SUMMARY MicroRNAs (miRNAs), ~22-nucleotide noncoding RNAs, assemble into RNA-induced silencing complexes (RISC) and localize to cytoplasmic substructures called P-bodies. Dictated by base-pair complementarity between miRNA and a target mRNA, miRNAs specifically repress posttranscriptional expression of several mRNAs. Here, we report that HIV-1 mRNA interacts with RISC proteins and that disrupting P-body structures enhances viral production and infectivity. In HIV-1-infected human T lymphocytes, we identified a highly abundant miRNA, miR-29a, which specifically targets the HIV-1 3’-UTR region. Inhibiting miR-29a enhanced HIV-1 viral production and infectivity, whereas expressing a miR-29 mimic suppressed viral replication. We also found that specific miR-29a-HIV-1 mRNA interactions enhance viral mRNA association with RISC and P-body proteins. Thus, we provide an example of a single host miRNA regulating HIV-1 production and infectivity. These studies highlight the significance of miRNAs and P-bodies in modulating host cell interactions with HIV-1 and possibly other viruses.

The HIV-1 protein Vif, essential for in vivo viral replication1–4, targets the human DNA-editing enzyme, APOBEC3G (A3G)5, which inhibits replication of retroviruses and hepatitis B virus6,7. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G8, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule–based new therapies for HIV infection or for enhancing innate immunity against viruses.

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Background: SMURF1 ubiquitin ligase controls ubiquitination and stability of diverse cellular protein substrates. Results: Deubiquitinase USP9X interacts with SMURF1 and stabilizes SMURF1 through deubiquitination. Conclusion: USP9X is novel regulator of SMURF1 and is required for SMURF1-dependent cellular physiology. Significance: Association between deubiquitinase and ubiquitin ligase may serve as a common strategy to control the cellular protein dynamics through modulating ubiquitin ligase activity.

A series of novel HIV-1 protease inhibitors based on two pseudosymmetric dipeptide isosteres have been synthesized and evaluated. The inhibitors were designed by incorporating N-phenyloxazolidinone-5-carboxamides into the hydroxyethylene and (hydroxyethyl)hydrazine dipeptide isosteres as P2 and P2' ligands. Compounds with (S)-phenyloxazolidinones attached at a position proximal to the central hydroxyl group showed low nM inhibitory activities against wild-type HIV-1 protease. Selected compounds were further evaluated for their inhibitory activities against a panel of multidrug-resistant protease variants and for their antiviral potencies in MT-4 cells. The crystal structures of lopinavir (LPV) and two new inhibitors containing phenyloxazolidinone-based ligands in complex with wild-type HIV-1 protease have been determined. A comparison of the inhibitor-protease structures with the LPV-protease structure provides valuable insight into the binding mode of the new inhibitors to the protease enzyme. Based on the crystal structures and knowledge of structure-activity relationships, new inhibitors can be designed with enhanced enzyme inhibitory and antiviral potencies.