Although both cellular viral kinases encoded by endogenous HHVs, which are reactivated during the culture of human tissues ex vivo, are able to phosphorylate nucleotide analogs ; here, we decided to infect tissues with CMV, as GCV is specifically activated by UL97-encoded kinase expressed by CMV. To investigate whether exogenous CMV would confer better anti-HIV activity of GCV, we compared the HIV-inhibitory effect of GCV in tonsillar tissues that we infected with CMV and HIV and in donor-matched controls that were infected with HIV only. We found that the suppression of HIV-1 replication was dose dependent and not statistically different whether we coinfected HIV-infected tissues with CMV or not. The 50% effective concentrations of GCV in tonsillar tissue blocks were, respectively 4.7 μmol/l (95% confidence interval: 0.9–24.9) in HIV-1-singly infected tissues and 5.4 μmol/l (95% confidence interval: 1.5–19.0) in CMV-coinfected tissues (Fig. 1c).
Ganciclovir suppresses infection of HIV-1 of clades A, B, and C in human tissues ex vivo
We evaluated the susceptibility of three primary isolates to GCV: HIV-197USNG30 of clade C, HIV-196USNN20 of clade A, and HIV-1ME1 of clade B. Primary isolate HIV-196USNN20 has a broad coreceptor utilization, including CCR2B, CCR3, CCR4, CCR5, CXCR4, Bob and Bonzo, whereas HIV-197USNG30 and HIV-1ME1 are exclusively CCR5 tropic. Human lymphoid tissues (27 tissue blocks per experimental condition) were inoculated with each of these three isolates and then treated for the entire culture period with 5 or 50 μmol/l GCV (which is, respectively, the 50% effective concentration and a concentration that completely suppresses X4LAI.04 replication in this system). We found that GCV suppressed replication of all the three tested primary isolates and this suppression was not significantly different from that of laboratory isolate X4LAI.04 at both 5 and 50 μmol/l GCV (P > 0.05; Fig. 1d).
Ganciclovir suppresses replication of drug-resistant HIV-1 in human tissues ex vivo
First, we investigated the sensitivity of NRTI-resistant virus to GCV (5 and 50 μmol/l) in human tissue ex vivo. In particular, we evaluated the sensitivity to GCV of a azidothymidine-resistant HIV  and two NRTI-multidrug-resistant molecular clones (clones 10076-4 and 7303-3). The molecular clones belong to a panel of six prototypical infectious multi-NRTI-resistant HIV-1 molecular clones that carry several prototypical mutations conferring resistance to at least two of the following NRTIs: abacavir, zidovudine, stavudine, zalcitabine, didanosine, tenofovir, and lamivudine . The replication of azidothymidine-resistant virus, and of 10076-4 and 7303-3 molecular clones was, respectively, suppressed by 48.6 ± 21, 80.6 ± 12.2, 46.4 ± 14% at GCV concentration of 5 μmol/l and by 68.5 ± 3.9, 92.4 ± 4.3, and 73.5 ± 1.9% at GCV concentration of 50 μmol/l. No statistical difference between the inhibition of azidothymidine-resistant HIV-1 or multi NRTI-resistant molecular clones and the inhibition of the laboratory strain X4LAI.04 was observed (P > 0.05; Fig. 1d).
In a second set of experiments, we tested the sensitivity to GCV of the nNRTI-resistant HIV-1N119 (resistant to nevirapine), of a fusion inhibitor-resistant HIV [HIV-1pNL4-3 gp41(36G)/V38A/N42D; resistant to enfuvirtide (T-20)], and of two proteases inhibitor-resistant HIV-1 (HIV-1L10R/M46I/L63P/V82T/I84 V and HIV-1M46I/L63P/V82T/I84 V), which are resistant to the structurally diverse protease inhibitors MK-639, XM323, A-80897, Ro31-8959, VX-478, and SC-52151 . The replication of HIV-1N119, HIV-1pNL4-3 gp41(36G)/V38A/N42D, HIV-1L10R/M46I/L63P/V82T/I84 V, and HIV-1M46I/L63P/V82T/I84 V was, respectively, suppressed by 47.9 ± 4.9, 52.5 ± 23.3, 38.2 ± 16.6, and 36.2 ± 18.1% at GCV concentration of 5 μmol/l and by 88.3 ± 3.2, 96.1 ± 2.1, 86.1 ± 1.7, and 80.6 ± 4.5% at GCV concentration of 50 μmol/l (Fig. 1d).
Incorporation of ganciclovir triphosphate by HIV-1 reverse transcriptase results in downstream DNA synthesis inhibition
We hypothesized that GCV-TP interferes with the activity of HIV-1 reverse transcriptase and directly suppresses HIV-1 replication. To test this hypothesis, we evaluated the ability of GCV-TP to suppress HIV-1 reverse transcriptase by measuring the polymerizing activity of purified recombinant enzyme using an exogenously added primer-template hybrid (Fig. 2a). In particular, we assessed the extension of a GCV-monophosphate-terminated primer using a multiple nucleotide incorporation assay. Pausing in DNA synthesis was observed at positions n
+ 1 and n
+ 3 for HIV-1 reverse transcriptase when GCV monophosphate was located at position n. These pausing sites were overcome with increasing concentrations of nucleotides, suggesting that either compromised nucleotide binding following these pausing sites or hybrid dissociation was caused by GCV monophosphate that was located in the primer strand.
Kinetic comparison of ganciclovir triphosphate and dGTP incorporation for HIV-1 reverse transcriptase
To probe the structural determinants governing the efficiency of GCV-TP incorporation, we employed a steady-state kinetic approach to compare GCV-TP incorporation with that of the natural dGTP substrate (Table 1). For reverse transcriptase, the rate of GCV-TP incorporation (denoted by kcat) was similar to that for the natural dGTP substrate; however, HIV-1 reverse transcriptase displayed marked increases in Km values for GCV-TP compared with dGTP (HIV-1 reverse transcriptase: 0.058 μmol/l for dGTP and 4.4 μmol/l for GCV-TP) .
Difference in efficiency of nucleotide incorporation downstream of ganciclovir monophosphate in the primer strand
In an effort to analyze the pattern of inhibition by GCV-TP, a steady-state kinetic analysis of single nucleotide incorporation by HIV reverse transcriptase was conducted; the positions for nucleotide incorporation selected were those immediately following the pausing sites seen in the full-length DNA synthesis experiment for HIV-1 reverse transcriptase (Fig. 2b), namely positions n
2 and n
4. The kinetic analysis for these two primer systems with internal GCV-monophosphate residues was compared with the control primer system having the natural dGMP located at position n. The results (Table 2) point to a distinct mechanism of inhibition for HIV-1 reverse transcriptase. Although nucleotide incorporation is 27-fold less efficient at position n
2 when GCV monophosphate is located at position n than the control with dGMP at position n, the efficiency of nucleotide incorporation is over 1000-fold less efficient at position n
4 when GCV monophosphate is found at position n, as compared with the control.
Ganciclovir does not decrease inflammation/activation in human tissues ex vivo
Although the data above indicates that GCV acts directly on HIV, we investigated alternative potential mechanisms of such an inhibition: the decrease of general inflammation of the tissue and the decrease of the activation status of tonsillar cells, in particular of CD4+ T cells. We addressed these two possibilities in two different sets of experiments.
First, we measured the levels of 32 cytokines/chemokines in the supernatants of human tissues treated or not with GCV. Tissues were treated with 5 μmol/l GCV (50% effective concentrations of X4LAI.04) or 50 μmol/l GCV (10-fold higher than 50% effective concentrations). Among the 22 cytokines/chemokines detectable in tonsillar tissue cultures, none were significantly different in tissues treated with GCV (whether tissues were treated with 5 μmol/l or 50 μmol/ GCV) compared to their counterparts in untreated tissue cultures (Table 3).
In the second set of experiments, we specifically investigated whether GCV affected the viability of tissue cells, in particular the viability of HIV target cells (CD4+ T cells expressing CXCR4 or CCR5) including activated T cells (CD25+ or CD38+; Fig. 1d). After 12 days in culture, we isolated cells from tissue blocks treated with 5 or 50 μmol/l GCV and from donor-matched untreated tissue and stained them for CD3, CD4+, CD8−, CXCR4, CCR5, CD25, and CD38. We evaluated the cytopathicity of GCV by comparing the number of cells in GCV-treated tissues with those in matched untreated tissues using a novocyte flow cytometer, which allowed us to acquire a similar volume for each experimental condition. To account for size differences in tissue blocks, we normalized the data by the weight of the tissues. Figure 1e showed that GCV, even at the concentration of 50 μmol/l, did not deplete any cell subset, including activated CD4+ T cells that are primary targets for HIV.
It is commonly accepted that persistent immune activation is a driving force of HIV disease. In HIV-1-infected individuals, CMV prevalence is around 90%, and a growing body of evidence suggests that asymptomatic replication of CMV, a member of the HHV family, may be an important contributor to this persistent immune activation (reviewed in [8,21]) accelerating the development of HIV-triggered immunological abnormalities [8,38–41]. In particular, CMV shedding in the genital tract has been associated with increased T-cell immune activation and proliferation in peripheral blood [9–11,40], higher HIV-1 shedding in semen [13,15–17,19], and higher risk of HIV-1 transmission [3,6,7]. Both CMV and HIV are associated with inflammation-related morbidities . Several studies have suggested that both direct and indirect interactions between CMV and HIV could influence their replication and the resulting disease pathogenesis. Freeman et al. reported on several of such interactions making the contribution of CMV to HIV pathogenesis and vice versa difficult to tease apart.
ART has affected the course of HIV infection in a revolutionary way, dramatically decreasing the mortality of HIV-1-infected individuals, and reducing complications associated with opportunistic infections. However, even with sustained suppression of HIV replication, abnormal immune activation persists in most of these individuals. This immune activation is associated with subsequent morbidity and earlier mortality . It has been suggested that CMV contributes to immune activation even when ART has suppressed HIV replication [10,19,43–46].
These observations suggested that treating asymptomatic CMV coinfection may be beneficial by suppressing general T-cell activation, and ultimately decreasing HIV-1 viral load, as well as delaying HIV disease progression [10,22]. In this context, Shin et al. investigated the effect of anti-CMV therapy using valganciclovir on immune activation and HIV-1 viral load in semen. They found that GCV did indeed reduce HIV-1 viral load. However, this reduction was surprisingly not associated with decreases in T-cell activation and in production of inflammatory cytokines.
On the basis of GCV-mediated reduction of HIV-1 genital viral load in the absence of significant genital changes in immunoactivation, we hypothesized along with Shin et al. that GCV has a direct effect on HIV replication provided it is phosphorylated by cellular or HHV TK.
Here, we tested this hypothesis in human tissues ex vivo. These are adequate models for preclinical drug testing, as they support productive HIV infection without exogenous activation and stimulation. Moreover, ex vivo tissues retain cytoarchitecture as well as the pattern of expression of key cell-surface molecules relevant to HIV infection . First, we tested the sensitivity of two laboratory isolates of HIV-1, of CXCR4-tropicX4LAI.04 and of CCR5-tropic R5BaL. We demonstrated that GCV inhibited HIV-1 in both lymphoid and cervicovaginal tissues ex vivo and that GCV inhibited both X4 and R5 HIV-1. HIV-1 inhibition was not limited to laboratory isolates as three HIV-1 primary isolates of different subtypes (A, B, and C) were also suppressed by GCV. Also, GCV sensitivity was not limited to the wild-type HIV-1 as NRTI, nNRTI, fusion inhibitor, and protease inhibitor-resistant viruses were all sensitive to GCV.
To investigate whether HIV inhibition by GCV was the result of a direct inhibition on the virus itself rather than the result of a GCV-mediated decrease of inflammation of the tissue, we measured the levels of 32 cytokines/chemokines (including but not limited to inflammatory cytokines) in the supernatants of tonsillar tissues treated or not with GCV. No significant difference in any levels of cytokines/chemokines was found between treated and untreated tissues. Neither did we find that GCV affected the viability or the number of tonsillar cells, in particular of CD4+ T cells, which are the primary target cells for HIV infection. Thus, it seems that GCV inhibited HIV-1 replication by directly affecting the virus and not by decreasing inflammation or activation of the tissue.
GCV is a synthetic purine nucleoside analogue of guanine and also has to undergo triphosphorylation to become active, with the initial monophosphorylation catalyzed more efficiently by HHV-encoded kinase than by cellular kinases. HHVs seem to be necessary to inhibit HIV-1, as GCV did not inhibit HIV-1 in MT-4 cell cultures (data not shown), which are free of endogenous HHVs . Moreover, we showed here that the 50% effective concentrations of GCV for HIV-1 was approximately 5 μmol/l, whether human tissues were exogenously coinfected with CMV or not. Thus, it seems that kinases expressed by endogenous HHVs activate GCV by adding the first phosphate. HHV-6 U69-encoded kinase is one such enzyme that has been shown to phosphorylate ganciclovir .
Both the multisite incorporation assay and the steady-state kinetic analysis suggest that GCV monophosphate inhibits HIV-1 reverse transcriptase by acting as a delayed chain terminator. It is possible that the inhibitor's interaction with reverse transcriptase is more strongly compromised by the lack of the sugar ring stacking with the enzyme that is seen with the natural dGTP substrate.
Using a steady-state kinetic approach, we found that the major mechanism of inhibition of HIV-1 reverse transcriptase by GCV was through a block in nucleotide incorporation at position n+4, as the efficiency of nucleotide incorporation was over 1000-fold less efficient at position n+4 when GCV-monophosphate is found at position n. In comparison, the efficiency of nucleotide incorporation was over 27-fold less efficient at position n+2 when GCV monophosphate was found at position n.
GCV is not the first delayed chain terminator to be described, as entecavir has been shown to block DNA synthesis through inhibition at positions n
1 (minor) and n
4 (major) in HIV-1 reverse transcriptase . However, both the chemical structures and the mechanisms of action of GCV and entecavir are different, indicating that diverse mechanisms of delayed chain termination can be obtained from chemical modification of the inhibitor.
The effect of GCV on HIV viral load in CMV-coinfected tissues resembles that of acyclovir (ACV) in HSV-2-coinfected individuals. As with the inhibition of HIV by GCV, the inhibition of HIV by ACV was thought to proceed exclusively via an indirect mechanism. Indeed, the effect of ACV on HIV viral load was initially attributed to the suppression of HSV-2 replication followed by the suppression of HSV-2-triggered inflammation [49,50]. However, we and others [30,51,52] have found that ACV also has a direct effect on HIV-1 after the drug is phosphorylated by HHV. The direct inhibitory effect of ACV on HIV viral load observed ex vivo was recently confirmed in vivo in a double-blind crossover clinical trial . In this trial, we showed that valacyclovir (a prodrug of ACV) suppresses HIV-1 viral load in HIV-1-infected HSV-2 seronegative persons, proving that the effects of valacyclovir on HIV-1 are consistent with a direct effect of ACV on HIV-1 replication .
As with what we previously described for ACV/valACV, here, we found that GCV inhibited HIV in human tissues ex vivo but not in cell lines. Indeed, no anti-HIV effect of GCV was observed here in MT-4 cell lines or in H9 cells as reported by Medina et al.. The anti HIV activity of GCV may be directly related to endogenous HHVs present in tissues  but not in cell lines . Indeed, HHVs are ubiquitous in the tonsillar tissues. Earlier we found that 96% of tonsillar tissues (received from the same hospital as in the current study) were positive for one or several HHVs. In particular, real-time PCR analysis revealed CMV in 15% of tissues; EBV in 52%; HHV-7 in 89%; and HHV-6 in 96% of tissues.
The anti-HIV activity of the GCV occurs at clinically relevant concentrations. Indeed, although GCV penetration efficiency and drug clearance were unknown for ex-vivo tissues, the calculated 50% effective concentrations of 4.7 μmol/l is in the range of what has been reported in vivo. For a dose of 900 mg of oral valganciclovir per day, the maximum mean plasma concentration is 26.3 μmol/l with a mean Area under the curve0–24 of 51.2 μgh/ml, and plasmatic concentrations remain higher than 4.9 μmol/l for the first 15 h after drug administration .
In conclusion, our results suggest that an anti-CMV strategy using valganciclovir in HIV-1-infected individuals may reduce HIV-1 viral load directly by inhibiting HIV-1 reverse transcriptase. Given the potential impact of CMV in immune activation and the potential role of CMV in various human pathologies, in particular in cardiovascular disease, T-cell senescence, and aging, strategies to reduce CMV replication in HIV-infected individuals may turn out to be beneficial.
We thank the entire staff of the Department of Pathology of Children's National Medical Center for their generous assistance in obtaining human tonsillar tissues. The work of C.V., A.L., A.A., E.S., and L.M. was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. The work of J.A.B. and M.G. was supported by grants from the Canadian Institutes of Health Research (CIHR). M.G. is the recipient of a career award from the FRSQ. J.A.B. is the recipient of a Doctoral Training Scholarship from the Fonds de la recherche en santé du Québec (FRSQ) and a Chemical Biology Training Program Scholarship from the CIHR.
C.V., A.L., A.A., E.S., and L.M. designed ex-vivo experiments performed by C.V., A.L., A.A., and E.S. J.A.B., and M.G. designed and performed the experiments on GCV-TP incorporation by HIV RT. C.V. and J.A.B. wrote the paper and L.M. edited it. All authors reviewed the manuscript.
Conflicts of interest
There are no conflicts of interest.
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cytomegalovirus; delayed-chain termination; ganciclovir; HIV-1; reverse transcriptase
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