ATPgammaS disrupts human immunodeficiency virus type 1 virion core integrity

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 ATPgammaS and GTPgammaS, nucleotide triphosphate analogues resistant to Hsp70 hydrolysis, dramatically reduced the infectivity of HIV-1 and murine leukemia virus virions. ATPgammaS-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 ATPgammaS. The effects of ATPgammaS 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, ATPgammaS-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.

FIG. 1.

Association of Hsp70 with HIV-1 cores and inhibition of HIV-1 assembly by an Hsp70 mutant with defective ATPase activity. (A) Purified HIV-1 virions or cores prepared from these virions were analyzed by immunoblotting with antibodies against Hsp70 and the indicated viral proteins. Viral genomic RNA was detected by dot blot with a ³²P-end-labeled DNA oligonucleotide probe (bottom panel). (B) 293T cells were transfected with the indicated quantities of pNL4-3 and either wild-type Hsp70 or mutant Hsp70(K71E) expression plasmids. Virion assembly was monitored by measuring reverse transcriptase activity in the culture supernatant 2 days posttransfection. Each transfection was performed in triplicate.

FIG. 2.

ATPγS inhibits HIV-1 virion infectivity. Virions treated with ATP or ATPγS for 6 h at 37°C were used to infect cells bearing a long terminal repeat-GFP reporter. GFP expression was analyzed by flow cytometry 2 days postinfection. Darker dots indicate GFP-positive cells. To demonstrate that the GFP signal resulted from de novo infection, the reverse transcriptase inhibitor lamivudine was added to the cells 2 h before infection, as indicated.

FIG. 3.

Inhibition of HIV-1 virion infectivity by ATPγS is independent of multiplicity of infection, env, and most viral accessory genes. VSV-G-pseudotyped HIV-1-GFP vector bearing inactivating mutations in vif, vpr, vpu, and nef was treated with 2 mM ATPγS and used to infect 293T cells over a range of multiplicities of infection. Cells were fixed 2 days later and analyzed by flow cytometry for GFP expression.

FIG. 4.

Incorporation of Hsp70 family members into MLV virions. MLV virions were harvested from the supernatant of transfected 293T cells (left panel) or chronically infected Rat-2 cells (right panel). Immunoblot analysis was performed on the cell lysates and purified virions with antibodies against Hsp70 or Hsc70. The same amount of MLV virions was used from both cell lines for immunoblot analysis, as determined by Coomassie blue staining of the viral capsid protein.

FIG. 5.

ATPγS and GTPγS inhibit MLV virion infectivity. VSV-G-pseudotyped MLV_NCA/IRES-GFP GFP reporter virions were treated with ATPγS or GTPγS and used to infect 293T cells. Two days postinfection, cells were analyzed for GFP expression by flow cytometry. Darker dots indicate GFP-positive cells.

FIG. 6.

ATPγS-treated HIV-1 virions fail to complete reverse transcription acutely after infection. VSV-G-pseudotyped HIV-1_{NL4-3/env(−)} virions treated with ATPγS were used to infect Jurkat T cells. As a control, the HIV-1 reverse transcriptase inhibitor lamivudine (3TC) was added to cells 2 h prior to infection. Twenty-four hours postinfection, total cellular DNA was isolated and viral cDNA was quantitated with real-time PCR and primers specific for the indicated products.

FIG. 7.

ATPγS inhibits retroviral natural endogenous reverse transcription. Purified HIV-1_NL4-3, SIV_MAC239, and MLV_NCA virions were incubated in natural endogenous reverse transcription buffer with MgCl₂, [α-³²P]dCTP, and unlabeled deoxynucleoside triphosphates in the presence of 5 mM ATPγS. Radiolabeled DNA was extracted from the virions and analyzed by alkaline agarose gel electrophoresis. The migration positions of size markers (in kilobases) is indicated on the left of the panels.

FIG. 8.

ATPγS does not inhibit the processivity of HIV-1 reverse transcriptase. With the low deoxynucleoside triphosphate extension assay, HIV-1 reverse transcriptase's ability to extend a radiolabeled primer on single-stranded M13mp18 DNA was assessed with low concentrations of all four deoxynucleoside triphosphates and increasing concentrations of ATPγS. ATP was used as a control. The migration of size markers (in base pairs) is shown on the left of the panel.

FIG. 9.

Electron micrographs showing thin sections of virions treated with ATP (A) or ATPγS (B to E). After treatment with nucleotides, virions were fixed with gluteraldehyde and mixed with uninfected 293T cells to provide contrast for electron microscopy staining.

FIG. 10.

ATPγS-treated HIV-1 VLPs are defective at abrogating HIV-1 restriction by owl monkey TRIM-Cyp. Owl monkey kidney cells were infected with VSV-G-pseudotyped HIV-1-GFP vector. The bottom left panel shows infection in the presence of mock-treated HIV-1 VLPs. The bottom right panel shows infection in the presence of HIV-1 VLPs treated with 5 mM ATPγS. At 48 h postinfection, the number of GFP-positive cells was determined by flow cytometry. Darker dots indicate GFP-positive cells.