Impaired Infectivity of Ritonavir-resistant HIV Is Rescued by Heat Shock Protein 90AB1

Pheroze Joshi,Cheryl A Stoddart

doi:10.1074/jbc.m111.248021

Pheroze Joshi, Cheryl A Stoddart

Open Access

https://doi.org/10.1074/jbc.m111.248021

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Abstract

Certain ritonavir resistance mutations impair HIV infectivity through incomplete Gag processing by the mutant viral protease. Analysis of the mutant virus phenotype indicates that accumulation of capsid-spacer peptide 1 precursor protein in virus particles impairs HIV infectivity and that the protease mutant virus is arrested during the early postentry stage of HIV infection before proviral DNA synthesis. However, activation of the target cell can rescue this defect, implying that specific host factors expressed in activated cells can compensate for the defect in ritonavir-resistant HIV. This ability to rescue impaired HIV replication presented a unique opportunity to identify host factors involved in postentry HIV replication, and we designed a functional genetic screen so that expression of a given host factor extracted from activated T cells would lead directly to its discovery by rescuing mutant virus replication in nonactivated T cells. We identified the cellular heat shock protein 90 kDa α (cytosolic), class B member 1 (HSP90AB1) as a host factor that can rescue impaired replication of ritonavir-resistant HIV. Moreover, we show that pharmacologic inhibition of HSP90AB1 with 17-(allylamino)-17-demethoxygeldanamycin (tanespimycin) has potent in vitro anti-HIV activity and that ritonavir-resistant HIV is hypersensitive to the drug. These results suggest a possible role for HSP90AB1 in postentry HIV replication and may provide an attractive target for therapeutic intervention.

Highlights

Ysis of Gag and studies with the HIV maturation inhibitor bevirimat [2] indicate that uncleaved CA-SP1 by itself is sufficient to impair viral replication
Our results suggest that the unprocessed Gag molecules disrupt CA maturation and that cellular activation is the intrinsic difference between thymocytes and mitogen-activated T cells in determining the replication capacity of RTV-resistant HIV
Cleavage of the MA and p6 proteins was not affected by the RTV-resistant HIV PR; a significant reduction in cleavage at the CA-SP1 and NC-SP2 junctions was observed in the presence of both PR mutations (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Cells and Viruses—293T and P4-Magi cell lines were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone), and Jurkat E6-1, Jurkat E6-1 CypAϪ/Ϫ, CEM T4, and Sup T1 were cultured at 37 °C in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS. Endogenous Reverse Transcriptase Assay and Total Viral RNA Content—WT HIV and HIV PRI54V/V82A virus (500 TCID50) were pretreated with DNase I and 10 mM MgCl2 for 1 h to remove contaminating plasmid DNA and concentrated as described above. Production and Titration of Retroviral cDNA Expression Library—To generate a library of transducing viruses, 20 ␮g of the pFB cDNA expression plasmid was transiently transfected into the AmphoPack-293 packaging cell line (Clontech) using Lipofectamine 2000 (Invitrogen). Because the pFB transducing vector does not carry a reporter, the titer of the retroviral cDNA expression library was determined by a onestep quantitative RT-PCR using a calibrated RNA standard curve generated with the pFB-GFP reference virus. Jurkat E6-1 cells were transduced at an m.o.i. of 0.1 with individual cDNA-expressing retroviral particles and inoculated with the HIV PRI54V/V82A-RFP reporter virus as described above. Inhibition of viral replication and cellular toxicity were assayed after 7 days, and results represent the mean of three independent assays

RESULTS

Viral RNA content

DISCUSSION