OMK cells per well in 24-well plates the day before infection. The cells were coinfected by spinoculation at 1,200 x g for 72 min with VSV G-pseudotyped HIV-1 vector and either mock- or ATPS-treated HIV-1 VLPs; 48 h postinfection, the number of GFP-positive cells was determined by flow cytometry.
Virion incubation with nucleotides and assessment of infectivity. Virion or vector stocks were concentrated by sedimentation through a 25% sucrose cushion as previously described (9). Pellets were resuspended in 10 mM Tris-HCl (pH 7.5) and 100 mM NaCl. The buffer was then adjusted to 5 mM MgCl2 and 5 mM of the indicated nucleotide and then incubated at 37°C for 6 h.
To assess HIV-1 virion infectivity after incubation with nucleotide, 4 x 104 GHOST cells were seeded per well in 24-well plates the day before infection. The cells were challenged with nucleotide-treated virions for 16 h, after which the medium was replaced with fresh DMEM containing dextran sulfate to block syncytium formation and further rounds of infection (44); 48 h postinfection, cells were fixed with 3% formaldehyde, and the number of GFP-positive cells was determined by flow cytometry (FACScalibur, Becton Dickinson).
To assess the infectivity of VSV-G-pseudotyped MLV or HIV-1 vector, 105 293T cells were seeded per well in 24-well plates the day before infection, and the cells were exposed to nucleotide-treated MLV virions or HIV-1 vector. At 48 h postinfection, cells were fixed with 3% formaldehyde, and the number of GFP-positive cells was determined by flow cytometry.
HIV-1 entry assay. The HIV-1 virion-based fusion assay was performed as described previously (19) with the GeneBLAzer in vivo detection kit (Invitrogen). Jurkat T cells were incubated for 4 h with HIV-1 virions carrying the ?-lactamase-Vpr chimeric protein. Cells were washed once with Hanks' balanced salt solution and resuspended in serum-free RPMI containing 2 μM CCF2-AM dye, 1% probenecid, and 1 mM HEPES. After an overnight incubation at room temperature, cells were washed twice with Hanks' balanced salt solution and fixed with 1.2% paraformaldehyde. The change in CCF2 fluorescence emission after cleavage by the ?-lactamase-Vpr chimera was detected by flow cytometry with a FACS Aria (Becton Dickinson). CCF2 dye was excited at 407 nm with a krypton laser; green fluorescence from uncleaved CCF2 was detected at an emission peak of 520 nm and blue fluorescence from cleaved CCF2 was detected at 450 nm. Data is analyzed with FACS Diva software, and results are reported as ratio of blue to green fluorescence.
Monitoring HIV-1 cDNA synthesis by real-time PCR. Jurkat T cells (107) were infected with VSV G-pseudotyped HIV-1NL4-3/env (–) at a multiplicity of infection of 1. Cells were washed with phosphate-buffered saline 24 h postinfection and total cellular DNA was extracted with the blood And cell culture DNA midi kit (Qiagen). PCR products (40 cycles: 94°C, 15 s; 65°C, 30 s; 72°C, 30 s) were generated with an ABI Prism 7700 sequence detector (Applied Biosystems), as previously described (5). Products were detected with SYBR Green (Molecular Probes, 1:100,000) and quantitated by comparison with standard curves generated by serial dilution of plasmid template containing the relevant amplicon. Full-length linear viral cDNA was detected with a primer pair which distinguishes between plasmid DNA and viral DNA synthesized de novo; this was possible due to nucleotide mismatches between the two long terminal repeats of pNL4-3 (9). All other viral cDNA products were detected with previously published primer sets (41).
Natural endogenous reverse transcription. Natural endogenous reverse transcription was performed as described previously (78). HIV-1, SIV, and MLV virions were pelleted through a 25% sucrose cushion and incubated in natural endogenous reverse transcription buffer composed of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl2, 50 μCi of [-32P]dCTP (3,000 Ci/mmol, Perkin Elmer) and 0.1 mM deoxynucleoside triphosphates (1 mM for MLV). After 6 h of incubation at 37°C, reactions were stopped by adding 10 mM EDTA, 1% SDS, and 50 μg of proteinase K per ml, for 1 h at 60°C. Viral DNA was extracted by phenol-chloroform (1:1), precipitated with ethanol, and free [-32P]dCTP was removed by passing the sample through a MicroSpin S-200 HR column (Amersham Pharmacia). The labeled viral DNA was then electrophoresed on an alkaline-denatured agarose gel, dried, and visualized by autoradiography.
Low deoxynucleoside triphosphate extension assay. HIV-1 reverse transcriptase was prepared with the expression plasmid pRT6H/NB-PROT, as described previously (8). For each extension assay, 2 pmol of the M13 –47 primer (New England Biolabs) was 5'-end labeled with [-32P]ATP with T4 polynucleotide kinase. After incubating at 37°C for 1 h, unincorporated nucleotide was removed by passing the primer through a MicroSpin S-200 HR column and the labeled primer was annealed to 0.25 μg of M13mp18 DNA (New England Biolabs) by heating to 96°C and slowly cooling to room temperature; 0.5 μg of reverse transcriptase was added to the labeled template-primer complex in 25 mM Tris-HCl (pH 8.0)-75 mM KCl-8 mM MgCl2-2 mM dithiothreitol-100 μg of bovine serum albumin per ml-10 mM CHAPS. The reactions were supplemented with 0.5 μM each deoxynucleoside triphosphate as well as the indicated concentrations of ATP or ATPS and allowed to proceed for 30 min at 37°C. After phenol-chloroform extraction and isopropanol precipitation, the labeled DNA was electrophoresed on a 6% polyacrylamide gel and processed for autoradiography.
Transmission electron microscopy. After incubation with ATP or ATPS, purified HIV-1 virions were mixed with uninfected 293T cells to provide contrast for electron microscopy staining. Samples were fixed with freshly made 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, embedded in Epon, and poststained with 1% uranyl acetate. Sections were approximately 60 nm thick to accommodate the volume of the capsid structure parallel to the plane of section. Electron microscopy was performed as described (40), and a series of electron micrographs were evaluated, focusing on virion core structure.
RESULTS
Intravirion Hsp70 is associated with HIV-1 cores. Previously, we showed that Hsp70 is incorporated into primate lentiviral virions, where it is protected within the virion membrane from exogenously added protease (37). To determine if virion-associated Hsp70 copurifies with HIV-1 core preparations, concentrated virion stocks were treated with detergent and cores were pelleted, as previously described (75). The protein content of these cores was analyzed by immunoblotting with antibodies against viral proteins and Hsp70. Viral genomic RNA content was examined by hybridization with an oligonucleotide probe. As expected, matrix and TM(gp41), two proteins associated with the virion membrane, were lost during core preparation (Fig. 1). Capsid, nucleocapsid, reverse transcriptase, integrase, and viral genomic RNA remained associated with the cores (Fig. 1). Hsp70 was also stably associated with the cores, suggesting that it plays a role in early steps of the virus life cycle.
Genetic approaches to study the function of intravirion Hsp70. HIV-1 Gag is sufficient for Hsp70 incorporation into HIV-1 particles (37). To determine if virion-associated Hsp70 is required for infectivity, we first attempted to identify an assembly-competent gag mutant that failed to incorporate Hsp70 into particles. A panel of gag mutations that delete the matrix, p6, much of the nucleocapsid, and the amino terminus of the capsid through the major homology region all produced particles that incorporate Hsp70 (data not shown). Larger deletions failed to assemble particles, as previously reported (1), and thus could not be evaluated for Hsp70 incorporation. We then evaluated HIV-1 capsid mutations P38A, E45A, T54A, Q63A/Q67A, L136D, D152A, D163A, K170A, and K203A, which were selected for assembly competence but lack of infectivity (73). Virions bearing these capsid mutations had no defect in Hsp70 incorporation, as determined by immunoblotting following subtilisin treatment (data not shown). Thus, we were unable to identify gag mutations that produce Hsp70-deficient virions.
Next, we tried to knockdown Hsp70 expression in virion-producing cells with RNA interference. Hsp70 family members are encoded by 11 genes, five of which encode cytoplasmic proteins with the potential to be incorporated into HIV-1 virions. Our efforts to simultaneously silence all five genes were not successful (data not shown).
Finally, we attempted to produce virions bearing Hsp70 mutants (K71E, D199S, or D206S) with defective ATPase activity (53, 76) to determine if they block infectivity of the released virions. While overexpression of wild-type Hsp70 did not affect HIV-1 virion release, expression of the mutants in HIV-1-producing cells disrupted virion assembly in a dose-dependent manner (Fig. 1B and data not shown), and thus we were unable to study the role of intravirion Hsp70 with the mutants.
Treatment of HIV-1 virions with ATPS or GTPS decreases infectivity. Hsp70 binds and releases target proteins in an ATP-hydrolytic reaction cycle in which ATP binding induces dissociation of Hsp70 from bound polypeptides (38). Since retroviral virion envelopes are reported to be permeable to nucleotides (7, 79), we purified HIV-1 virions by pelleting through a sucrose cushion and incubating them with ATP or various analogues, hoping to disturb the interaction of Hsp70 with putative substrates in the virion core. Incubation with ATP or ADP for 6 h at 37°C had no effect on virion infectivity in a single-cycle assay (Fig. 2 and data not shown). Incubation under identical conditions with ATPS, an ATP analogue that is resistant to hydrolysis by Hsp70, reduced infectivity by 10-fold (Fig. 2). The reduced infectivity resulted from effects of ATPS on the virions and was not due to toxicity to the target cells because no reduction in infectivity was observed when ATPS was added directly to the cells (data not shown). GTPS, another slowly hydrolysable nucleotide analogue, was as effective as ATPS at inhibiting HIV-1 virion infectivity (data not shown).
The effect of ATPS on HIV-1 infectivity is independent of HIV-1 env and most accessory genes. To determine which viral proteins are required for ATPS inhibition, we examined its effects on HIV-1 vectors with inactivating mutations in env as well as the accessory genes vif, vpr, vpu, and nef. GFP transducing HIV-1 vector particles pseudotyped with VSV G were treated with ATPS and used to infect 293T cells. Flow cytometric analysis of GFP-positive cells 2 days postinfection revealed that ATPS inhibited HIV-1 vector infectivity as efficiently as it inhibited HIV-1 virion infectivity (Fig. 3). Hence, the effect of the drug is independent of env and the deleted accessory genes. Additionally, ATPS was effective at inhibiting virion infectivity even at high multiplicities of infection (Fig. 3).
Hsp70 family members are incorporated into MLV virions. When virions from the prototypical oncoretrovirus Moloney murine leukemia virus (MLV) were purified from chronically infected Rat2 cells, we failed to detect Hsp70 in association with the particles, perhaps because Hsp70 is expressed at low levels in these cells (Fig. 4) (37). However, Rat-2-derived MLV particles incorporate the constitutively expressed paralogue, Hsc70 (Fig. 4). Furthermore, when 293T cells are transfected with an MLV proviral plasmid, Hsp70 is incorporated into the resulting virions as efficiently as it is incorporated into 293T-derived HIV-1 virions (Fig. 4). Additionally, we found that Hsp70 is associated with highly purified, subtilisin-treated virions from visna virus, bovine immunodeficiency virus, equine infectious anemia virus, and human T-cell leukemia virus (data not shown), which were provided by David Ott and Julian Bess.
ATPS and GTPS inhibit MLV virion infectivity. Having determined that MLV particles package Hsp70 family members, the effect of ATPS and GTPS on MLV infectivity was examined. Purified MLV-GFP reporter virions pseudotyped with VSV G were treated with ATPS or GTPS and then used to infect 293T cells. GFP-positive cells were counted by flow cytometry and both ATPS and GTPS were found to inhibit MLV virion infectivity (Fig. 5). These results suggest that these two nucleotide triphosphate analogues inhibit a step of the retroviral life cycle that is common to both HIV-1 and MLV. In side-by-side comparisons, ATPS inhibition curves were performed for HIV-1 virions, VSV G-pseudotyped HIV-1 vectors, and VSV G-pseudotyped MLV virions. The 50% inhibitory concentration for HIV-1 virus and vector was 750 μM, and for MLV virus it was 250 μM.
ATPS does not inhibit HIV-1 virion membrane fusion with the plasma membrane of susceptible target cells. To determine how ATPS inhibits virion infectivity, we first determined whether ATPS treatment inhibits HIV-1 fusion with target cells. We used an enzymatic assay in which HIV-1 virions incorporate a ?-lactamase-Vpr chimeric protein and the target cells are loaded with CCF2-AM, a fluorescent, membrane-permeating ?-lactamase substrate (19). Virions were treated with ATPS and used to infect target Jurkat cells. Entry of the virions into the cells was monitored by measuring the amount of CCF2-AM cleaved by the ?-lactamase-Vpr protein. ATPS-treated virions entered target cells as efficiently as mock-treated virions (Table 1).
ATPS-treated HIV-1 virions fail to complete reverse transcription. Next we determined if ATPS inhibited viral cDNA synthesis. VSV G-pseudotyped HIV-1NL4-3/env (–) virions were treated with ATPS and used to infect Jurkat T cells at an multiplicity of infection of 1. Total cellular DNA was subjected to real-time PCR analysis with sets of oligonucleotide primers that monitor discrete steps in reverse transcription, as shown in Fig. 6. Compared to control virions, a progressive defect in reverse transcription was observed after infection with virions exposed to ATPS: minus-strand strong stop was reduced 2.5-fold, and full-length linear cDNA was reduced 30-fold (Fig. 6). The reverse transcription defect was of sufficient magnitude to fully account for the decrease in infectivity associated with ATPS treatment.
ATPS inhibits natural endogenous reverse transcription. When supplied with deoxynucleoside triphosphates and Mg2+, purified retroviral virions are capable of viral cDNA synthesis in the absence of detergent, a process that has been called natural endogenous reverse transcription (78). Since ATPS blocked HIV-1 cDNA synthesis acutely after infection in cells, we examined the effect of the drug on natural endogenous reverse transcription. HIV-1NL4-3, SIVMac239, and MLV virions were incubated with deoxynucleoside triphosphates, Mg2+, and -32P-labeled dCTP. Following natural endogenous reverse transcription, radiolabeled DNA was extracted and the cDNA products were analyzed by alkaline agarose gel electrophoresis. The presence of ATPS during natural endogenous reverse transcription significantly reduced the amount of product for all three retroviruses (Fig. 7). Thus, ATPS treatment of virions inhibits both intracellular and intravirion reverse transcription.
ATPS does not inhibit the processivity of HIV-1 reverse transcriptase. Inhibition of retroviral reverse transcription by ATPS suggested that the ATP analogue might act directly on reverse transcriptase itself. To test this possibility, virions were lysed with Triton X-100, and reverse transcriptase activity was measured with a homopolymeric poly(rA)-oligo(dT) template. Reverse transcriptase activity in this assay was not inhibited by ATPS even when it was added at a 500-fold excess over the dTTP in the assay (data not shown).
To take a more careful look at possible effects of ATPS on HIV-1 reverse transcriptase processivity, we performed a low deoxynucleoside triphosphate extension assay with recombinant HIV-1 reverse transcriptase in vitro (8). Even at ATPS concentrations high enough to inhibit virion infectivity (2 mM) the drug had minimal effects on reverse transcriptase processivity (Fig. 8). At a concentration of 5 mM, 2,500 times the concentration of deoxynucleoside triphosphates in the reaction, some interference with reverse transcriptase processivity was observed. However, this inhibition was not specific to ATPS, since at this high concentration even ATP inhibited reverse transcriptase processivity, even though it had no observable effect on virion infectivity.
ATPS disrupts the core morphology in HIV-1 virions. Aberrant core morphology is often associated with defects in reverse transcription and loss of infectivity (60, 67, 72). Since treatment of HIV-1 virions with ATPS inhibited reverse transcription and virion infectivity, we wanted to determine if this inhibition correlated with morphological changes in the virion core. Purified HIV-1 virions were treated with either ATP or ATPS, and then the virions were fixed and examined by transmission electron microscopy. Compared with ATP treatment, a significant reduction in the number of mature, conical core structures (Fig. 9A) was observed when cell-free HIV-1 virions were incubated with ATPS (Table 2). A significant increase in grossly aberrant core morphology was also observed with ATPS, including partially filled, irregular, and empty core structures (Fig. 9B to E). These data suggest that the defects in reverse transcription and infectivity that we observed with ATPS-treated virions are secondary to the morphological changes that this drug induces in the virion core.
Biochemical and functional assessment of HIV-1 cores from ATPS-treated virions. In light of the electron microscopy results, we examined the effect of ATPS on HIV-1 virion core yield and stability with a previously described method (29). Cores were purified by exposure to detergent with subsequent acceleration through a sucrose density gradient. Cores purified on the gradient were incubated at 37°C for up to 3 h. Significant increases in core yield and stability were found when virions were treated with ATPS (data not shown). However, the same effect was observed when virions were treated with ATP, our control compound that does not inhibit virion infectivity or disrupt core morphology.
HIV-1 infection of owl monkey kidney cells is restricted by TRIMCyp, a gene that encodes a fusion protein of TRIM5 and cyclophilin A (52, 63). This restriction can be saturated by HIV-1 virus-like particles (VLPs) bearing the viral protease (70). Recently, it was demonstrated that capsid mutations that disrupt core stability and integrity decrease the ability of VLPs to abrogate HIV-1 restriction in OMK cells (28). To determine if ATPS disrupts the integrity of VLP cores, OMK cells were infected with the HIV-1-GFP reporter vector and either untreated HIV-1 VLPs or VLPs treated with ATP or ATPS. Challenge of OMK cells with modest quantities of HIV-1-GFP resulted in few infected cells (Fig. 10, two top panels). As expected, coinfection with a large quantity of VLPs released HIV-1 from restriction (Fig. 10). ATP treatment had no effect on the ability of VLPs to saturate owl monkey TRIMCyp (data not shown), but ATPS-treated VLPs were completely defective at blocking HIV-1 restriction (Fig. 10). Similarly, ATPS treated N-tropic MLV VLPs were defective at abrogating MLV restriction in TE671 rhabdomyosarcoma cells (data not shown). These results confirm that retroviral core structure is altered by ATPS treatment.
DISCUSSION
Incorporation of Hsp70 family members into retroviral virions. Our results demonstrate that Hsp70 is incorporated not only into primate lentiviral virions, as previously shown (37), but probably into all retroviruses. This pattern of incorporation is quite different from what is seen with another host cell protein, cyclophilin A, which is only incorporated into HIV-1 virions (10). Hsp70 is also distinguished from cyclophilin A in that it remains stably associated with detergent-stripped HIV-1 virion cores. Cyclophilin A is recruited to virions via contacts with capsid surface residues that are unique to HIV-1 (30, 32). The contacts are quite minimal, and so cyclophilin A is easily stripped from cores (75). Though we have shown that gag is sufficient for virion incorporation of Hsp70 (37), our genetic approaches to generate Hsp70-deficient virions have been thwarted by effects on virion assembly. Hence, we do not know which viral factors mediate Hsp70's stable interaction with purified cores.
Effects of Hsp70 on virion assembly. Though conditions have been found in which purified Gag fragments multimerize into ordered structures in vitro (12-14, 33, 35, 77), the formation of higher-order structures by full Gag polyprotein seems to require factors in mammalian cell lysate that include ATP (11, 26, 45, 71, 74). These results suggest that cellular ATP-binding proteins play an essential role in HIV-1 virion assembly. Indeed, one such ATPase, HP68, associates with Gag assembly intermediates, and a fragment lacking the ATP binding motif inhibits virion release from cells (26, 80). Our finding of similar defects when ATPase-deficient Hsp70 mutants are expressed in virion producer cells (Fig. 1B) suggests that Hsp70 is also crucial for virion assembly. Additionally, ATPase-defective Hsp70 mutants interfere with vesicular trafficking between endosomal compartments and the cell surface (51). Since a significant portion of HIV-1 Gag produced in cells buds into the endosomal compartment, from which it shuttles to the plasma membrane for virion release (54, 64), the block to assembly that we observed may be due to the fact that mutant Hsp70 inhibits transport of virus-laden endosomes to the plasma membrane.
ATPS disrupts virion core morphology. ATPS inhibition of HIV-1 virion infectivity is independent of the mode of virion fusion with target cells and does not require viral accessory genes, since minimal HIV-1 vectors pseudotyped with VSV-G are as susceptible to ATPS as is full-length HIV-1. MLV virions are also inhibited by ATPS, indicating that the target of the drug is either a viral protein with a function shared by all retroviruses or a cellular factor that all retroviruses require for infectivity, perhaps Hsp70.
ATPS-treated virions fuse normally with susceptible target cells and presumably release the virion core into the host cell cytoplasm, yet the reverse transcription complex is somehow defective. ATPS does not act directly on reverse transcriptase, as determined by the in vitro processivity assay, but on another factor required for both intravirion and intracellular reverse transcription.
Avian sarcoma and leukosis virus uncoating and reverse transcription in a cell-free system require ATP hydrolysis and are inhibited by ATPS (50). In this system it is not known if ATP is required by a molecular chaperone that aids uncoating or if it is used for some other purpose, such as the phosphorylation of a viral or cellular protein. When HIV-1 virions were treated with ATPS, few conical cores were detected by electron microscopy (Fig. 9 and Table 2). In many instances it has been found that amino acid changes in HIV-1 capsid have no effect on viral assembly, but these virions fail to form proper cores and are noninfectious, with defects in reverse transcription (60, 67, 72). Thus, a properly formed HIV-1 conical core seems necessary for infectivity, and the irregular core structures that result from ATPS treatment of virions are likely to be causally related to the block in infectivity and reverse transcription.
We found no significant defect in the stability of cores from ATPS-treated virions, but noninfectious capsid mutants have been reported with hypo-, hyper-, or relatively normal core stability (29, 68). Saturation of retroviral restriction factors by VLPs requires that the Gag polyprotein be processed by the viral protease (65) and that the capsid be reasonably intact (28). Our finding that ATPS disrupts the ability of VLPs to saturate restriction factors suggests that capsid conformation is altered by this treatment.
How does ATPS inhibit HIV-1 infectivity? The morphological changes in HIV-1 cores reported here may result from effects of ATPS on the peptide binding properties of virion-associated Hsp70. While bound to ATP, Hsp70 rapidly associates with and dissociates from peptide substrates. Upon ATP hydrolysis, Hsp70 binds substrate tightly and is not released until ADP is replaced by ATP (38, 39). When bound to ATPS, the conformation of Hsp70 resembles that of Hsp70 bound to ADP (49), and it binds to peptides with an especially low dissociation rate, since ATPS cannot be hydrolyzed (34). Thus, the changes in core structure may result from the Hsp70-ATPS complex locking onto a virion core protein in a nonphysiological manner, leading to collapse of the core and defects in reverse transcription.
Alternatively, decreased infectivity from ATPS treatment might result from thiophosphorylation of a virion protein. ATPS is an excellent substrate for protein kinases. Since thiophosphorylated proteins are resistant to phosphatases, using ATPS as a kinase substrate would result in the irreversible transfer of the thiophosphate residue to proteins that are normally regulated by reversible phosphorylation (27). Thiophosphorylation of glycogen phosphorylase and myosin light chain leads to constitutive activation of these proteins, while thiophosphorylation of the epidermal growth factor receptor blocks its degradation (17, 18). At least three kinases, cyclic AMP-dependent protein kinase A, ERK-1, and ERK-2, are packaged into HIV-1 virions (15, 16). It is thus possible that irreversible thiophosphorylation of a virion component may be responsible for the defects in infectivity and the morphological changes in core structure.
ACKNOWLEDGMENTS
We thank Marianne Ljungkvist for valuable contribution to this study and Julian Bess, Heinrich Gottlinger, Warner Green, Ned Landau, David Ott, David Sayah, Elena Sokolskaja, Gilda Tachedjian, and Uta von Schwedler for reagents and technical advice.
This work was supported by National Institutes of Health grant RO1AI41857 to J.L. and core facilities of the Columbia-Rockefeller Center for AIDS Research.
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