Susceptibility of the Human Retrovirus XMRV to Antiretroviral Inhibitors

Robert A Smith; Geoffrey S Gottlieb; A Dusty Miller

Disclosures

Retrovirology. 2010;7(70) 

In This Article

Results

Comparison of HIV-1 and XMRV Drug Susceptibilities

We used a previously-described marker rescue assay[7,25] in conjunction with a Tat-inducible, β-gal-expressing HeLa cell line (MAGIC-5A)[26] to quantify the susceptibility of XMRV to antiretroviral inhibitors. Our XMRV stocks were derived from two independently-isolated strains of the virus: XMRVVP62 and XMRV22Rv1. XMRVVP62 was produced from a full-length molecular clone (pVP62) that was previously constructed by joining two overlapping cDNA fragments amplified from prostate tumor tissues.[3,11] For our experiments, high-titer XMRVVP62 stocks were generated by transfecting pVP62 into LNCaP prostate cancer cells.[11] XMRV22Rv1 was originally discovered in a prostate carcinoma cell line (22Rv1) that had been grown by xenotransplantation in nude mice.[7,27] 22Rv1 cells contain multiple integrated copies of the XMRV genome and release high titers of infectious XMRV into the culture supernatant.[7]

To generate viruses for drug susceptibility testing, HTX human fibrosarcoma cells were transduced with an MLV vector encoding HIV-1 tat (LtatSN) and were subsequently infected with either XMRVVP62 or XMRV22Rv1 (Figure 1). The resultant stocks (XMRV+LtatSN) were mixtures of native XMRV and XMRV-pseudotyped virions [LtatSN(XMRV)] in which LtatSN RNA was packaged together with XMRV Gag, Pol and Env proteins; only the LtatSN(XMRV) fraction was detected in subsequent culture steps. To quantify drug susceptibility, MAGIC-5A cultures were treated with varying concentrations of NRTIs, NNRTIs, or integrase inhibitors, and infected with XMRVVP62+LtatSN or XMRV22Rv1+LtatSN (Figure 1). Entry of XMRV occurs through the interaction of the virus with xenotropic and polytropic retrovirus receptor 1 (XPR1), which is endogenously expressed in HeLa cell lines.[28] XMRV+LtatSN infection of MAGIC-5A cells induced the expression of β-galactosidase (β-gal) via Tat-mediated transactivation of an upstream HIV-1 LTR, thereby enabling us to quantify the dose-dependent reduction of β-gal+ foci in infected indicator cell cultures. For assays of protease inhibitor (PI) susceptibility, XMRV-infected HTX/LtatSN cells were seeded in microtiter plates and immediately treated with PIs. Following a two-day incubation period, samples from the PI-treated HTX cultures were transferred to MAGIC-5A cells for FFU determination. MAGIC-5A cells also express receptors and coreceptors for HIV-1 entry (CD4, CXCR4 and CCR5; Figure 1), and thus, we were able to perform side-by-side comparisons of the drug susceptibilities of XMRV and HIV-1 in the same host cell type. In both cases, viral replication was limited to a single cycle of infection.

Figure 1.

Drug susceptibility assays for XMRV and HIV-1. For XMRV, HTX/LtatSN cells were infected (solid arrows) with XMRV22Rv1 or XMRVVP62, resulting in the release of native XMRV (gray virions) as well as XMRV-pseudotyped virions that contain LtatSN RNA (LtatSN(XMRV); blue virions). Infection of MAGIC-5A cells with XMRV+LtatSN results in transfer of the HIV-1 tat marker gene, thereby inducing β-gal expression through Tat-dependent transactivation of an upstream HIV-1 LTR promoter. β-gal+ (blue) cells are detected by staining the MAGIC-5A monolayers with X-gal (dashed arrows). Entry of XMRV into HTX/LtatSN and MAGIC-5A cells is mediated by the endogenously-expressed xenotropic and polytropic retrovirus receptor 1 (XPR1). For HIV-1, virus stocks were produced by transient transfection (dotted arrow) of 293T/17 cells with pNL4-3. As with XMRV+LtatSN, infection of MAGIC-5A cells with HIV-1NL4-3 (red virions) results in Tat expression and β-gal+ focus formation. MAGIC-5A cells were previously engineered to express the CD4 receptor and CCR5 coreceptor for HIV-1 entry; these cells also express the endogenous CXCR4 coreceptor.[26] Dashed vertical lines indicate the stages at which protease inhibitors (left) and reverse transcriptase or integrase inhibitors (right) were added to the culture supernatants.

XMRV is Susceptible to a Specific Subset of NRTIs

We initially measured the susceptibility of XMRV to each of seven different NRTIs that are FDA-approved for treating HIV-1 infection. AZT showed the most potent anti-XMRV activity of all the nucleoside analogs tested (Table 1); EC50 values for XMRVVP62+LtatSN, XMRV22Rv1+LtatSN and HIV-1NL4-3 were similar for AZT, indicating that these viruses are comparably susceptible to the analog. These results agree with a previous comparison of the AZT sensitivity of HIV-1 and XMRV using a reporter virus-based assay.[22] We also found that, relative to HIV-1NL4-3, XMRVVP62+LtatSN and XMRV22Rv1+LtatSN were fully sensitive to tenofovir (the active form of TDF), as the observed EC50 values were not significantly different between these three viruses (Table 1). In contrast, XMRV was 13-34-fold resistant to ddI, d4T and abacavir relative to HIV-1NL4-3. Higher levels of resistance were observed for 3TC and FTC, which failed to inhibit XMRV infection at doses that were 100-fold greater than the corresponding EC50s for HIV-1NL4-3.

To further characterize the nucleoside analog susceptibility of XMRV, we determined the antiviral activities of additional NRTIs that are active against HIV-1 and other retroviruses, but that are not currently approved for treating HIV-1 infection. AZddA and AZddG contain an azido group at the 3' position of the ribosyl sugar, and thus, are structurally related to AZT. AZddA and AZddG have been shown to inhibit HIV-1 replication in culture, and the 5'-triphosphate forms of these analogs inhibit the DNA polymerase activity of HIV-1 RT in cell-free assays.[29] EC50 values for the inhibition of XMRV and HIV-1 by AZddA and AZddG were comparable, although the EC50 for XMRV22Rv1+LtatSN with AZddG was fourfold greater than that of HIV-1NL4-3 (Table 1). Importantly, the concentrations of AZddA, AZddG and AZT required to inhibit XMRV infection were at least 100-fold lower than the 50% cytotoxic concentrations (CC50 values) of these analogs in HeLa-CD4 cell cultures (> 270 μM for all three inhibitors;[29]). We also measured the anti-XMRV activity of adefovir, an acyclic nucleoside phosphonate that is used in prodrug form (adefovir dipivoxil) to treat hepatitis B virus infection. EC50 measurements for the activity of adefovir against XMRVVP62+LtatSN, XMRV22Rv1+LtatSN and HIV-1NL4-3 varied by a factor of twofold or less; these differences were not statistically significant (Table 1).

Taken together, these data show that XMRV is sensitive to AZT, AZddA, AZddG, tenofovir and adefovir at doses that are comparable to those required to inhibit HIV-1 replication. At the highest concentrations of the drugs used in our assays (10 μM for AZT, 40 μM for AZddA and AZddG and 100 μM for adefovir and tenofovir), the mean numbers of cells in the fixed and stained cultures were 80-100% of untreated controls, indicating that the EC50 values obtained for these analogs were not influenced by drug-mediated cytotoxicity.

XMRV is Resistant to NNRTIs and to the Pyrophosphate Analog Foscarnet

Nevirapine, efavirenz and other NNRTIs inhibit HIV-1 RT by binding to a small hydrophobic pocket located near the polymerase active site.[30] Although wild-type strains of HIV-1 Group M are sensitive to NNRTIs, HIV type 2 (HIV-2), simian immunodeficiency virus and many Group O isolates of HIV-1 are intrinsically resistant to this drug class. Consistent with the relatively narrow spectrum of NNRTI-mediated antiviral activity, both strains of XMRV were >18-fold and >200-fold resistant to nevirapine and efavirenz, respectively, relative to HIV-1NL4-3 (Table 1). In contrast, the pyrophosphate analog foscarnet (PFA) is active against many DNA viruses and retroviruses including HIV-1 and -2, Rauscher MLV, Moloney MLV, hepatitis B virus, cytomegalovirus and herpes simplex virus.[31] Despite this broad spectrum of antiviral activity, XMRVVP62+LtatSN and XMRV22Rv1+LtatSN were resistant to PFA (Table 1). Concentrations of PFA as high as 400 μM had no effect on XMRV infection; increasing the drug level to 900 μM produced visible cytotoxic effects in MAGIC-5A indicator cell cultures (data not shown).

XMRV is Intrinsically Resistant to PIs but is Sensitive to Integrase Inhibitors

To identify antivirals that inhibit XMRV targets other than RT, we assessed the ability of nine different HIV-1 PIs to block the production of newly-formed, infectious XMRVVP62+LtatSN in chronically-infected HTX cultures. In these experiments, we screened each PI for anti-XMRV activity using a single drug concentration that was approximately equal to the EC95 for HIV-1NL4-3, as determined in our concurrent studies of HIV-1 and HIV-2 (range = 0.1–1 μM; see Methods section for details). As seen in our previous assays, these PI doses reduced the infectious titer of HIV-1NL4-3 in pNL4-3-transfected 293T/17 cultures by 94% or greater, relative to untreated controls (Figure 2). In contrast, each of the nine PI treatments had no detectable effect on the infectious titer of XMRVVP62+LtatSN, indicating that XMRVVP62 is intrinsically resistant to this inhibitor class. These results are consistent with a recent report showing that XMRV is relatively insensitive to PIs (EC50 values ≥34 μM) in cultures of immortalized human breast cancer cells.[23]

Figure 2.

Intrinsic resistance of XMRV to protease inhibitors (PIs). For XMRVVP62+LtatSN (shaded bars), HTX/LtatSN cells were infected with virus derived from the pVP62 clone, seeded into microtiter plates, and immediately treated with the indicated doses of PIs. For HIV-1NL4-3 (solid bars), 293T/17 cells were seeded into microtiter plates, transfected with plasmid DNA encoding the full-length NL4-3 molecular clone, and treated with the indicated concentrations of each PI. The same PI stocks were used to treat both sets of virus-producing cultures. Supernatants from PI-treated HTX and 293T/17 cultures were then diluted and plated onto MAGIC-5A indicator cells to quantify infectious particles. Bars represent the percentage of β-gal+ FFU in supernatants from the PI-treated cultures, relative to untreated controls, and are the means ± standard deviations from two independent experiments with two or more determinations of FFU per drug treatment per experiment. See List of Abbreviations for drug names.

We also examined the susceptibility of XMRV to two different inhibitors of HIV-1 integrase strand-transfer activity: raltegravir and elvitegravir. Of the 24 antiretroviral drugs tested in our analysis, raltegravir was the most potent inhibitor of XMRV infection. XMRV and HIV-1 exhibited comparable sensitivity to raltegravir, as the EC50 values for XMRVVP62+LtatSN and XMRV22Rv1+LtatSN were similar to that of HIV-1NL4-3 (Table 2). Elvitegravir also inhibited XMRV infection in our indicator cell assays, but higher doses of the drug were required to observe this activity. EC50 measurements for XMRVVP62+LtatSN and XMRV22Rv1+LtatSN were 71- and 40-fold greater for elvitegravir relative to raltegravir and 79- and 46-fold higher than the EC50 for elvitegravir-mediated inhibition of HIV-1NL4-3, respectively (Table 2). Although these data show that elvitegravir is less potent than raltegravir against XMRV, we note that elvitegravir inhibited the virus at concentrations in the nanomolar range, and thus, was comparable to AZT with respect to anti-XMRV activity (Tables 1 and 2). For both raltegravir and elvitegravir, no statistically-significant declines in mean target cell number were observed at the highest doses of drugs tested (10 μM; p > 0.05, Student's two-sided t-test). This result agrees with previously-published CC50 values for raltegravir and elvitegravir in PBMC (> 100 μM and 40 μM, respectively;[23,32]) and excludes cytotoxicity as a potential confounder in our measurements of integrase inhibitor susceptibility.

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