Schlafen 12 restricts HIV-1 latency reversal by a codon-usage dependent post-transcriptional block in CD4+ T cells.

Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.

Importance of structure-based studies for the design of a novel HIV-1 inhibitor peptide.

Based on the structure of an HIV-1 entry inhibitor peptide two stapled- and a retro-enantio peptides have been designed to provide novel prevention interventions against HIV transmission. The three peptides show greater inhibitory potencies in cellular and mucosal tissue pre-clinical models than the parent sequence and the retro-enantio shows a strengthened proteolytic stability. Since HIV-1 fusion inhibitor peptides need to be embedded in the membrane to properly interact with their viral target, the structural features were determined by NMR spectroscopy in micelles and solved by using restrained molecular dynamics calculations. Both parent and retro-enantio peptides demonstrate a topology compatible with a shared helix-turn-helix conformation and assemble similarly in the membrane maintaining the active conformation needed for its interaction with the viral target site. This study represents a straightforward approach to design new targeted peptides as HIV-1 fusion inhibitors and lead us to define a retro-enantio peptide as a good candidate for pre-exposure prophylaxis against HIV-1.

Labyrinthopeptins Exert Broad-Spectrum Antiviral Activity through Lipid-Binding-Mediated Virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

Peptide Assembly on the Membrane Determines the HIV-1 Inhibitory Activity of Dual-Targeting Fusion Inhibitor Peptides.

Novel strategies in the design of HIV-1 fusion/entry inhibitors are based on the construction of dual-targeting fusion proteins and peptides with synergistic antiviral effects. In this work we describe the design of dual-targeting peptides composed of peptide domains of E2 and E1 envelope proteins from Human Pegivirus with the aim of targeting both the loop region and the fusion peptide domains of HIV-1 gp41. In a previous work, we described the inhibitory role of a highly conserved fragment of the E1 protein (domain 139-156) which interacts with the HIV-1 fusion peptide at the membrane level. Here, two different dual-targeting peptides, where this E1 peptide is located on the N- or the C-terminus respectively, have been chemically synthesized and their antiviral activities have been evaluated with HIV pseudotyped viruses from different clades. The study of the functional behaviour of peptides in a membranous environment attending to the peptide recognition of the target sites on gp41, the peptide conformation as well as the peptide affinity to the membrane, demonstrate that antiviral activity of the dual-targeting peptides is directly related to the peptide affinity and its subsequent assembly into the model membrane. The overall results point out to the necessity that fusion inhibitor peptides that specifically interfere with the N-terminal region of gp41 are embedded within the membrane in order to properly interact with their viral target.

HIV LTR-Driven Antisense RNA by Itself Has Regulatory Function and May Curtail Virus Reactivation From Latency.

Latently infected T lymphocytes are an important barrier toward eliminating a persistent HIV infection. Here we describe an HIV-based recombinant fluorescent-lentivirus referred to as "rfl-HIV" that enables to analyze sense and antisense transcription by means of fluorescence reporter genes. This model virus exhibited similar transcriptional and functional properties of the antisense transcript as observed with a wild type HIV, and largely facilitated the generation of latently-infected T cells clones. We show that latently-infected cells can be divided into two types, those with and those without antisense transcription. Upon addition of latency reversal agents, only the cells that lack antisense transcripts are readily reactivated to transcribe HIV. Thus, antisense transcripts may exhibit a dominant suppressor activity and can lock an integrated provirus into a non-reactivatable state. These findings could have important implications for the development of strategies to eradicate HIV from infected individuals.

The Myxobacterial Metabolite Soraphen A Inhibits HIV-1 by Reducing Virus Production and Altering Virion Composition.

Soraphen A is a myxobacterial metabolite that blocks the acetyl-coenzyme A carboxylase of the host and was previously identified as a novel HIV inhibitor. Here, we report that soraphen A acts by reducing virus production and altering the gp120 virion content, impacting entry capacity and infectivity. These effects are partially reversed by addition of palmitic acid, suggesting that inhibition of HIV envelope palmitoylation is one of the mechanisms of antiviral action.

Benzyl-2-Acetamido-2-Deoxy-α-d-Galactopyranoside Increases Human Immunodeficiency Virus Replication and Viral Outgrowth Efficacy In Vitro.

Glycosylation of host and viral proteins is an important posttranslational modification needed to ensure correct function of glycoproteins. For this reason, we asked whether inhibition of O-glycosylation during human immunodeficiency virus (HIV) in vitro replication could affect HIV infectivity and replication rates. We used benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN), a compound that has been widely used to inhibit O-glycosylation in several cell lines. Pretreatment and culture of PHA-blast target cells with BAGN increased the percentage of HIV-infected cells (7.6-fold, p = 0.0115), the per-cell amount of HIV p24 protein (1.3-fold, p = 0.2475), and the viral particles in culture supernatants (7.1-fold, p = 0.0029) compared to BAGN-free cultures. Initiating infection with virus previously grown in the presence of BAGN further increased percentage of infected cells (30-fold, p < 0.0001), intracellular p24 (1.5-fold, p = 0.0433), and secreted viral particles (74-fold, p < 0.0001). BAGN-treated target cells showed less CD25 and CCR5 expression, but increased HLA-DR surface expression, which positively correlated with the number of infected cells. Importantly, BAGN improved viral outgrowth kinetics in 66% of the samples tested, including samples from HIV controllers and subjects in whom no virus could be expanded in the absence of BAGN. Sequencing of the isolated virus indicated no skewing of viral quasi-species populations when compared to BAGN-free culture conditions. BAGN also increased virus production in the ACH2 latency model when used together with latency-reversing agents. Taken together, our results identify BAGN treatment as a simple strategy to improve viral outgrowth in vitro and may provide novel insights into host restriction mechanisms and O-glycosylation-related therapeutic targets for HIV control strategies.

Position effects influence HIV latency reversal.

The main obstacle to curing HIV is the presence of latent proviruses in the bodies of infected patients. The partial success of reactivation therapies suggests that the genomic context of integrated proviruses can interfere with treatment. Here we developed a method called Barcoded HIV ensembles (B-HIVE) to map the chromosomal locations of thousands of individual proviruses while tracking their transcriptional activities in an infected cell population. B-HIVE revealed that, in Jurkat cells, the expression of HIV is strongest close to endogenous enhancers. The insertion site also affects the response to latency-reversing agents, because we found that phytohemagglutinin and vorinostat reactivated proviruses inserted at distinct genomic locations. From these results, we propose that combinations of drugs targeting all areas of the genome will be most effective. Overall, our data suggest that the insertion context of HIV is a critical determinant of the viral response to reactivation therapies.

Homeostatically Maintained Resting Naive CD4+ T Cells Resist Latent HIV Reactivation.

Homeostatic proliferation (HSP) is a major mechanism by which long-lived naïve and memory CD4+ T cells are maintained in vivo and suggested to contribute to the persistence of the latent HIV-1 reservoir. However, while many in vitro latency models rely on CD4+ T cells that were initially differentiated via T-cell receptor (TCR) stimulation into memory/effector cells, latent infection of naïve resting CD4+ T cells maintained under HSP conditions has not been fully addressed. Here, we describe an in vitro HSP culture system utilizing the cytokines IL-7 and IL-15 that allows studying latency in naïve resting CD4+ T cells. CD4+ T cells isolated from several healthy donors were infected with HIV pseudotypes expressing GFP and cultured under HSP conditions or TCR conditions as control. Cell proliferation, phenotype, and GFP expression were analyzed by flow cytometry. RNA expression was quantified by qRT-PCR. Under HSP culture conditions, latently HIV-1 infected naïve cells are in part maintained in the non-dividing (= resting) state. Although a few HIV-1 provirus+ cells were present in these resting GFP negative cells, the estimated level of GFP transcripts per infected cell seems to indicate a block at the post-transcriptional level. Interestingly, neither TCR nor the prototypic HDAC inhibitor SAHA were able to reactivate HIV-1 provirus from these cells. This lack of reactivation was not due to methylation of the HIV LTR. These results point to a mechanism of HIV control in HSP-cultured resting naïve CD4+ T cells that may be distinct from that in TCR-stimulated memory/effector T cells.

Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

Low Seroprevalence of West Nile Virus in Blood Donors from Catalonia, Spain.

West Nile virus (WNV) is an emerging arbovirus first recognized in Europe in the 1950s. Since then, outbreaks have been reported in several European countries. In 2010, the first WNV outbreak was recorded in Spain, affecting the southern part of the country. We conducted a seroprevalence study in the Catalonia region (northeastern Spain), an area considered at high risk of arbovirus transmission. A total of 800 serum samples from blood donors were collected and screened for antibodies against WNV by enzyme-linked immunosorbent assay (ELISA) and confirmed by a microneutralization assay. More than 50 samples tested positive by ELISA, but only one sample contained neutralizing antibodies against WNV and was obtained from a donor native of Pakistan. The low seroprevalence detected may serve as reference baseline data for monitoring WNV activity in our region in future years.

Soraphen A: A broad-spectrum antiviral natural product with potent anti-hepatitis C virus activity.

BACKGROUND & AIMS: Soraphen A (SorA) is a myxobacterial metabolite that inhibits the acetyl-CoA carboxylase, a key enzyme in lipid biosynthesis. We have previously identified SorA to efficiently inhibit the human immunodeficiency virus (HIV). The aim of the present study was to evaluate the capacity of SorA and analogues to inhibit hepatitis C virus (HCV) infection. METHODS: SorA inhibition capacity was evaluated in vitro using cell culture derived HCV, HCV pseudoparticles and subgenomic replicons. Infection studies were performed in the hepatoma cell line HuH7/Scr and in primary human hepatocytes. The effects of SorA on membranous web formation were analysed by electron microscopy. RESULTS: SorA potently inhibits HCV infection at nanomolar concentrations. Obtained EC50 values were 0.70 nM with a HCV reporter genome, 2.30 nM with wild-type HCV and 2.52 nM with subgenomic HCV replicons. SorA neither inhibited HCV RNA translation nor HCV entry, as demonstrated with subgenomic HCV replicons and HCV pseudoparticles, suggesting an effect on HCV replication. Consistent with this, evidence was obtained that SorA interferes with formation of the membranous web, the site of HCV replication. Finally, a series of natural and synthetic SorA analogues helped to establish a first structure-activity relationship. CONCLUSIONS: SorA has a very potent anti-HCV activity. Since it also interferes with the membranous web formation, SorA is an excellent tool to unravel the mechanism of HCV replication.

The myxobacterial metabolite ratjadone A inhibits HIV infection by blocking the Rev/CRM1-mediated nuclear export pathway.

BACKGROUND: The nuclear export of unspliced and partially spliced HIV-1 mRNA is mediated by the recognition of a leucine-rich nuclear export signal (NES) in the HIV Rev protein by the host protein CRM1/Exportin1. This makes the CRM1-Rev complex an attractive target for the development of new antiviral drugs. Here we tested the anti-HIV efficacy of ratjadone A, a CRM1 inhibitor derived from myxobacteria. RESULTS: Ratjadone A inhibits HIV infection in vitro in a dose-dependent manner with EC50 values at the nanomolar range. The inhibitory effect of ratjadone A occurs around 12 hours post-infection and is specific for the Rev/CRM1-mediated nuclear export pathway. By using a drug affinity responsive target stability (DARTS) assay we could demonstrate that ratjadone A interferes with the formation of the CRM1-Rev-NES complex by binding to CRM1 but not to Rev. CONCLUSION: Ratjadone A exhibits strong anti-HIV activity but low selectivity due to toxic effects. Although this limits its potential use as a therapeutic drug, further studies with derivatives of ratjadones might help to overcome these difficulties in the future.

Identification of myxobacteria-derived HIV inhibitors by a high-throughput two-step infectivity assay.

BACKGROUND: Drug-resistance and therapy failure due to drug-drug interactions are the main challenges in current treatment against Human Immunodeficiency Virus (HIV) infection. As such, there is a continuous need for the development of new and more potent anti-HIV drugs. Here we established a high-throughput screen based on the highly permissive TZM-bl cell line to identify novel HIV inhibitors. The assay allows discriminating compounds acting on early and/or late steps of the HIV replication cycle. RESULTS: The platform was used to screen a unique library of secondary metabolites derived from myxobacteria. Several hits with good anti-HIV profiles were identified. Five of the initial hits were tested for their antiviral potency. Four myxobacterial compounds, sulfangolid C, soraphen F, epothilon D and spirangien B, showed EC50 values in the nM range with SI > 15. Interestingly, we found a high amount of overlapping hits compared with a previous screen for Hepatitis C Virus (HCV) using the same library. CONCLUSION: The unique structures and mode-of-actions of these natural compounds make myxobacteria an attractive source of chemicals for the development of broad-spectrum antivirals. Further biological and structural studies of our initial hits might help recognize smaller drug-like derivatives that in turn could be synthesized and further optimized.

Screening of small molecules affecting mammalian P-body assembly uncovers links with diverse intracellular processes and organelle physiology.

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

Myxobacteria: natural pharmaceutical factories.

Myxobacteria are amongst the top producers of natural products. The diversity and unique structural properties of their secondary metabolites is what make these social microbes highly attractive for drug discovery. Screening of products derived from these bacteria has revealed a puzzling amount of hits against infectious and non-infectious human diseases. Preying mainly on other bacteria and fungi, why would these ancient hunters manufacture compounds beneficial for us? The answer may be the targeting of shared processes and structural features conserved throughout evolution.

Fitness ranking of individual mutants drives patterns of epistatic interactions in HIV-1.

Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1) resistance to azidothymidine (AZT) requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41-215 cluster) of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions.