A Role for microRNA-155 Modulation in the Anti-HIV-1 Effects of Toll-Like Receptor 3 Stimulation in Macrophages

Abstract

HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I∶C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood. We found that poly(I∶C)- and LPS-stimulation of MDMs abrogated infection by CCR5-using, macrophage-tropic HIV-1, and by vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 virions, while TLR2, TLR7 or TLR9 agonists only partially reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines or β-chemokines. Integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). Using microarray analysis and quantitative reverse transcription (RT)-PCR, we found increased microRNA (miR)-155 levels in MDMs upon TLR3/4- but not TLR7-stimulation, and a miR-155 specific inhibitor (but not a scrambled control) partially restored infectivity in poly(I∶C)-stimulated MDMs. Ectopic miR-155 expression remarkably diminished HIV-1 infection in primary MDMs and cell lines. Furthermore, poly(I∶C)-stimulation and ectopic miR-155 expression did not alter detection of early viral RT products, but both resulted in an accumulation of late RT products and in undetectable or extremely low levels of integrated pro-viruses and 2-LTR circles. Reduced mRNA and protein levels of several HIV-1 dependency factors involved in trafficking and/or nuclear import of pre-integration complexes (ADAM10, TNPO3, Nup153, LEDGF/p75) were found in poly(I∶C)-stimulated and miR-155-transfected MDMs, and a reporter assay suggested they are authentic miR-155 targets. Our findings provide evidence that miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry, pre-integration events, leading to severely diminished HIV-1 infection. The infection of macrophages by HIV-1 is a crucial event in the pathogenesis of AIDS. Toll-like receptors (TLR) are a family of receptors present in macrophages – among other cells – that detect various components of microbes and trigger host defenses. It is known that stimulation of macrophages through TLR3 or TLR4 reduces their susceptibility to HIV-1 infection, but the mechanism is not well understood. Here we show for the first time in primary human macrophages that TLR3 and TLR4 but not other TLRs induce higher levels of microRNA-155 – a key regulator of inflammatory and immune responses – and that microRNA-155 has a remarkable anti-HIV-1 effect. MicroRNAs are small, non-coding RNAs that bind to target mRNAs based on sequence complementarity, and lead to reduced protein output. We also show that the anti-HIV-1 effects of microRNA-155 seem to be mediated through targeting the mRNAs of several cellular proteins needed by the virus for trafficking and/or nuclear import of the viral DNA, which is required for integration into the host DNA and successful infection. These studies provide evidence of novel microRNA-155 targets and may serve as the basis for an innovative approach to reduce cellular susceptibility to HIV-1 infection.