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.
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