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HIV-1 Nef is an attractive target for antiretroviral drug discovery because of its roles promoting HIV-1 infectivity, replication, and host immune system avoidance. Here we applied a screening strategy in which recombinant HIV-1 Nef protein was coupled to activation of the Src-family tyrosine kinase Hck, which enhances the HIV-1 life cycle in macrophages. Nef stimulates recombinant Hck activity in vitro, providing a robust assay for chemical library screening. High-throughput screening of more than 730,000 compounds using the Nef•Hck assay identified six unique hit compounds that bound directly to recombinant Nef by SPR in vitro and inhibited HIV-1 replication in primary macrophages in the 0.04 to 5 μM range without cytotoxicity. Eighty-four analogs were synthesized around an isothiazolone scaffold from this series, many of which bound to recombinant Nef and inhibited HIV-1 infectivity in the low to submicromolar range. Compounds in this series restored MHC-I to the surface of HIV-infected primary cells and disrupted a recombinant protein complex of Nef with the C-terminal tail of MHC-I and the μ1 subunit of the AP-1 endocytic trafficking protein. Nef inhibitors in this class have the potential to block HIV-1 replication in myeloid cells and trigger recognition of HIV-infected cells by the adaptive immune system in vivo.

The substantial impact of acyclic nucleoside phosphonates (ANPs) on human medicine encourages the synthesis of new ANP analogues with a potentially differentiated antiviral spectrum. Herein, we demonstrate the functionalization of the 2-position of the (R,S)-3-hydroxy-2-(phosphonomethoxy)propyl side-chain of an inactive ANP with a polar cyano group to generate a thymine analogue with selective inhibition of hepatitis B virus (HBV) replication (SI > 302; EC50 = 0.33 μM), without significant antiretroviral activity. These findings suggest new strategies to synthesize unique ANPs with a targeted antiviral profile. ispartof: RSC MEDICINAL CHEMISTRY vol:12 issue:5 pages:804-808 ispartof: location:England status: published

Approximately 257 million people chronically infected with hepatitis B virus (HBV) worldwide are at risk of developing hepatocellular carcinoma (HCC). However, despite the availability of potent nucleoside/tide inhibitors, currently there are no curative therapies for chronic HBV infections. To identify potential new antiviral molecules, a select group of compounds previously evaluated in clinical studies were tested against 12 different viruses. Amongst the compounds tested, SRI-32007 (CYT997) demonstrated antiviral activity against HBV (genotype D) in HepG2.2.2.15 cell-based virus yield assay with 50% effective concentration (EC50) and selectivity index (SI) of 60.1 nM and 7.2, respectively. Anti-HBV activity of SRI-32007 was further confirmed against HBV genotype B in huh7 cells with secreted HBe antigen endpoint (EC50 40 nM and SI 250). To determine the stage of HBV life cycle inhibited by SRI-32007, time of addition experiment was conducted in HepG2-NTCP cell-based HBV infectious assay. Results indicated that SRI-32007 retained anti-HBV activity even when added 72 hours postinfection (72 h). Additional mechanism of action studies demonstrated potent inhibition of HBV core promoter activity by SRI-32007 with an EC50 of 40 nM and SI of >250. This study demonstrates anti-HBV activity of a repurposed compound SRI-32007 through inhibition of HBV core promoter activity. Further evaluation of SRI-32007 in HBV animal models is needed to confirm its activity in vivo. Our experiments illustrate the utility of repurposing strategy to identify novel antiviral chemical leads. HBV core promoter inhibitors such as SRI-32007 might enable the development of novel therapeutic strategies to combat HBV infections.

Background Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The “classic” quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 “next-generation” viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. Methods Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy–suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. Results Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1–3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6–5.4-fold). Frozen storage did not substantially alter infectious units per million values (−18%; 95% CI, −52% to 39%). Conclusions The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays., We demonstrated similar estimates of variation for classic and next-generation quantitative viral outgrowth assays (QVOAs), with enhanced sensitivity supporting the use of next-generation QVOAs as a proxy for classic QVOAs in measuring the latent human immunodeficiency virus reservoir.

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we developed a cell-based ‘gain of function’ assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. It was conceivable that the compounds inhibited the formation of infectious virions by targeting host cell proteins instead of Vpu directly, so we developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu.

In previous work, we described 6–6’-bisindole compounds targeting a hydrophobic pocket on the N-heptad repeat region of viral glycoprotein-41 as effective inhibitors of HIV-1 fusion. Two promising compounds with sub-micromolar IC(50)’s contained a benzoic acid group and a benzoic acid ester attached at the two indole nitrogens. Here we have conducted a thorough SAR study evaluating the contribution of each of the ring systems and various substituents to compound potency. Hydrophobicity, polarity and charge were varied to produce 35 new compounds that were evaluated in binding, cell-cell fusion and viral infectivity assays. We found that (a) activity based solely on increasing hydrophobic content plateaued at ~ 200 nM; (b) the bisindole scaffold surpassed other heterocyclic ring systems in efficacy; (c) a polar interaction possibly involving Gln575 in the pocket could supplant less specific hydrophobic interactions; and (d) the benzoic acid ester moiety did not appear to form specific contacts with the pocket. The importance of this hydrophobic group to compound potency suggests a mechanism whereby it might interact with a tertiary component during fusion, such as membrane. A promising small molecule 10b with sub-μM activity was discovered with molecular weight < 500 da and reduced logP compared to earlier compounds. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion.

The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus, (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC50, of 7.6 &plusmn, 5.8 &micro, M), (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC50, &gt, 100 &micro, (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells, (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity, (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.

Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.

The HIV nucleocapsid NCp7-SL2 RNA interaction is interrupted in the presence of a formally substitution-inert gold(dien)-nucleobase/N-heterocycle AuN(4) compound where the N-heterocycle serves the dual purposes of a template for “non-covalent” molecular recognition of the essential tryptophan of the protein, mimicking the natural reaction and subsequent “fixation” by Au-Cys bond formation providing a chemotype for a new distinct class of nucleocapsid-nucleic acid antagonist.

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40–60 times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells., Author summary Quantifying the number of cells harboring HIV-1 provirus is critical to evaluating HIV cure interventions, but precise quantification of the latent reservoir has proven to be technically challenging. Our data demonstrates that targeted differentiation of CD4+ T cells to an effector memory phenotype is a successful strategy for promoting latency reversal in vitro, and significantly enhances the performance of existing protocols to quantify the frequency of replication-competent HIV-1 in resting CD4+ T cells from virally-suppressed individuals. Using peripheral blood samples from well-established and characterized cohorts, we show the presence of a significantly higher frequency of replication-competent provirus than has previously been reported, raising the average estimated frequency 18-fold over the established QVOA measures. These results demonstrate that ex vivo effector memory differentiation has moved reservoir measurements closer to what may be the bona fide inducible replication-competent reservoir frequency, thus beginning to bridge the gap between viral outgrowth and molecular-based quantification. Taken together, these data support accumulating evidence that effector memory differentiation is a key pathway to HIV-1 latency reversal that may be exploited for assay development, mechanistic understanding, and therapeutic interventions.

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a “gold standard” for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates—as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir—rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points., Author summary The latent reservoir of resting CD4+ T cells is a major, if not the primary, obstacle to curing HIV. Quantitative viral outgrowth assays (QVOAs) are used to measure the latent reservoir in ART-suppressed HIV-infected people. Using QVOA is difficult, however, as the fraction of cells constituting the latent reservoir is typically about one in one million, far lower than other infectious disease biomarkers. To study reliability of these assays, we distributed 75 PBMC samples from five ART-suppressed HIV-infected participants among four labs, each conducting QVOA and following prespecified sample batching procedures. Using a Bayesian statistical method, we analyzed detailed assay output to understand how results varied within batches, between batches, and between labs. We found that, if batch variation can be controlled (i.e., a lab assays all samples in one batch), typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. We also found that freezing, storing, and thawing samples for later analysis caused no more than a 2-fold change in results. These outcomes, and the statistical methods developed to obtain them, should lead towards more precise and powerful assessments of HIV cure strategies.

The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, β-octyl glucoside (βOG). X-ray crystallography revealed that a single βOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-βOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54–66. Nef dimer formation is stabilized in solution by the addition of βOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.

The removal of one cell from cleaving embryos (blastomere biopsy, BB) is the main component of pre-implantation genetic diagnosis (PGD), a diagnostic test aimed to select only healthy embryos before transfer in utero. Studies on animals have suggested BB as risk factor for impaired development during both pre- and postnatal life. However, we still have incomplete knowledge on the real side effects of BB and the mechanisms underlying them. The present study was designed to evaluate whether BB or miscellaneous factors (in vitro culture, IVC; and embryo transfer, ET) may affect developmental potential of conceptuses obtained following BB (i.e. pregnancy rate/loss, fetal/placental weight, feto:placenta ratio) and placental epigenetic programming. To this aim, 3-month-old C57 females, naturally mated without hormonal stimulation, were used as donors. At 2.5 days post-coitum (dpc), 8-cell stage embryos were collected and subjected to BB in PBS (without Ca2+/Mg2+) supplemented with 0.4% BSA and antibiotics. Embryos were cultured in KSOM medium in an incubator at 37.5°C and 5% CO2. Two in vitro control groups were used: (1) IVC, embryos cultured in vitro, not subjected to biopsy; (2) ET, blastocysts collected at 3.5 dpc and directly transferred into recipient females. At 3.5 dpc, 10 to 12 blastocysts were transferred into pseudo pregnant females (n ≥ 7/group). An additional control group consisted of naturally conceived pregnancy (n = 8, in vivo control, CTR). At 18.5 dpc, conceptuses were collected and subjected to gross morphological evaluations (n > 20 conceptuses/group). Placentae (n ≥ 4/group) were analysed for 5-methylation and hydroxymethylation content by ELISA assay. Decimal variables were analysed using the Mann-Whitney test, and percentages were analysed with the Fisher exact test. Assessment of development to term revealed reduced survival rate in BB, IVC, and ET v. CTR (45.23, 47.82, 60, and 98.15% respectively; P

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC50 as low as 8.4 μm and a TC50 of >100 μm when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.

Author(s): Stone, M; Rosenbloom, D; Bacchetti, P; Deng, X; Busch, M; Dimapasoc, M; Keating, S; Richman, D; Mellors, J; Deeks, S; Siliciano, J; Chomont, N; Ptak, R

Acyclic nucleosides containing a 3-fluoro-2-(phosphonomethoxy)propyl (FPMP) side chain are known to be moderately potent antihuman immunodeficiency virus (HIV) agents, while being completely devoid of antiviral activity against a wide range of DNA viruses. The derivatization of the phosphonic acid functionality of FPMPs with a diamyl aspartate phenoxyamidate group led to a novel generation of compounds that not only demonstrate drastically improved antiretroviral potency but also are characterized by an expanded spectrum of activity that also covers hepatitis B and herpes viruses. The best compound, the (S)-FPMPA amidate prodrug, exerts anti-HIV-1 activity in TZM-bl and peripheral blood mononuclear cells at low nanomolar concentrations and displays excellent potency against hepatitis B virus (HBV) and varicella-zoster virus (VZV). This prodrug is stable in acid and human plasma media, but it is efficiently processed in human liver microsomes with a half-life of 2 min. The (R) isomeric guanine derivative emerged as a selectively active anti-HIV and anti-HBV inhibitor, while being nontoxic to human hepatoblastoma cells. Notably, the pyrimidine containing prodrug (S)-Asp-FPMPC is the only congener within this series to demonstrate micromolar antihuman cytomegalovirus (HCMV) potency.

Zika virus (ZIKV) is transmitted by mosquitoes and causes Dengue-like illness, neurological symptoms such as Guillain-Barre Syndrome and microcephaly in children born to infected pregnant mothers. Recently, the World Health Organization (WHO) declared ZIKV infection as a Global Health Emergency. However, there are no known prophylactic or therapeutic measures against this virus. As a proof of concept toward combination therapeutic strategy against ZIKV, combinations of host-targeted (Interferon-α and Interferon-β) and direct acting (Sofosbuvir) antivirals were evaluated in a hepatic cell line (Huh7) using a Cytoprotection (CP) assay. The combination of these antivirals resulted in synergistic inhibition of ZIKV infection in the in vitro CP assay. Additional testing in a ZIKV yield assay demonstrated that combination treatment of these antivirals conferred >2-log reduction in the release of viral RNA. Measurement of ZIKV proteins in the cells infected with multiple ZIKV strains isolated from different geographical regions (Americas, Asia, and Africa) using an immunofluorescence assay confirmed the effective antiviral activity of this combination against ZIKV. These results demonstrate the in vitro proof of concept (POC) for using a combination approach utilizing the strengths of both virus and host-targeted antivirals. These results suggest the effectiveness of the combination strategy in combating ZIKV, in the in vitro systems. Further evaluation of such combination therapies in vivo might provide an impetus for the development of effective ZIKV therapeutic strategies.

The ‘Human Immunodeficiency Virus Type 1 (HIV-1), Human Interaction Database’, available through the National Library of Medicine at http://www.ncbi.nlm.nih.gov/genome/viruses/ retroviruses/hiv-1/interactions, serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. Each HIV-1 human protein interaction can be retrieved without restriction by web-based downloads and ftp protocols and includes: Reference Sequence (RefSeq) protein accession numbers, National Center for Biotechnology Information Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. In addition to specific HIV-1 protein–human protein interactions, included are interaction effects upon HIV-1 replication resulting when individual human gene expression is blocked using siRNA. A total of 3142 human genes are described participating in 12 786 protein–protein interactions, along with 1316 replication interactions described for each of 1250 human genes identified using small interfering RNA (siRNA). Together the data identifies 4006 human genes involved in 14 102 interactions. With the inclusion of siRNA interactions we introduce a redesigned web interface to enhance viewing, filtering and downloading of the combined data set.

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672–681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD's virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC 50 ) of PD, when diluted in seminal plasma, is ∼1 μM, similar to the IC 50 determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC 50 ], >300 μM), freshly activated human peripheral blood mononuclear cells (CC 50 , ∼200 μM), and primary CD4 + T cells, macrophages, and dendritic cells (CC 50 , >300 μM). PD also exhibited high stability in pH-adjusted Dulbecco's phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD's microbicidal candidacy in animals and humans.

DEB025 (alisporivir) is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV). It binds to cyclophilin A (CypA) and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA) region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96), as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent.

The synthesis of four l-2′-deoxy-threose nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine, and guanosine has been performed. Especially the adenine containing analogue (PMDTA) was endowed with potent antiviral activity displaying an EC50 of 4.69 μM against HIV-1 and an EC50 value of 0.5 μM against HBV, whereas completely lacking cytotoxicity. The synthesis of a number of phosphonomonoamidate and phosphonobisamidate prodrugs of PMDTA led to a boost in antiviral potency. The most potent congeners were a l-aspartic acid diisoamyl ester phenoxy prodrug and a l-phenylalanine propyl ester phosphonobisamidate prodrug that both display anti-HIV and anti-HBV activities in the low nanomolar range and selectivity indexes of more than 300.

In an attempt to identify novel nucleoside phosphoramidate analogues for improving the anti-HCV activity of 2’-C-Me-uridine, we have synthesized for the first time a series of L-glutamic acid, L-serine, L-threonine and L-tyrosine containing aryloxyphosphoramidate prodrugs of 2’-C-Me-uridine. Evaluation of their activity against HCV revealed that they displayed very potent anti-HCV activity, with EC50 values that are in the same range as of Sofosbuvir. ispartof: Organic & Biomolecular Chemistry vol:14 issue:37 pages:8743-8757 ispartof: location:England status: published

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag–Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90 ), most of the Gag and Gag–Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.

Despite the rapid spread of Zika virus (ZIKV) infection and associated neurological complications in the America's, prophylactic or therapeutic countermeasures are not currently available. This is mostly due to the fact that until recently there was no presumed need for medical intervention since there was no association between ZIKV infection and significant human morbidity. Consequently, there are currently no tools due mostly to the lack of sensitive cell based assays amenable for identification of ZIKV inhibitors. To address this unmet need we have developed a cell based virus yield assay suitable for testing antivirals against Zika virus. Using bioinformatics, several isolates of ZIKV from the Americas, Africa, and Asia were analyzed for sequence similarity. The alignment data were then used to design primers targeting a ZIKV genomic region that was highly conserved among all the ZIKV isolates. Subsequently, primers were used in a sensitive, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to detect ZIKV RNA. The qRT-PCR assay was found to be highly sensitive (lower limit of detection between-10-100 copies) and reproducible. Evaluation of the primers and probes used for ZIKV against another flavivirus (Dengue virus) demonstrated specificity of detection. To evaluate potential of qRT-PCR assay as an antiviral screening tool against ZIKV, Vero cells pretreated with Type I Interferons (IFN α) were infected with virus, followed by measurement of ZIKV RNA found in the cell culture supernatants using qRT-PCR assay. Dose-dependent antiviral activity of Type I Interferons and mycophenolic acid (MPA) against Zika virus in this cell culture system was confirmed using qRT-PCR. Due to reproducible assay performance, qPCR associated higher sensitivity and short duration of the assay time, this novel cell based assay will be very useful for confirming the activity of antivirals against ZIKV.

Nonpeptide inhibition of fusion remains an important goal in anti-HIV research, due to its potential for low cost prophylaxis or prevention of cell-cell transmission of the virus. We report here on a series of indole compounds that have been identified as fusion inhibitors of gp41 through a structure-based drug design approach. Experimental binding affinities of the compounds for the hydrophobic pocket were strongly correlated to fusion inhibitory data (R(2) = 0.91), and corresponding inhibition of viral replication confirmed the hydrophobic pocket as a valid target for low molecular weight fusion inhibitors. The most active compound bound to the hydrophobic pocket and inhibited cell-cell fusion and viral replication at submicromolar levels. A common binding mode for the inhibitors in this series was established by carrying out docking studies using structures of gp41 in the Protein Data Bank. The molecules were flexible enough to conform to the contours of the pocket, and the most active compound was able to adopt a structure mimicking the hydrophobic contacts of the D-peptide PIE7. The results enhance our understanding of indole compounds as inhibitors of gp41.

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag / gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis -packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5′-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag , providing further support for the trans- packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.

Blocking HIV-1 cell entry has long been a major goal of anti-HIV drug development. Here, we report a successful design of two highly potent chimeric HIV entry inhibitors composed of one CCR5-targeting RANTES (regulated on activation normal T cell expressed and secreted) variant (5P12-RANTES or 5P14-RANTES (Gaertner, H., Cerini, F., Escola, J. M., Kuenzi, G., Melotti, A., Offord, R., Rossitto-Borlat, I., Nedellec, R., Salkowitz, J., Gorochov, G., Mosier, D., and Hartley, O. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17706-17711)) linked to a gp41 fusion inhibitor, C37. Chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed extremely high antiviral potency in single cycle and replication-competent viral assays against R5-tropic viruses, with IC(50) values as low as 0.004 nm. This inhibition was somewhat strain-dependent and was up to 100-fold better than the RANTES variant alone or in combination with unlinked C37. The chimeric inhibitors also fully retained the antiviral activity of C37 against X4-tropic viruses, and this inhibition can be further enhanced significantly if the target cell co-expresses CCR5 receptor. On human peripheral blood mononuclear cells, the inhibitors showed very strong inhibition against R5-tropic Ba-L strain and X4-tropic IIIB strain, with IC(50) values as low as 0.015 and 0.44 nm, which are 45- and 16-fold better than the parent inhibitors, respectively. A clear delivery mechanism requiring a covalent linkage between the two segments of the chimera was observed and characterized. Furthermore, the two chimeric inhibitors are fully recombinant and are easily produced at low cost. These attributes make them excellent candidates for anti-HIV microbicides. The results of this study also suggest a potent approach for optimizing existing HIV entry inhibitors or designing new inhibitors.

The development of an anti-HIV microbicide is critical in the fight against the spread of HIV. It is shown here that the covalent linking of compounds that bind gp120 with compounds that bind gp41 can inhibit HIV entry even more potently than individual inhibitors or noncovalent combinations. The most striking example involves griffithsin, a potent HIV inhibitor that binds to the surface of HIV gp120. While griffithsin inhibits HIV Env-mediated fusion in a CCR5-tropic cell-cell fusion assay with a 50% inhibitory concentration (IC 50 ) of 1.31 ± 0.87 nM and the gp41-binding peptide C37 shows an IC 50 of 18.2 ± 7.6 nM, the covalently linked combination of griffithsin with C37 (Griff37) has an IC 50 of 0.15 ± 0.05 nM, exhibiting a potency 8.7-fold greater than that of griffithsin alone. Similarly, in CXCR4-tropic cell-cell fusion assays, Griff37 is 5.2-fold more potent than griffithsin alone. In viral assays, both griffithsin and Griff37 inhibit HIV replication at midpicomolar levels, but the linked compound Griff37 is severalfold more potent than griffithsin alone against both CCR5- and CXCR4-tropic virus strains. Another example of this strategy is the covalently linked combination of peptide C37 with a variant of the gp120-binding peptide CD4M33 (L. Martin et al., Nat. Biotechnol. 21:71-76, 2003). Also, nuclear magnetic resonance (NMR) spectra for several of these compounds are shown, including, to our knowledge, the first published NMR spectrum for griffithsin.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Moloney leukemia virus 10 (MOV10) protein is a superfamily-1 RNA helicase, and it is also a component of the RNA-induced silencing complex. Recent studies have shown that MOV10 plays an active role in the RNA interference pathway. Here, we report that MOV10 inhibits retrovirus replication. When it was overexpressed in viral producer cells, MOV10 was able to reduce the infectivity of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and murine leukemia virus. Conversely, when MOV10 expression was reduced by small interfering RNAs, HIV-1 infectivity was increased. Consistently, silencing of MOV10 expression in a human T cell line enhanced HIV-1 replication. Furthermore, we found that MOV10 interacts with HIV-1 nucleocapsid protein in an RNA-dependent manner and is packaged into virions. It blocks HIV-1 replication at a postentry step. In addition, we also found that HIV-1 could suppress MOV10 protein expression to counteract this cellular resistance. All of these results indicate that MOV10 has a broad antiretroviral activity that can target a wide range of retroviruses, and it could be actively involved in host defense against retroviral infection.

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5′ monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1H10found five point mutations in the HIV-1 genenef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1H10. Transfer of the mutations from TCN-resistant isolate HIV-1H10to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations innefare necessary and sufficient for TCN resistance.

Since the 1980s, the rapid progression of the HIV/AIDS pandemic has prompted a major international research effort. As a result, the current knowledge on HIV biology, its evolution, and origins [1–5] exceeds that of many, if not all, other viruses. One of the most important areas of HIV research is the detailed understanding of HIV replication. As with all viruses, HIV must exploit the host’s cellular machinery and metabolism to copy its genetic material, synthesize viral proteins, and assemble new virions. The viral replication cycle is thus dependent on an intricate network of direct and indirect protein interactions: between the viral proteins, between the virus and the host, and ultimately between the various host proteins that constitute the subverted cellular systems. When we also take into account the host’s immune response and intrinsic antiviral factors, there are clearly a large number of host–pathogen relationships that are important to our full understanding of HIV biology. However, until recently [6], this valuable information has remained ‘locked’ in the published literature, making it time-consuming to study by individual researchers and inaccessible to computational analysis, thus hindering the progress of research. Here we highlight new developments in the area of host–pathogen systems biology that in our opinion will provide helpful insights to the HIVresearch community.

The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in developing a viable AIDS vaccine. We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of such antibodies. In an attempt to develop HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp41(89.6) was fused to a human IgG(1) Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp140(89.6). Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies. Further research is needed to confirm and extend these results which may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies.

The 'Human Immunodeficiency Virus Type 1 (HIV-1), Human Protein Interaction Database', available through the National Library of Medicine at www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions, was created to catalog all interactions between HIV-1 and human proteins published in the peer-reviewed literature. The database serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. To facilitate this discovery approach, the following information for each HIV-1 human protein interaction is provided and can be retrieved without restriction by web-based downloads and ftp protocols: Reference Sequence (RefSeq) protein accession numbers, Entrez Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. Currently, 2589 unique HIV-1 to human protein interactions and 5135 brief descriptions of the interactions, with a total of 14,312 PMID references to the original articles reporting the interactions, are stored in this growing database. In addition, all protein-protein interactions documented in the database are integrated into Entrez Gene records and listed in the 'HIV-1 protein interactions' section of Entrez Gene reports. The database is also tightly linked to other databases through Entrez Gene, enabling users to search for an abundance of information related to HIV pathogenesis and replication.

Entry inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been the focus of much recent research. C34, a potent fusion inhibitor derived from the HR2 region of gp41, was engineered into a 1:1 human serum albumin conjugate through stable covalent attachment of a maleimido-C34 analog onto cysteine 34 of albumin. This bioconjugate, PC-1505, was designed to require less frequent dosing and less peptide than T-20 and was assessed for its antifusogenic activity both in vitro and in vivo in the SCID-hu Thy/Liv mouse model. PC-1505 was essentially equipotent to the original C34 peptide and to T-20 in vitro. In HIV-1-infected SCID-hu Thy/Liv mice, T-20 lost activity with infrequent dosing, whereas the antiviral potency of PC-1505 was sustained, and PC-1505 was active against T-20-resistant ("DIV") virus with a G36D substitution in gp41. The in vivo results are the direct result of a significantly improved pharmacokinetic profile for the C34 peptide following albumin conjugation. Contrary to previous reports that the gp41 NHR trimer is poorly accessible to C34 fused to protein cargoes of increasing size (Hamburger, A. E., Kim, S., Welch, B. D., and Kay, M. S. (2005) J. Biol. Chem. 280, 12567-12572), these results are the first demonstration of the capacity for a large, endogenous serum protein to gain unobstructed access to the transient gp41 intermediates that exist during the HIV fusion process, and it supports further development of albumin conjugation as a promising approach to inhibit HIV-1 entry.