Non-HIV microbes affect HIV pathogenesis by mechanisms that have yet to be elucidated. In general, host factors, in particular imunoactivation together with viral evolution are factors that largely determine the course of HIV infection and non-HIV microbes may affect these phenomena. We addressed these problems by studying pathogenesis of HIV and its interaction with other viruses in various human tissues infected ex vivo.? ? 1. Critical role of tissue activation in HIV infection ? First, we focused on the role of immunoactivation in HIV-1 pathogenesis. Paradoxically, HIV infection that leads to immunodeficiency depends on tissue immunoactivation. We propose that immune activation in secondary lymphoid tissues is central to the pathogenesis of immune deficiency in chronic HIV-1 infection. In vivo it may predict the pace of CD4 depletion and the onset of clinical immune deficiency. Earlier we found that in HIV-infected lymph nodes there is a significant perturbation of cytokine secretion in particular of interleukin-1beta, interleukin-2, interferon-gamma and interleukin-15 as well as MIP-1alpha and SDF-1beta. Also, there was a profound upregulation of the activation marker, CD38, in naive, central memory, and effector CD4+ and CD8+ T cells. To study this phenomenon we used a system of blocks of human lymphoid tissue and of cervico-vaginal tissue cultured ex vivo. HIV-1 infection was associated with the activation of both HIV-1-infected and uninfected (bystanders) T cells in infected tissues creating a vicious cycle that facilitates HIV own production by creating new cellular targets for viral infection and therefore contributing to the T cell depletion. ? ? 2. Effect of non-HIV pathogens on HIV infection in human lymphoid tissue? Co-pathogens may contribute to immunoactivation, thus affecting HIV disease. We investigated HIV response to coinfection with some of HIV-1 copathogens, human cytomegalovirus (HCMV or HHV-5) and the cellular parasite Toxoplasma gondii. ? This required optimization of the human tonsillar tissue to support hCMV replication. hCMV DNA, flow cytometry of viral-positive cells and expression of the green fluorescent protein CMVPT30-gfp in a recombinant strain of hCMV demonstrated that hCMV readily replicated in tissue blocks. Immunophenotyping of HCMV-infected cells showed the preferential infection of activated lymphocytes (CD3+ HLA-DR+ and CD3+ CD25+) and also of plasmocytoid and myeloid dendritic cells (CD3- CD123+ CD11c+). The number of these cells significantly increased in HIV-1-coinfected tissues. Accordingly, HCMV replication was enhanced 2- to-3 fold. This upregulation occurred in tissues infected with either CXCR4- or CCR5-utilizing HIV-1. Thus, HIV-1 creates new targets for hCMV, which may explain the strong association of hCMV with HIV-1 infection in vivo. Human lymphoid tissue ex vivo also provides a model to study HIV interaction with non-viral HIV pathogens in particular of a parasite Toxoplasma gondii. We found that live T. gondii preferentially inhibits CCR5-utilizing HIV-1 replication in coinfected tissues. This effect was reproduced by treatment of the tissue blocks with recombinant C-18, a T. gondii-encoded cyclophilin that binds to CCR5. Thus, T.gondii may influence the outcome of viral infection by preferentially suppressing CCR5-utilizing HIV-1 variants. ? These studies revealed that HIV- copathogens may differentially generate positive and negative signals for HIV replication, thus affecting selection of particular HIV variants eventually changing the course of HIV disease.? ? 3. Up- and downregulation of HIV-1 replication in cervico-vaginal tissue ? Endogenous factors also generate such signals. We participated in the identification of a semen factor that facilitates HIV- replication, and we showed that it upregulates HIV replication in infected T cells, macrophages, ex vivo human tonsillar tissues, and cervico-vaginal tissues. This factor consists of naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase, which form amyloid fibrils. Thus, these fibrils may play an important role in sexual transmission of HIV and could represent new targets for its prevention. Whether non-HIV microbes affect formation of such fibrils now has to be investigated.? Natural infection with non-HIV-1 microbes can be used for development of new anti-HIV strategies. Here, we exploit the unique ability of herpesvirus (HHV) kinases to phosphorylate acyclovir (ACV), the first step in transforming this otherwise inert compound into an inhibitor of HHV DNA polymerases. Surprisingly, we found that in a cell-free system, phosphorylated ACV directly suppresses HIV reverse transcriptase. We showed that HIV-1 RT incorporates ACV-TP into the nascent HIV-1 DNA chain with a level of efficiency similar to that of its natural equivalent dGTP. Incorporation of ACV-TP results in the formation of a dead-end complex and traps RT at the site of termination leading to the complete termination of reverse transcription. Accordingly, we found that in tissues coinfected with HHVs that are capable of phosphorylating ACV, HIV-1 was inhibited. That was demonstrated in cervico-vaginal tissue as well as in other tissues, where the critical events of HIV-1 pathogenesis and transmission occurs in vivo, tonsils, lymph nodes, and colorectal tissue. Various HHVs, including the ubiquitous HHV-6 detected in all but one tissue ex vivo, may mediate HIV-1 suppression by ACV. No anti-HIV ACV activity was detected in either the HHV-free tissue or in HHV-free cell cultures. However, when HHV-6-infected cells were added to the HIV-1-infected (HHV-free) cell cultures, ACV became a potent suppressor of HIV-1 replication. In summary, the following mechanism seems to be responsible for ACV suppression of HIV-1 in human tissues ex vivo, the majority of which carry one or several HHVs, including HHV-6: ACV is monophosphorylated by herpesviral enzymes in HHV-infected cells and is then cellular kinases convert it into ACV-TP, which suppresses HIV in coinfected tissues by inhibiting HIV-1 RT. Our results suggest that ACV may be therapeutically beneficial for various HIV-1-infected patients, since the majority of humans are already infected with various HHVs that transform ACV at least during reactivation. However, clinical trials are needed to test this concept in vivo. In general, the combination of ACV with an endogenous HHV infection to suppress HIV may constitute a new principle of anti-HIV therapy; a binary weapon in which one inert component is converted by another, endogenous component, into an active therapeutic compound.
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