Deubiquitinating Enzyme USP21 Inhibits HIV-1 Replication by Downregulating Tat Expression.

Ubiquitination plays an important role in human immunodeficiency virus 1 (HIV-1) infection. HIV proteins such as Vif and Vpx mediate the degradation of the host proteins APOBEC3 and SAMHD1, respectively, through the proteasome pathway. However, whether deubiquitylating enzymes play an essential role in HIV-1 infection is largely unknown. Here, we demonstrate that the deubiquitinase USP21 potently inhibits HIV-1 production by indirectly downregulating the expression of HIV-1 transactivator of transcription (Tat), which is essential for transcriptional elongation in HIV-1. USP21 deubiquitylates Tat via its deubiquitinase activity, but a stronger ability to reduce Tat expression than a dominant-negative ubiquitin mutant (Ub-KO) showed that other mechanisms may contribute to USP21-mediated inhibition of Tat. Further investigation showed that USP21 downregulates cyclin T1 mRNA levels by increasing methylation of histone K9 in the promoter of cyclin T1, a subunit of the positive transcription elongation factor b (P-TEFb) that interacts with Tat and transactivation response element (TAR) and is required for transcription stimulation and Tat stability. Moreover, USP21 had no effect on the function of other HIV-1 accessory proteins, including Vif, Vpr, Vpx, and Vpu, indicating that USP21 was specific to Tat. These findings improve our understanding of USP21-mediated functional suppression of HIV-1 production. IMPORTANCE Ubiquitination plays an essential role in viral infection. Deubiquitinating enzymes (DUBs) reverse ubiquitination by cleaving ubiquitins from target proteins, thereby affecting viral infection. The role of the members of the USP family, which comprises the largest subfamily of DUBs, is largely unknown in HIV-1 infection. Here, we screened a series of USP members and found that USP21 inhibits HIV-1 production by specifically targeting Tat but not the other HIV-1 accessory proteins. Further investigations revealed that USP21 reduces Tat expression in two ways. First, USP21 deubiquitinates polyubiquitinated Tat, causing Tat instability, and second, USP21 reduces the mRNA levels of cyclin T1 (CycT1), an important component of P-TEFb, that leads to Tat downregulation. Thus, in this study, we report a novel role of the deubiquitinase, USP21, in HIV-1 infection. USP21 represents a potentially useful target for the development of novel anti-HIV drugs.

Biological evaluation of molecules of the azaBINOL class as antiviral agents: Inhibition of HIV-1 RNase H activity by 7-isopropoxy-8-(naphth-1-yl)quinoline.

Inspired by bioactive biaryl-containing natural products found in plants and the marine environment, a series of synthetic compounds belonging to the azaBINOL chiral ligand family was evaluated for antiviral activity against HIV-1. Testing of 39 unique azaBINOLs and two BINOLs in a single-round infectivity assay resulted in the identification of three promising antiviral compounds, including 7-isopropoxy-8-(naphth-1-yl)quinoline (azaBINOL B#24), which exhibited low-micromolar activity without associated cytotoxicity. The active compounds and several close structural analogues were further tested against three different HIV-1 envelope pseudotyped viruses as well as in a full-virus replication system (EASY-HIT). The in vitro studies indicated that azaBINOL B#24 acts on early stages of viral replication before viral assembly and budding. Next we explored B#24's activity against HIV-1 reverse transcriptase (RT) and individually tested for polymerase and RNase H activity. The azaBINOL B#24 inhibits RNase H activity and binds directly to the HIV-1 RT enzyme. Additionally, we observe additive inhibitory activity against pseudotyped viruses when B#24 is dosed in competition with the clinically used non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz. When tested against a multi-drug resistant HIV-1 isolate with drug resistance associated mutations in regions encoding for HIV-1 RT and protease, B#24 only exhibits a 5.1-fold net decrease in IC50 value, while efavirenz' activity decreases by 7.6-fold. These results indicate that azaBINOL B#24 is a potentially viable, novel lead for the development of new HIV-1 RNase H inhibitors. Furthermore, this study demonstrates that the survey of libraries of synthetic compounds, designed purely with the goal of facilitating chemical synthesis in mind, may yield unexpected and selective drug leads for the development of new antiviral agents.

Antiretroviral (HIV-1) activity of azulene derivatives.

The antiretroviral activity of azulene derivatives was detected for the first time. A series of eighteen diversely substituted azulenes was synthesized and tested in vitro using HIV-1 based virus-like particles (VLPs) and infectious HIV-1 virus in U2OS and TZM-bl cell lines. Among the compounds tested, the 2-hydroxyazulenes demonstrated the most significant activity by inhibiting HIV-1 replication with IC50 of 2-10 and 8-20 µM for the VLPs and the infectious virus, respectively. These results indicate that azulene derivatives may be potentially useful candidates for the development of antiretroviral agents.

Glucose conjugation of anti-HIV-1 oligonucleotides containing unmethylated CpG motifs reduces their immunostimulatory activity.

Antisense oligodeoxynucleotides (ODNs) are short synthetic DNA polymers complementary to a target RNA sequence. They are commonly designed to halt a biological event, such as translation or splicing. ODNs are potentially useful therapeutic agents for the treatment of different human diseases. Carbohydrate-ODN conjugates have been reported to improve the cell-specific delivery of ODNs through receptor mediated endocytosis. We tested the anti-HIV activity and biochemical properties of the 5'-end glucose-conjugated GEM 91 ODN targeting the initiation codon of the gag gene of HIV-1 RNA in cell-based assays. The conjugation of a glucose residue significantly reduces the immunostimulatory effect without diminishing its potent anti-HIV-1 activity. No significant effects were observed in either ODN stability in serum, in vitro degradation of antisense DNA-RNA hybrids by RNase H, cell toxicity, cellular uptake and ability to interfere with genomic HIV-1 dimerisation.

A multivalent HIV-1 fusion inhibitor based on small helical foldamers.

The peptide sequence AcNH-TEG-Glu-Aib-Trp-AibAib-Trp-AibAib-Ile-Asp-OH (1), designed to display the WWI epitope found near the C-terminus of gp41, an envelope glycoprotein decorating the surface of the HIV-1 virus, has been synthesized and proved to have a relevant content of helical conformation because of the presence of five α-aminoisobutyric acid (Aib) units. Three copies of it have been connected to a tripodal platform based on 2,4,6-triethylbenzene-1,3,5-trimethylamine. The tripodal template 2 is even more structured than 1 thus suggesting a significant interaction between the three sequences connected to the platform. Preliminary inhibition assays of HIV-mediated cell fusion indicated that while the single peptide 1 is inactive within the concentration range of our assay, when it is conjugated to the tripodal platform, it is moderately active. These promising results suggest that our approach constitute a valid alternative to those reported so far.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4+ lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Synthesis, structure-activity relationship studies and biological evaluation of novel 2,5-disubstituted indole derivatives as anticancer agents.

Three novel series of 2,5-disubstituted indole derivatives were synthesized and evaluated in vitro for their antiproliferative activity against human cancer cells and HIV-1 inhibition activity used as a readout of cellular activity. Most compounds were found to have potent anticancer activity. In particular, 2c and 3b which showed effectively to repress HIV-1 transcription had a pan antiproliferative activity in cervical cancer cells (HeLa), breast cancer cells (MCF-7), liver cancer cells (HepG2), and lung cancer cells (H460 and A549). While 3b exhibited high sensitivity to A549 cells with the IC50 value 0.48 ± 0.15 µm, 2c showed high selectivity toward HepG2 cells with the IC50 value 13.21 ± 0.30 µm. With respect to the cellular mechanism of action, HepG2 cells treated with 2c and A549 cells treated with 3b for 24 h were studied by annexin V/PI staining and Western blot analysis, and results revealed that 2c and 3b may induce cancer cells apoptosis through inhibiting the phosphorylation at Ser2 of RNAPII CTD which can be phosphorylated by cyclin-dependent kinase 9. These studies indicated that 2c and 3b may develop as potent lead compounds in the therapy of cancer. However, determining their roles in preventing HIV-1 still requires further intensive study.