MicroRNAs: Innate Immune Modulated Restriction Factors of SIV Infection

Abstract Macrophages play a major role in the innate immune response to infection. CD4+ T cells and macrophages are the target cells for HIV, the virus that causes AIDS, and cause establishment of infection in the brain. HIV infection of the brain often leads to chronic inflammation and HIV-associated neurocognitive disorders (HAND). Our lab has developed an SIV macaque model of HIV to study CNS disease and the role of macrophages and other cell types involved in these disorders. Host cellular restriction factors inhibit HIV replication, and miRNAs are a member of this group. These small RNA molecules fine-tune gene expression through targeting 3’ UTRs of mRNAs and inhibiting translation and/or causing mRNA degradation. Recently, there has been increased interest in miRNA regulation of viruses due to their role in pathogenesis of HIV-1 and other viruses. In our studies, microarray analysis demonstrated levels of specific miRNAs that differ between monocytes (HIV-restricted) and macrophages (HIV-permissive) immune cells. These results suggest miRNAs may restrict virus infection, and also limit infection within target cell populations. Several miRNAs affect IFNβ, a cytokine induced early after HIV infection. Many genes are activated by IFNβ, and its regulation is critical for controlling immune signaling. miRNAs, miR-26a, -34a, -145 and –let7b have predicted binding sites in the IFNβ 3’ UTR. An ELISA showed each of these miRNAs regulates IFNβ protein levels and RT-qPCR data demonstrates that mature miRNAs miR-26a, -34a and –let7b were induced by IFNβ, indicating that IFNβ signaling induces miRNAs that then regulate expression of the protein. We also identified miRNAs that target SIV RNAs and inhibit virus replication. The SIV 3’ UTR contains functional binding sites for four different miRNAs, miR-29a, -29b, -9 and -146a, determined by RNA immunoprecipitation. Using primary macrophages, we found each miRNA reduced intracellular viral RNAs, as well as production of virus in supernatants of infected cells. RT-qPCR assays indicated modulation of mature miRNAs and primary miRNA transcripts by IFNβ and TNFα. These results demonstrate transcriptional regulation of miR-29a, -29b, -9 and -146a, four miRNAs that regulate virus replication in cells essential for immune signaling. Thesis Advisor: Janice E. Clements, Ph.D. Thesis Reader: Mollie K. Meffert, M.D., Ph.D