1. Analysis of the conformation of HIV Env on individual virions HIV Env plays a major role in HIV infection, as the correct trimeric conformation of Env is critical for the virus to bind and to fuse with plasma membrane. Dysfunctional forms of Env render virions incapable of binding/fusion with cells. Each HIV-1 virion carries 10-14 Env spikes, and in principle it is possible that on a given virion all spikes are either defective or functional, rendering the former virion defective and the latter virion infectious. Alternatively, virions may carry both functional and non-functional Envs in different conformations. Determining which of these possibilities are true, we analyzed the distribution of different forms of Env on single HIV-1 virions, using flow virometry. Specifically, individual HIV virions were captured with 15-nm magnetic nanoparticles decorated with (capture) antibodies that recognize different conformations of Env. We found that only a minor fraction of virions carry both trimeric and defective Envs, while most of the virions carry either exclusively trimeric or exclusively defective Envs. Accordingly, the depletion of the virions that carry defective Envs only mildly decreased the infection of human lymphoid tissues. In conclusion, using flow virometry, we demonstrated that most HIV virions carry either only functional or only defective Envs. The observed lack of Env mosaicism suggests that this all-or-nothing viral strategy likely aids immune evasion by subverting the focus of humoral responses to generate multiple non-neutralizing antibodies at no cost to infectious virions. 2. Extracellular vesicles containing HIV Env facilitate HIV infection Various cells in vivo and in vitro release extracellular vesicles (EVs), many of which are generated along pathways similar to the ones used by retroviruses, in particular HIV. Consequently, EVs are of the same size and physical properties as this virus and it is therefore almost impossible to separate such EVs from HIV virions. Here, we overcame some of these problems by segregating EVs through CD45 and/or acetylcholinesterase (AChE), two proteins that are not incorporated into HIV membranes and thus can be used to distinguish EVs from HIV virions. To capture and further identify these EVs, we applied our flow virometry nanotechnology (see #1). In the present work, we addressed two questions regarding EVs released by HIV-infected cells: (i) whether these EVs carry viral Envs, and (ii) whether EVs affect HIV infection. Using the tetraspanin CD81 that is shared by EVs and HIV, we captured both HIV virions and EVs with magnetic nanoparticles coupled with anti-CD81 antibodies and then identified EVs from the presence of either CD45 or AChE. When we stained our preparation with fluorescent anti-Env antibodies, approximately 50% of the events were positive both for EV markers and for Env. The results were similar whether we used CD45 or AChE for identification of EVs, and whether we used prototypical CXCR4 or CCR5 HIV viral preparations. Thus, EVs released by HIV-infected cells carry HIV Env. Do these EVs, which seem to be a part of HIV preparations, affect viral infection? We addressed this question by inoculating human lymphoid tissue ex vivo with a viral suspension depleted of specific EVs. Depletion of viral preparations of EVs, in particular of those that carry Env, decreased viral infection of human lymphoid tissue ex vivo. Thus, EVs that carry Env identified in our work seem to facilitate HIV infection and therefore may constitute a new therapeutic target for antiviral strategy. 3. Mechanisms of inhibitory activity of an anti-CMV drug against HIV Valganciclovir, a common anti-CMV drug, has been shown to be beneficial to CMV/HIV-coinfected individuals by decreasing HIV viral load. The anti-HIV effect of this anti-herpetic drug was considered to be indirect and was ascribed to decreasing immunoactivation caused by CMV infection. We investigated whether there was a direct effect of the drug on HIV infection. We used ganciclovir (GCV), the active form of valganciclovir, and tested the effect of GCV on HIV replication. We treated tonsillar and cervico-vaginal tissues ex vivo with GCV and then inoculated tissues with HIV. GCV suppressed replication of HIV-1 in these tissues by 85-90%. We deciphered the mechanism of this suppression. GCV is a synthetic purine nucleoside analogue of guanine that must undergo triphosphorylation to become active, with the initial monophosphorylation catalyzed more efficiently by herpesvirus (HHV)-encoded kinase than by cellular kinases. HHVs seem to be necessary to inhibit HIV-1, as GCV did not inhibit HIV-1 in HHV-free MT-4 cells. Moreover, the EC50 of GCV for HIV-1 was approximately 5 microM, whether human tissues were exogenously co-infected with CMV or not. Thus, it seems that kinases expressed by endogenous HHVs present in human tissues activate GCV by adding the first phosphate. Using an exogenous template reverse transcriptase assay, we showed that GCV-MP inhibits HIV-1 reverse transcriptase (RT) by acting as a delayed chain terminator. Our results suggest that an anti-CMV strategy using GCV in HIV-1-infected individuals may reduce HIV-1 viral load directly by inhibiting HIV-1 RT. Future trials should evaluate the relative contributions both of indirect mechanisms of HIV-1 and of the direct suppression of HIV-1 RT by phosphorylated GCV. 4. Lactobacillus-mediated prevention of HIV vaginal transmission The vaginal microbiota of healthy reproductive-age women is generally dominated by Lactobacillus species that are involved in maintaining vaginal homeostasis and have been reported to protect against vaginal transmission of HIV. However, the exact mechanism of HIV inhibition by vaginal lactobacilli remains to be fully elucidated. We investigated the effects of vaginal lactobacilli on HIV-1 infection of human lymphoid tissues and of human cervico-vaginal tissues ex vivo. To address these effects in the context of human tissues, we first colonized them ex vivo with different strains of Lactobacillus that were isolated from vaginal swabs of healthy premenopausal women. To investigate whether lactobacilli release suppressive factors that inhibit HIV-1 replication, we applied bacteria-conditioned culture medium on human tissues ex vivo and inoculated tissues with HIV. Conditioned medium significantly inhibited HIV-1 replication in human cervico-vaginal and tonsillar tissues. Although such a medium may contain multiple inhibitory factors, we first focused on two of them, pH and lactic acid. The pH of conditioned medium of all tested lactobacilli ranged from 6.3 to 6.9. Although acidification may be directly responsible for HIV-1 inhibition, no HIV-1 suppression was observed when pH was adjusted to 6.9 in control experiments. This suggested that other factors beyond lowered pH may also be important for HIV-1 inhibition. One of these factors may be the major lactobacillus metabolite, namely lactic acid. We found that the addition of lactic acid isomers D and L to tissue culture medium at concentrations that corresponded to amounts released by lactobacilli resulted in HIV-1 inhibition. Moreover, lactobacillus have a direct virucidal effect on HIV-1. We incubated an HIV-1 preparation in lactobacillus-conditioned medium and then tested HIV-1 infectivity in human tissue culture and found that HIV-1 infectivity in cervico-vaginal tissues was significantly reduced. Finally, we detected that a direct interaction of lactobacilli with HIV affected this virus and found that virions adhere to lactobacilli. Thus, in ex vivo systems we identified several mechanisms by which lactobacilli inhibit HIV infection. Extrapolated to in vivo conditions, these mechanisms may explain the protective effect of vaginal lactobacillus on HIV infection.
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