Basic Science: Concise Communications
Targeting host nucleotide biosynthesis with resveratrol inhibits emtricitabine-resistant HIV-1
Heredia, Alonsoa; Davis, Charlesa; Amin, Mohammed N.a; Le, Nhut M.a; Wainberg, Mark A.b; Oliveira, Maureenb; Deeks, Steven G.c; Wang, Lai-Xia; Redfield, Robert R.a
aInstitute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA
bMcGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
cSan Francisco VA Medical Center and University of California, San Francisco, California, USA.
Correspondence to Alonso Heredia, Institute of Human Virology, 725 W. Lombard St, Baltimore, MD 21201, USA. Tel: +1 410 7064594; fax: +1 410 7064992; e-mail: email@example.com
Received 15 August, 2013
Revised 27 November, 2013
Accepted 27 November, 2013
Objective: The M184V mutation in the HIV-1 reverse transcriptase gene is frequent (>50%) in patients, both in resource-rich and resource-limited countries, conferring high-level resistance (>100-fold) to the cytosine analog reverse transcriptase inhibitors lamivudine and emtricitabine. The reverse transcriptase enzyme of M184V HIV-1 mutants has reduced processivity, resulting in reduced viral replication, particularly at low deoxynucleotide (dNTP) levels. We hypothesized that lowering intracellular dNTPs with resveratrol, a dietary supplement, could interfere with replication of M184V HIV-1 mutants.
Design and methods: Evaluation of the activity of resveratrol on infection of primary peripheral blood lymphocytes by wild-type and M184V mutant HIV-1. We assayed both molecular clones and primary isolates of HIV-1, containing M184V alone and in combination with other reverse transcriptase mutations. Viral infection was quantified by p24 ELISA and by quantitative real-time PCR analysis. Cell viability was measured by colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays.
Results: In virus-infectivity assays, resveratrol did not inhibit replication of wild-type NL4-3 (resveratrol EC50 > 10 μmol/l), but it inhibited NL4-3 184V mutant (resveratrol EC50 = 5.8 μmol/l). These results were confirmed by real-time PCR analysis of early and late products of reverse transcription. Resveratrol inhibited molecular clones and primary isolates carrying M184V, alone or in combination with other reverse transcriptase mutations (resveratrol EC50 values ranging from 2.5 to 7.7 μmol/l).
Conclusions: Resveratrol inhibits HIV-1 strains carrying the M184V mutation in reverse transcriptase. We propose resveratrol as a potential adjuvant in HIV-1 therapy, particularly in resource-limited settings, to help control emtricitabine-resistant M184V HIV-1mutants.
Thanks to currently available antiretrovirals, HIV suppression is achieved in many patients. However, some patients still fail treatment because of drug resistance [1,2]. Treatment failure is particularly worrisome in resource-limited regions, where the prevalence of drug mutations has increased over time and resistance testing is not readily available [3–6]. Targeting cellular ribonucleotide reductase, which catalyzes synthesis of dNTPs, with hydroxyurea enhances the anti-HIV activities of nucleoside analog reverse transcriptase inhibitors (NRTIs), including drug-resistant strains [7–9]. However, potential toxicity has limited the use of hydroxyurea in HIV-1 patients.
Resveratrol (3,5,4’-trihydroxystilbene; Fig. 1a) is a natural ingredient in certain plants and plant products that inhibits ribonucleoside reductase . Resveratrol also arrests the cell cycle at the S/G2 transition, prolonging the S phase and increasing nuclear nucleoside utilization . We have previously reported that resveratrol, which lacks activity against wild-type HIV-1 or against HIV-1 carrying thymidine or adenosine analog resistance mutations, enhances the antiviral activity of NRTIs . Resveratrol antiviral enhancement is highest (up to 10-fold) with the adenosine analogs didanosine (DDI) and tenofovir (TDF), consistent with resveratrol preferential depletion of deoxyadenosine-5’-triphosphate (dATP), natural competitor of DDI and TDF . Importantly, resveratrol restores the TDF sensitivity of TDF-resistant HIV-1 .
The cytosine analogs lamivudine (3TC) and emtricitabine (FTC) select for the M184V mutation, the most prevalent NRTI-associated mutation in treated patients [15,16]. M184V confers high-level drug resistance (100−1000-fold) through steric hindrance between the beta-branched side chain of Val and the oxathiolane ring of the drug . However, the gain of function in the mutated M184V reverse transcriptase enzyme is offset by a decreased processivity (i.e. average number of nucleotides incorporated each time reverse transcriptase engages a primer) and thereby reduced replicative capacity of the mutant virus . Decreased processivity of the mutant reverse transcriptase is further reduced at low dNTP levels [19,20]. Thus, we tested the hypothesis that reduction of dNTP levels with resveratrol might impair the replication of HIV-1 carrying the M184V mutation. Our data demonstrate that resveratrol by itself has activity against viruses with the M184V mutation, unlike what has been shown with wild-type HIV-1 or with HIV-1 carrying thymidine/adenosine analog resistance mutations [12,14].
Viruses and drugs
The HIV-1 molecular clone NL4-3 and its derivatives carrying reverse transcriptase sequences amplified from the plasma of patients with drug-resistant HIV-1 were obtained from Dr Robert Shafer (Stanford University School of Medicine, Stanford, California, USA) through the National Institutes of Health (NIH) AIDS Repository (Germantown, Maryland, USA). Molecular clone NL4-3184I and isolates 4742, BG05 and BG15were provided by Dr Mark Wainberg [21,22]. Multidrug-resistant isolates were provided by Dr Steven Deeks. FTC was from the NIH AIDS Repository and resveratrol (trans form) from Sigma (St Louis, Missouri, USA).
Cell infectivity and viability assays
Peripheral blood lymphocytes (PBLs) from healthy donors were infected as described . On day 7 after infection, HIV-1 p24 antigen production in the culture supernatant was assayed by ELISA (Coulter, Hialeah, Florida, USA). Cell viability was measured by the MTT kit (Roche Applied Science, Mannheim, Germany), following the manufacturer's directions.
DNA was isolated from HIV-1-infected cells using Miniblood kit (Qiagen, Germantown, Maryland, USA). Real-time PCR amplification was performed with a Quantitect SYBR Green PCR Kit (Qiagen) and primer pairs specific for early (R/U5) and late (R/gag region) transcripts, as described .
Resveratrol inhibits emtricitabine-resistant HIV-1 carrying the M184V mutation
We evaluated the activity of resveratrol against wild-type NL4-3 and mutant NL4-3/184V infectious molecular clones in PBLs. As expected, in the absence of resveratrol, 10 μmol/l FTC completely inhibited wild-type NL4-3, but not NL4-3/184V (Fig. 1b). Also as expected, resveratrol treatment alone did not have activity against wild-type NL4-3. In contrast, resveratrol alone inhibited NL4-3/184V by approximately 80% (Fig. 1b). Resveratrol inhibition of NL4-3/184V was slightly increased by FTC. We confirmed the resveratrol activity against NL4-3/184V in PBLs by real-time PCR (Fig. 1c). At 72 h after infection, resveratrol did not inhibit DNA synthesis of wild-type HIV-1 (as expected), but 5 μmol/l resveratrol markedly (>10-fold) and 10 μmol/l resveratrol completely inhibited DNA synthesis of NL4-3/184V (both R-U5 and R-gag transcripts). These data show that resveratrol has antiviral activity against NL4-3/184V. The PCR data indicate that inhibition occurs at an early step in the virus life cycle (at or before DNA synthesis), which is consistent with the dNTP-depleting activity of resveratrol. That resveratrol inhibits mutant 184V, but not wild-type HIV, suggests that resveratrol depletion of dNTPs is sufficient to reduce the enzymatic activity of the former, but not of the latter, reverse transcriptase, in agreement with reduced processivity of 184V mutant reverse transcriptase enzymes at low dNTPs [18–20]. Consistent with these data, resveratrol also inhibited 3TC/FTC-resistant NL4-3/184I (Fig. 1d). The M184I mutation generally precedes M184V in patients and, similar to M184V, it also reduces reverse transcriptase processivity at low dNTPs [18,20]. Next, we evaluated NL4-3/184V inhibition by hydroxyurea, which inhibits HIV by decreasing dNTPs [7,23,24]. Hydroxyurea concentrations of up to 100 μmol/l did not affect PBL viability (not shown); yet, it had a dose–response activity against mutant, but not wild type, inhibiting NL4-3/184V by up to approximately 60% (Fig. 1e). Together, these data support the idea that decreases in dNTPs account for resveratrol inhibition of HIV carrying the 184V mutation.
We next evaluated resveratrol against HIV-1 primary isolate pairs, with and without the M184V mutation as the sole mutation in reverse transcriptase, derived from the same patient (4742, BG05 and BG15) . We chose viruses with M184V as single mutation to avoid confounding of the data by additional mutations that might compensate for the viral growth disadvantage conferred by M184V. In each wild type–M184V virus pair, resveratrol inhibited M184V mutant (EC50 values ranging between 2.5 and 6.7 μmol/l), but not wild type (EC50 > 10 μmol/l) (Table 1). These results demonstrate that resveratrol has antiviral activity against viruses with the M184V mutation as the sole mutation in reverse transcriptase.
Resveratrol inhibits multidrug-resistant viruses carrying the M184V mutation
The M184V mutation is frequently observed in conjunction with reverse transcriptase mutations conferring resistance to other NRTIs. We tested the activity of resveratrol against NL4-3 clones containing the reverse transcriptase region amplified from plasma of patients with multidrug resistance (Table 1). Resveratrol failed to inhibit drug-resistant NL4-3 clones lacking the M184V mutation, but inhibited all drug-resistant clones containing M184V (EC50 values ranging between 2.7 and 5.8 μmol/l). To further confirm inhibition of resveratrol against multidrug-resistant HIV-1, we evaluated resveratrol against isolates from patients with multidrug resistance (isolates 3212, 6061 and 6017). Similar to the data with NL4-3 molecular clones, resveratrol inhibited the multidrug-resistant isolates with the M184V mutation, but not those lacking it. Collectively, the data demonstrate antiviral activity of resveratrol against HIV-1 carrying the M184V mutation, alone or in combination with other mutations in reverse transcriptase.
The dietary supplement resveratrol modulates cell proliferation by inhibiting ribonucleotide reductase, preferentially depleting dATP and prolonging the cellular S phase [10,11,13]. Resveratrol inhibition of cell proliferation is more potent in cancer cells (EC50 < 10 μmol/l) than in normal cells or PBLs (EC50 > 10 μmol/l) [25,26]. We have previously demonstrated that resveratrol enhances the antiviral activity of NRTIs [12,14]. Clouser et al. have shown that resveratrol and nucleoside analog decitabine are synergistic against HIV-1.
We now demonstrate that treatment with resveratrol alone is sufficient to inhibit HIV-1 mutants with the M184V mutation, present in more than 50% of treated patients . Resveratrol inhibited viruses carrying M184V, singly or in combination with other mutations. Mechanistically, resveratrol inhibited synthesis of HIV184V DNA products, presumably by lowering dNTPs and thereby decreasing processivity of the HIV184V reverse transcriptase enzyme. This contention is supported by the following observations. First, mutant reverse transcriptase enzymes carrying the M184V mutation have reduced processivity at low dNTP levels [18–20]. Second, resveratrol maximally inhibited M184V mutants at 5–10 μmol/l, concentrations at which resveratrol depletion of dNTPs is highest . Third, mutant M184I, which also has reduced processivity at low dNTPs [18,20], was similarly inhibited by resveratrol. However, one limitation of our study is that we did not measure intracellular levels of dNTPs in resveratrol-treated cells, precluding more definitive conclusions about mechanisms.
The antiviral effect of resveratrol against multidrug-resistant HIV-1 carrying M184V, but not against mutants without it, might be explained by the relatively higher impact of M184V on reverse transcriptase processivity. The M184V mutation is associated with a several-fold higher reduction in viral fitness, which is determined in part by processivity [18,28], compared to other nucleoside analog mutations .
Strains of HIV-1 carrying the M184V mutation in reverse transcriptase display a replication disadvantage that has clinical benefit . Our data suggest that resveratrol could be used as an adjuvant in the treatment of HIV-1, helping to control replication of drug-resistant M184V mutants. Control of M184V mutants with resveratrol could help increase the overall potency of drug regimens and subsequent immune recovery; increase the antiviral effect of antiretrovirals, other than FTC and 3TC, whose activity against multidrug-resistant viruses carrying M184V is reduced, such as abacavir [31,32]; and reduce the likelihood of mutant M184V HIV-1 transmissions in the population. Because resveratrol targets a cellular (rather than viral) protein, resistance may not easily occur. In agreement, HIV-1 failed to develop resistance to resveratrol after 13 serial passages in PBLs . Although other ribonucleotide reductase inhibitors might also help control M184V HIV-1 mutants in patients [7–9,24,33,34], resveratrol may provide a better option. First, resveratrol lacks significant toxicity in humans [35,36]. Second, resveratrol might be unique in that it decreases oxidative stress induced by thymidine analogs , and reduces protease inhibitor toxicity . Future studies comparing different ribonucleotide reductase inhibitors will be needed to identify the most potent and safest in HIV-1 patients.
One caveat with resveratrol use is low bioavailability, with rapid metabolism into glucuronides and sulfates [39,40]. In humans, daily oral administration of 5 g resveratrol gives plasma Cmax of approximately 4–5 μmol/l [41,42]. These resveratrol levels may not be sufficient to explain the in-vivo anticancer activity reported previously [35,36], or the in-vivo anti-HIV activity predicted by our studies. It is possible that resveratrol metabolites (mainly glucuronides and sulfates) may be converted back to active resveratrol inside of cells [39,40]. Indeed, a recent study demonstrates that sulfate metabolites, which reach Cmax of approximately 20–30 μmol/l in plasma following oral doses of 5 g resveratrol, enter colon cancer cells, where they regenerate parent resveratrol at biologically active levels . We have previously reported that resveratrol glucuronides lacked anti-HIV activity . We are currently evaluating the anti-HIV activity of resveratrol and resveratrol sulfates (3-O-sulfate and 4’-O-sulfate) in humanized mice. Alternatively, resveratrol bioavailability might be improved using encapsulation systems  or other delivery methods currently in development .
In summary, adjuvant treatment with resveratrol may increase antiretroviral treatment success in patients by helping control replication of highly prevalent FTC-resistant M184V HIV-1 mutants.
Authors contributions: A.H., C.D., M.A., and R.R.R. designed the experiments; A.H., M.A., M.O., and N.M.L. performed the experiments; A.H., L.W., M.A. S.G.D., M.A.W., and R.R.R. contributed to data analysis, discussion of results, and writing of the manuscript.
We thank Dr Rafael de Cabo (Laboratory of Experimental Gerontology, National Institute on Aging, NIH) for helpful suggestions in the use of resveratrol.
Sources of support: This work was supported in part by research funds from the Institute of Human Virology. HIV-1 primary isolates were from patients in the SCOPE cohort, which was supported by the NIAID (K24AI069994), the UCSF/Gladstone Institute of Virology & Immunology CFAR (P30 AI027763), the UCSF Clinical and Translational Research Institute Clinical Research Center (UL1 RR024131), the Center for AIDS Prevention Studies (P30 MH62246), and the CFAR Network of Integrated Systems (R24 AI067039).
Conflicts of interest
The authors report no conflict of interest.
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antiretroviral therapy; cytosine analogs; drug resistance; emtricitabine; HIV-1; M184V mutation; nucleoside analog reverse transcriptase inhibitors; resource-limited setting
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