Sensitivity of V75I HIV-1 reverse transcriptase mutant selected in vitro by acyclovir to anti-HIV drugs.
McMahon, Moira Aa; Siliciano, Janet Db; Kohli, Rahul Mb; Siliciano, Robert Fb,c
aDepartment of Pharmacology and Molecular Sciences, USA
bDepartment of Medicine, Johns Hopkins University School of Medicine, USA
cHoward Hughes Medical Institute, Baltimore, Maryland, USA.
Received 16 September, 2009
Revised 7 October, 2009
Accepted 13 October, 2009
Trials of acyclovir for herpes simplex virus 2 infection in herpes simplex virus 2/HIV-1 coinfected patients not on antiretroviral therapy demonstrated a decrease in herpes simplex virus 2 and HIV-1 replication. Recent studies indicated that acyclovir has direct anti-HIV-1 activity and can select for the HIV-1 V75I reverse transcriptase variant in vitro. We show that the V75I variant has decreased sensitivity to some nucleoside analogs but an increased sensitivity to zidovudine, results that may guide selection of highly active antiretroviral therapy regimens in patients harboring this variant.
Herpes simplex virus 2 (HSV-2) is a common co-pathogen in HIV-1 infected individuals, and coinfection is widespread in regions such as sub-Saharan Africa . Each pathogen can enhance severity of symptoms of the other infection [1–4]. Recent clinical trials with the antiherpetic drug acyclovir or its prodrug valacyclovir have been designed to modulate HIV-1 disease by control of HSV-2 outbreaks in HSV-2/HIV-1 coinfected patients not on highly active antiretroviral therapy (HAART). These trials demonstrated an approximately 0.5 log10 decrease in HIV-1 plasma levels [5,6]. It is known that HIV-1 disease progression is strongly correlated with plasma viral load , and thus the modest decrease in HIV-1 plasma levels observed with acyclovir may be beneficial in prolonging time to initiation of HAART or the onset of AIDS. Acyclovir trials in large cohorts of HIV-1 infected participants are currently underway including HSV-2 seronegative patients who may also benefit from acyclovir therapy . Acyclovir has also been studied as a means of prophylaxis against the acquisition of HIV-1 in HSV-2-infected individuals [9,10]. Although acyclovir did not decrease the risk of acquisition, HIV-1 viral loads in acyclovir-treated patients were lower than those of the control group. Therefore, the benefit of acyclovir treatment in coinfected individuals continues to be explored.
Interestingly, in-vitro data revealed that the clinical observations cited above may be a result of direct inhibition of HIV-1 replication by acyclovir. Biochemical studies have shown that acyclovir triphosphate is a substrate of HIV-1 reverse transcriptase [11,12]. In addition, the V75I multidrug resistance mutation in HIV-1 reverse transcriptase was selected for in vitro under the selective pressure of acyclovir and pseudotyped virus containing this mutation was less sensitive to acyclovir than wild-type virus . These data provide an alternative hypothesis to the generally accepted theory that the control of HSV-2 infection indirectly decreases HIV-1 replication. Although HIV-1 resistance to acyclovir has yet to be reported in patients, in-vitro data suggest this is a possibility. In the similar case of entecavir, the unexpected discovery of the anti-HIV-1 activity of this hepatitis B drug and selection for the M184V reverse transcriptase mutation in patients  has led to modifications of treatment recommendations in coinfected individuals . Similarly, monitoring HIV-1 genotypes in patients taking acyclovir monotherapy may be critical for optimizing future treatment.
Importantly, the WHO recommends a first line regimen of two nucleoside reverse transcriptase inhibitors (NRTIs), and one nonnucleoside reverse transcriptase inhibitor (NNRTI), for resource limited countries including sub-Saharan Africa. Zidovudine or tenofovir are the preferred NRTIs in this regimen in combination with lamivudine or emtricitabine . As the majority of participants in the acyclovir clinical trials were from resource limited countries, a better understanding of treatment and resistance may be helpful in guiding future HAART regimens.
Because V75I is the only HIV-1 mutation associated with acyclovir, we evaluated the sensitivity of the V75I variant to FDA approved NRTIs. To do this, we used a sensitive single round HIV infectivity assay [13,16,17]. Wild-type and V75I HIV-1 proviral constructs with green fluorescent protein (GFP) gene in place of HIV envelope were pseudotyped with the chemokine receptor CXCR4 tropic HIV envelope, and the resulting pseudoviruses were used to infect primary activated CD4+ lymphoblasts isolated from normal donors in the presence of increasing concentrations of the indicated NRTIs. The level of HIV-1 replication was determined by quantifying the number of GFP+ cells 3 days post infection by flow cytometry.
Surprisingly, V75I mutant virus was hypersusceptible to zidovudine compared with wild-type virus (Fig. 1a). Current biochemical investigations into the mechanism of this hypersusceptibility are underway. However, as illustrated in Fig. 1b, the V75I mutant virus was modestly less sensitive to lamivudine, emtricitabine, and didanosine compared with wild-type virus, slightly less sensitive to abacavir, and there was a relative equal sensitivity to tenofovir and stavudine. Biochemical data suggest that the main mechanism of resistance of V75I reverse transcriptase to acyclovir triphosphate is an increased discrimination against the analog compared with 2'-deoxyguanosine 5'-triphosphate (dGTP) . It will be interesting if this is the mechanism as well that explains the resistance to some of the NRTIs. Nevertheless, if the V75I mutation is selected in patients taking acyclovir monotherapy, a first line HAART regimen containing zidovudine may exceed expectations.
One of the most important remaining questions is whether acyclovir monotherapy will continue to be beneficial for HIV-1 infected patients or will selection of resistant virus, possibly the V75I variant, affect treatment options. Most clinical trials so far have administered lower doses of acyclovir or valacyclovir to study participants, whereas selection of the V75I variant in vitro was seen at higher acyclovir concentrations. As illustrated in Fig. 1c, a comparison of fitness of wild-type versus V75I virus in the presence of acyclovir in the single round infectivity assay shows that replication of wild-type virus is favored over mutant at peak plasma concentrations (∼20 μmol/l) determined from standard dosing. Thus, the V75I variant may not be selected in patients at this dose. In contrast, a comparison of fitness of wild-type virus with M184V virus in the presence of lamivudine (Fig. 1c) clearly shows that replication of M184V virus is favored at clinical concentrations of drug, consistent with the rapid selection of this mutant in patients [19–21]. Nonetheless, acyclovir has shown to be a well tolerated drug and, at a higher 2 g/day dose of the prodrug valacyclovir, the steady state plasma concentrations of acyclovir can reach approximately 38 μmol/l . At these concentrations, low level resistant virus, not detectable by clinical genotypes, could exist and potentially be archived affecting future treatment.
In conclusion, the available clinical data demonstrate a benefit to treatment with acyclovir in HSV-2/HIV-1 coinfected patients not on HAART [5,6]. The V75I mutant virus, selected by acyclovir treatment in vitro, is hypersusceptible to zidovudine, a drug that is currently recommended as the first line NRTI for treatment of HIV-1 in resource limited countries. Continued investigation into the interplay between HSV-2 and HIV-1 as well as the effect of acyclovir with both viruses will facilitate the development of treatment options for HIV-1 infected patients with the continued goal of increasing patient survival and decreasing side effects of drug burden and toxicity.
The present study was funded by the US National Institutes of Health grants AI43222 and AI51178. M.A.M. and R.F.S. wrote the article. J.D.S. and R.M.K. contributed to discussion of data and revisions of the article.
1. Corey L, Wald A, Celum CL, Quinn TC. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J Acquir Immune Defic Syndr 2004; 35:435–445.
2. Schacker T, Hu HL, Koelle DM, Zeh J, Saltzman R, Boon R, et al
. Famciclovir for the suppression of symptomatic and asymptomatic herpes simplex virus reactivation in HIV-infected persons. A double-blind, placebo-controlled trial. Ann Intern Med 1998; 128:21–28.
3. Krone MR, Wald A, Tabet SR, Paradise M, Corey L, Celum CL. Herpes simplex virus type 2 shedding in human immunodeficiency virus-negative men who have sex with men: frequency, patterns, and risk factors. Clin Infect Dis 2000; 30:261–267.
4. Schacker T, Zeh J, Hu H, Shaughnessy M, Corey L. Changes in plasma human immunodeficiency virus type 1 RNA associated with herpes simplex virus reactivation and suppression. J Infect Dis 2002; 186:1718–1725.
5. Nagot N, Ouedraogo A, Foulongne V, Konate I, Weiss HA, Vergne L, et al
. Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. N Engl J Med 2007; 356:790–799.
6. Zuckerman RA, Lucchetti A, Whittington WL, Sanchez J, Coombs RW, Zuniga R, et al
. Herpes simplex virus (HSV) suppression with valacyclovir reduces rectal and blood plasma HIV-1 levels in HIV-1/HSV-2-seropositive men: a randomized, double-blind, placebo-controlled crossover trial. J Infect Dis 2007; 196:1500–1508.
7. Mellors JW, Rinaldo CR Jr, Gupta P, White RM, Todd JA, Kingsley LA. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science
8. Vanpouille C, Lisco A, Margolis L. Acyclovir: a new use for an old drug. Curr Opin Infect Dis
2009 [Epub ahead of print].
9. Celum C, Wald A, Hughes J, Sanchez J, Reid S, Delany-Moretlwe S, et al
. Effect of aciclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomised, double-blind, placebo-controlled trial. Lancet 2008; 371:2109–2119.
10. Watson-Jones D, Weiss HA, Rusizoka M, Changalucha J, Baisley K, Mugeye K, et al
. Effect of herpes simplex suppression on incidence of HIV among women in Tanzania. N Engl J Med 2008; 358:1560–1571.
11. Lisco A, Vanpouille C, Tchesnokov EP, Grivel JC, Biancotto A, Brichacek B, et al
. Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues. Cell Host Microbe 2008; 4:260–270.
12. McMahon MA, Siliciano JD, Lai J, Liu JO, Stivers JT, Siliciano RF, et al
. The antiherpetic drug acyclovir inhibits HIV replication and selects the V75I reverse transcriptase multidrug resistance mutation. J Biol Chem 2008; 283:31289–31293.
13. McMahon MA, Jilek BL, Brennan TP, Shen L, Zhou Y, Wind-Rotolo M, et al
. The HBV drug entecavir: effects on HIV-1 replication and resistance. N Engl J Med 2007; 356:2614–2621.
14. Hammer SM, Eron JJ Jr, Reiss P, Schooley RT, Thompson MA, Walmsley S, et al
. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA 2008; 300:555–570.
15. Gilks C, Vitoria M. Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach. WHO
16. Zhang H, Zhou Y, Alcock C, Kiefer T, Monie D, Siliciano J, et al
. Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1. J Virol 2004; 78:1718–1729.
17. Shen L, Peterson S, Sedaghat AR, McMahon MA, Callender M, Zhang H, et al
. Dose-response curve slope sets class-specific limits on inhibitory potential of anti-HIV drugs. Nat Med 2008; 14:762–766.
18. Tchesnokov EP, Obikhod A, Massud I, Lisco A, Vanpouille C, Brichacek B, et al
. Mechanisms associated with HIV-1 resistance to acyclovir by the V75I mutation in reverse transcriptase. J Biol Chem 2009; 284:21496–21504.
19. Schinazi RF, Lloyd RM Jr, Nguyen MH, Cannon DL, McMillan A, Ilksoy N, et al
. Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. Antimicrob Agents Chemother 1993; 37:875–881.
20. Tisdale M, Kemp SD, Parry NR, Larder BA. Rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3′-thiacytidine inhibitors due to a mutation in the YMDD region of reverse transcriptase. Proc Natl Acad Sci USA 1993; 90:5653–5656.
21. Gao Q, Gu Z, Parniak MA, Cameron J, Cammack N, Boucher C, et al
. The same mutation that encodes low-level human immunodeficiency virus type 1 resistance to 2′,3′-dideoxyinosine and 2′,3′-dideoxycytidine confers high-level resistance to the (-) enantiomer of 2′,3′-dideoxy-3′-thiacytidine. Antimicrob Agents Chemother 1993; 37:1390–1392.
22. Weller S, Blum MR, Doucette M, Burnette T, Cederberg DM, de Miranda P, et al
. Pharmacokinetics of the acyclovir pro-drug valaciclovir after escalating single- and multiple-dose administration to normal volunteers. Clin Pharmacol Ther 1993; 54:595–605.
© 2010 Lippincott Williams & Wilkins, Inc.
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