Treatment with highly active antiretroviral therapy suppresses, but does not eradicate, HIV-infected cells. Most studies of the persistent viral reservoir have focused on detecting latently infected CD4 T cells in the blood. However, an important HIV-infected cell type is the macrophage, which resides in tissues. Its precursor, the monocyte, circulates in the blood, but is relatively resistant to HIV infection until it differentiates into a macrophage at tissue sites. The macrophage may be an important HIV reservoir because infection of macrophages is not lytic. Unlike T cells, which die within a few days of infection, infected macrophages survive and can become viral factories. Moreover, there have been suggestions, which we have confirmed, that HIV prolongs the life span of infected macrophages. Understanding the role of host factors in HIV infection and persistence in macrophages has received relatively little attention. microRNAs (miRNA) are small noncoding RNAs that are processed to ~22 nt imperfectly paired double- stranded RNAs that regulate expression of genes that control cell differentiation, survival and responses to the environment by a gene silencing process called RNA interference (RNAi). Viruses can take advantage of this endogenous pathway by encoding for small RNAs that utilize the RNAi gene silencing machinery to control the expression of viral or host genes or by manipulating the expression of host miRNAs. Since HIV-infected macrophages do not die rapidly and since miRNA-mediated gene silencing generally takes a few days to be optimal (a time frame by which HIV-infected T cells usually die), we focused on the role of miRNA modulation in HIV infection of macrophages. miRNA expression was compared in HIV-infected and uninfected macrophages by miRNA microarray. 51 host miRNAs are differentially expressed after HIV infection - 44 are up-regulated and 7 are down-regulated at least 2-fold. Three miRNAs (miR-191, miR-23a, miR-22) that are upregulated 8-11 fold after infection, significantly enhance the survival of uninfected monocyte-derived macrophages (MDM). Conversely, inhibition of each of these miRNAs reduces survival of HIV-infected MDM to that of uninfected MDM. Based on these preliminary data, we hypothesize that miR-191, miR-23a and miR-22 facilitate HIV persistence by prolonging the lifespan of infected macrophages. We also hypothesize that these 3 miRNAs might also affect HIV replication and macrophage immune function to enhance the ability of HIV- infected macrophages to produce virus and escape immune control. The goal of this proposal is to investigate the role of these 3 miRNAs on HIV replication, macrophage survival and apoptosis, and immune activation. An important goal will be to define key genes that these miRNAs regulate to influence these processes.
Our specific aims are to: (1) define the role of miRNAs in general and of miR-191, miR-23a and miR-22 in particular on HIV replication and immune function and survival of infected macrophages;and (2) identify target genes and pathways regulated by miR-191, miR-23a and miR-22 in HIV-infected macrophages.

Public Health Relevance

HIV infection enhances the in vitro lifespan of macrophages. Our preliminary data suggest that 3 cellular microRNAs, up-regulated following HIV infection of monocyte-derived macrophages, are responsible for the extended life span of HIV-infected macrophages. The goal of this proposal is to confirm this finding and determine whether these microRNAs also directly affect HIV replication or macrophage function. The mechanism by which these microRNAs promote macrophage survival will also be defined by identifying the genes whose expression they regulate in HIV-infected macrophages. A better understanding of how HIV infection prolongs the survival of macrophages, converting them into reservoirs of persistent viral production, is key to efforts to improve HIV therapy, either to obtain a """"""""functional cure"""""""" in which immune responses control the infection in the absence of drugs or to eradicate the virus. It has become increasingly clear that microRNAs regulate not only cell survival and differentiation, but also regulate HIV latency in T cells and de novo infection of cells of the macrophage lineage. Understanding the molecular basis for microRNA manipulation of macrophage survival in HIV infection will potentially provide routes for therapeutic intervention, either by identifying host gene targets for small molecule drug development or by suggesting small oligonucleotide drugs that can interfere with persistence of HIV-infected macrophages.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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AIDS Molecular and Cellular Biology Study Section (AMCB)
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Salzwedel, Karl D
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Immune Disease Institute, Inc.
United States
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Huang, Linfeng; Lieberman, Judy (2013) Production of highly potent recombinant siRNAs in Escherichia coli. Nat Protoc 8:2325-36