Susceptibility of the Human Retrovirus XMRV to Antiretroviral Inhibitors

Robert A Smith; Geoffrey S Gottlieb; A Dusty Miller


Retrovirology. 2010;7(70) 

In This Article



AZT (generic name: zidovudine; 3'-azido-3'-deoxythymidine), ddI (didanosine; 2',3'-dideoxyinosine), D4T (stavudine; 2',3'-didehydro-3'-deoxythymidine) and PFA (foscarnet; phosphonoformic acid) were obtained commercially (Sigma-Aldrich), as were adefovir ((R)-9-(2-phosphonylmethoxyethyl)adenine), tenofovir ((R)-9-(2-phosphonylmethoxypropyl)adenine) and abacavir ((1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol) (Moravek Biochemicals), AZddA (3'-azido-2',3'-dideoxyadeonsine) and AZddG (3'-azido-2',3'-dideoxyguanosine) (Berry and Associates), and elvitegravir (Selleck Chemicals). Nevirapine and efavirenz were a gift from Koronis Pharmaceuticals (Seattle, Washington). 3TC (lamivudine; (-)-β-L-2',3'-dideoxy-3'-thiacytidine) and FTC (emtricitabine; (-)-β-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine) were kindly provided by Raymond Schinazi (Emory University) or were purchased from Moravek. All HIV-1 PIs used in this study, as well as the integrase inhibitor raltegravir, were obtained from the National Institutes of Health AIDS Reference Reagent Program.

Cell Culture and Virus Production

HTX cells are a pseudodiploid subclone of HT-1080 human fibrosarcoma cells.[52] The LtatSN vector was created by inserting the tat coding region of HIV strain SF2 into the retroviral expression vector LXSN.[53] HTX/LtatSN cells were generated by infecting HTX cells with helper-virus free LtatSN virus that was produced in PA317 amphotropic packaging cells[54] and then treating the cells with G418 (geneticin) to select for the presence of the vector. 22Rv1 cells[27] and 293T/17 cells[55] were obtained from the American Type Culture Collection. MAGIC-5A indicator cells (CD4+/CCR5+ HeLa cells that express β-galactosidase (β-gal) under the control of an HIV-1 LTR promoter)[26] were a kind gift from Dr. Michael Emerman (Fred Hutchinson Cancer Research Center). Cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum.

XMRV-pseudotyped LtatSN virus (XMRV+LtatSN) was generated by infecting HTX/LtatSN cells with virus produced from the VP62 molecular clone of XMRV (a kind gift from Robert Silverman, Cleveland Clinic)[11] or with virus harvested from XMRV-infected 22Rv1 cells.[7] HIV-1NL4-3 was produced using the full-length pNL4-3 HIV-1 plasmid molecular clone.[56] Plasmid DNA was isolated from pNL4-3-transformed E. coli JM109 using an Endo-Free™ maxiprep kit (Qiagen) and introduced into cultured 293T/17 cells via chloroquine-mediated transfection as previously described.[57] XMRVVP62+LtatSN, XMRV22Rv1+LtatSN and HIV-1NL4-3 stocks were harvested from confluent monolayers of producer cells, passed through 0.45-micron filters (XMRV+LtatSN) or centrifuged at 500 × g for 10 min at room temperature (HIV-1NL4-3) to remove host cells, and frozen in multiple aliquots at -70°C. Titers of the resultant stocks were 7.3 × 105, 1.2 × 105, and 3.0 × 106 MAGIC-5A focus forming units (FFU)/ml for XMRVVP62+LtatSN, XMRV22Rv1+LtatSN and HIV-1NL4-3, respectively.

Drug Susceptibility Assays-RT and Integrase Inhibitors

To compare the susceptibilities of XMRV and HIV-1 to NRTIs, NNRTIs and PFA, MAGIC-5A cells were seeded into 48-well plates at 1.5 × 104 cells/well. After 20-22 h of incubation, the cultures were dosed with varying drug concentrations and returned to the incubator for an additional 2.5 h. Immediately before infection, virus stocks were diluted to 3,000 FFU/ml in complete DMEM supplemented with 20 μg/ml diethylaminoethyl (DEAE) dextran. Supernatants from the drug-treated MAGIC-5A cultures were then aspirated and replaced with 100 μl of each diluted virus stock/well. To maintain drug pressure, a second dose of inhibitor was added to the inocula (at the same concentration as the first dose), and the plates were returned to the incubator for 2.5 h. After this time, an additional 300 μl of complete DMEM was added, a third dose of drug was added, and incubation was continued for 40 h. Individual dose-response experiments for each virus strain involved 2-3 solvent-only control cultures plus 2-3 cultures for each of seven different drug concentrations.

To score β-gal-positive (β-gal+) foci, 100 μl of fixative solution [1% formaldehyde, 0.2% glutaraldehyde in 1× phosphate-buffered saline (PBS)] was added to each culture well, and the plates were incubated at 37°C for 10 min. After washing the fixed monolayers twice with 100 μl of PBS, 100 μl of staining solution [4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl2 and 0.4 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) in PBS] was added to each well, and the plates were placed in the incubator for 1 h. The cultures were then aspirated to remove the X-gal staining solution, rinsed with 100 μl of PBS per well, aspirated again and stored in 200 μl of PBS per well. Foci (individual β-gal+ cells plus groups of 2-8 contiguous β-gal+ cells) were counted using a CTL Immunospot Analyzer (Cellular Technology Ltd.) or were manually counted by light microscopy. Untreated control cultures typically contained 200-500 foci per well.

To quantify viral susceptibility to integrase inhibitors, we adopted our MAGIC-5A-based assay to a 96-well format and used an expanded range of drug concentrations. These changes were necessitated by the shallow slopes observed in dose-response plots with raltegravir and elvitegravir relative to inhibitors from other drug classes.[58] Culture conditions and times of drug addition were identical to those used for the RT inhibitor assays, except that each culture well was seeded with 5 × 103 MAGIC-5A cells in 100 μl of medium, was infected with 200 FFU of virus in 50 μl of dextran-containing medium, and received an additional 150 μl of complete medium following the 2.5 h incubation period. Fixing and X-gal-staining steps were performed with one half of the volumes of solutions used in RT inhibitor assays, and β-gal+ foci were counted using the CTL Immunospot Analyzer.

Drug concentrations that inhibited focus formation by 50% (EC50 values) were calculated from dose-response plots by sigmoidal regression analysis (GraphPad Software). EC50 measurements for HIV-1NL4-3 were comparable to the values obtained in other single-cycle drug sensitivity assays.[26,59,60]

Potential drug-mediated cytotoxicity was assessed by comparing the number of cells in untreated control cultures to those in cultures that received the maximal dosage of drug used in our assays. Fixed cells were stained by exposing the MAGIC-5A monolayers to 10 μg/ml ethidium bromide in PBS for 5 min, then de-staining for 5 min in deionized water. Cell nuclei were visualized by fluorescence microscopy using a Texas red filter set (560 nm excitation, 645 nm emission). Images were acquired from 3-4 culture wells for each drug treatment and corresponding no-drug controls, and nuclei were enumerated using ImageJ software.[61]

Drug Susceptibility Assays-protease Inhibitors

To measure PI susceptibility, cultured cells that were producing either HIV-1 or XMRV were treated with varying doses of PIs, and the numbers of infectious virions released by each drug-treated or no-drug control culture were quantified in MAGIC-5A indicator cells. For HIV-1NL4-3, 293T/17 cells grown in 75 cm2 flasks were digested with trypsin, seeded into 48-well plates at 6 × 104 cells/well, and placed in an incubator. The following day (20-24 h), CaPO4-DNA co-precipitates were prepared by mixing 5 μg of HIV-1NL4-3 plasmid DNA with 900 μl of 0.2 M CaCl2, adding the solution dropwise with mixing into 900 μl of 2× Hepes-buffered saline, and then incubating the suspension at room temperature for 10 min. During this time, chloroquine was added to each 293T/17 culture well to a final concentration of 50 μM. Co-precipitate suspensions were then mixed by pipetting and added directly to the chloroquine-treated cultures (20 μl/well), and the plates were placed in the incubator for 10-12 h. Following this incubation period, the supernatants were aspirated and replaced with 400 μl of fresh medium per well, and PIs were added to the culture wells. The plates were then returned to the incubator for 30-35 h. Supernatants (20 μl) from the transfected 293T/17 cultures were removed without disturbing the cell monolayer and diluted 1:10, 1:100 and 1:1,000 in complete medium supplemented with 20 μg/ml DEAE dextran. Infectious titers in the diluted supernatants were measured in MAGIC-5A cells as described above, except that inhibitors were omitted from this phase of the assay.

For PI susceptibility assays with XMRV, HTX/LtatSN cells that were infected with XMRVVP62 were trypsinized, rinsed twice with 1× PBS, resuspended in complete medium and seeded into 48-well plates at approximately 5 × 104 cells/well. The cultures were then immediately treated with PIs as described above for HIV-1NL4-3. Following a 40-h incubation period, 180 μl of culture supernatant was harvested from each well, and DEAE-dextran was added to the samples to a final concentration of 20 μg/ml. The supernatants were diluted 1:4 and 1:16 in medium containing 20 μg/ml DEAE dextran, and 100 μl each of the undiluted, 1:4- and 1:16-diluted samples were transferred to MAGIC-5A cultures for FFU determination as described above.


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