Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection.

G-quadruplexes (G4s) are four-stranded nucleic acid structures formed by the stacking of G-tetrads. Here we investigated their formation and function during HIV-1 infection. Using bioinformatics and biophysics analyses we first searched for evolutionary conserved G4-forming sequences in HIV-1 genome. We identified 10 G4s with conservation rates higher than those of HIV-1 regulatory sequences such as RRE and TAR. We then used porphyrin-based G4-binders to probe the formation of the G4s during infection of human cells by native HIV-1. The G4-binders efficiently inhibited HIV-1 infectivity, which is attributed to the formation of G4 structures during HIV-1 replication. Using a qRT-PCR approach, we showed that the formation of viral G4s occurs during the first 2 h post-infection and their stabilization by the G4-binders prevents initiation of reverse transcription. We also used a G4-RNA pull-down approach, based on a G4-specific biotinylated probe, to allow the direct detection and identification of viral G4-RNA in infected cells. Most of the detected G4-RNAs contain crucial regulatory elements such as the PPT and cPPT sequences as well as the U3 region. Hence, these G4s would function in the early stages of infection when the viral RNA genome is being processed for the reverse transcription step.

Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Template requirements of Zika RNA polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA.

As ZIKV continues to spread, many "unknowns" remain and research is needed to advance the understanding of this important pathogen. Viral RNA dependent-RNA polymerases (RdRp) are validated targets for inhibitors of the replication of several viruses. Several studies have set up in vitro enzymatic assays of the RdRp of the Zika virus for testing of candidate inhibitors. While most of these studies use short synthetic polymers, we have shown in a previous work that the Zika polymerase domain is capable of a de novo synthesis of the viral genome using the natural viral RNA as template. Here we have studied the role of the sequences at the 3'end of the minus-strand RNA in the initiation of the RNA synthesis by the Zika isolated RdRp. Our results strongly suggest that the region containing the 105 first nucleotides from the 3' end of the minus-strand RNA is important for initiation of the positive RNA synthesis. This indicates that this region displays all the primary and secondary structures to be efficiently recognized by the recombinant RdRp in vitro. Moreover, we show that the 46 nucleotides are sufficient to initiate RNA synthesis. In addition, the ZIKV polymerase domain poorly replicated the RNA of other RNA viruses and appeared highly selective for its own RNA.

Absence of Resistance Mutations in the Integrase Coding Region among ART-Experienced Patients in the Republic of the Congo.

BACKGROUND: HIV infects around one hundred thousand patients in the Republic of the Congo. Approximately 25% of them receive an antiretroviral treatment; current first-line regimens include two NRTIs and one NNRTI, reverse transcriptase inhibitors. Recently, protease inhibitors (PIs) were also introduced as second-line therapy upon clinical signs of treatment failure. Due to the limited number of molecular characterizations and amount of drug resistance data available in the Republic of the Congo, this study aims to evaluate the prevalence of circulating resistance mutations within the pol region. METHODS: HIV-positive, ART-experienced patients have been enrolled in four semi-urban localities in the Republic of the Congo. Plasma samples were collected, and viral RNA was extracted. The viral load for each patient was evaluated by RT-qPCR, following the general diagnostic procedures of the University Hospital of Bordeaux. Finally, drug resistance genotyping and phylogenetic analysis were conducted following Sanger sequencing of the pol region. RESULTS: A high diversity of HIV-1 strains was observed with many recombinant forms. Drug resistance mutations in RT and PR genes were determined and correlated to HAART. Because integrase inhibitors are rarely included in treatments in the Republic of the Congo, the prevalence of integrase drug resistance mutations before treatment was also determined. Interestingly, very few mutations were observed. CONCLUSIONS: We confirmed a high diversity of HIV-1 in the Republic of the Congo. Most patients presented an accumulation of mutations conferring resistance against NRTIs, NNRTIs and PIs. Nonetheless, the absence of integrase mutations associated with drug resistance suggests that the introduction of integrase inhibitors into therapy will be highly beneficial to patients in the Republic of the Congo.

The properties of hot household hygroscopic materials and their potential use for non-medical facemask decontamination.

The widespread use of facemasks throughout the population is recommended by the WHO to reduce transmission of the SARS-CoV-2 virus. As some regions of the world are facing mask shortages, reuse may be necessary. However, used masks are considered as a potential hazard that may spread and transmit disease if they are not decontaminated correctly and systematically before reuse. As a result, the inappropriate decontamination practices that are commonly witnessed in the general public are challenging management of the epidemic at a large scale. To achieve public acceptance and implementation, decontamination procedures need to be low-cost and simple. We propose the use of hot hygroscopic materials to decontaminate non-medical facemasks in household settings. We report on the inactivation of a viral load on a facial mask exposed to hot hygroscopic materials for 15 minutes. As opposed to recent academic studies whereby decontamination is achieved by maintaining heat and humidity above a given value, a more flexible procedure is proposed here using a slow decaying pattern, which is both effective and easier to implement, suggesting straightforward public deployment and hence reliable implementation by the population.

Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration.

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

Quinolinonyl Non-Diketo Acid Derivatives as Inhibitors of HIV-1 Ribonuclease H and Polymerase Functions of Reverse Transcriptase.

Novel anti-HIV agents are still needed to overcome resistance issues, in particular inhibitors acting against novel viral targets. The ribonuclease H (RNase H) function of the reverse transcriptase (RT) represents a validated and promising target, and no inhibitor has reached the clinical pipeline yet. Here, we present rationally designed non-diketo acid selective RNase H inhibitors (RHIs) based on the quinolinone scaffold starting from former dual integrase (IN)/RNase H quinolinonyl diketo acids. Several derivatives were synthesized and tested against RNase H and viral replication and found active at micromolar concentrations. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, and Mg2+ titration experiments demonstrated that our compounds coordinate the Mg2+ cofactor and interact with amino acids of the RNase H domain that are highly conserved among naïve and treatment-experienced patients. In general, the new inhibitors influenced also the polymerase activity of RT but were selective against RNase H vs the IN enzyme.

Pyrrolyl Pyrazoles as Non-Diketo Acid Inhibitors of the HIV-1 Ribonuclease H Function of Reverse Transcriptase.

Due to the biological liability of diketo acid (DKA) chain, we transferred this element of our previously reported anti-HIV-1 pyrrolyl derivatives to a non-DKA scaffold, obtaining a series of pyrrolyl-pyrazole carboxylic acids as new RNase H inhibitors. Among the newly synthesized derivatives, oxyphenylpyrrolyl-pyrazoles demonstrated inhibitory activities within the low micromolar/submicromolar range with compound 11b being the most potent. Interestingly, all tested compounds showed up to 2 orders of magnitude of selectivity for RNase H vs integrase. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, showed the key structural features that could confer the ability to establish specific interactions within RNase H. Furthermore, they proved the ability of our compounds to interact with amino acids highly conserved among HIV-1 subspecies isolated among patients carrying drug-resistant variants. In the end, the newly discovered pyrazole carboxylic acid derivatives feature promising serum stability with respect to their corresponding DKAs.

Discovery of Novel Integrase Inhibitors Acting outside the Active Site Through High-Throughput Screening.

Currently, an increasing number of drugs are becoming available to clinics for the treatment of HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients, due to resistance with or without treatment adherence concerns. Accordingly, it is important to continue to discover small molecules that have a novel mechanism of inhibition. In this work, HIV integrase inhibitors were selected by high-throughput screening. Chemical structure comparisons enabled the identification of stilbene disulfonic acids as a potential new chemotype. Biochemical characterization of the lead compound stilbenavir (NSC34931) and a few derivatives was performed. Stilbene disulfonic acid derivatives exhibit low to sub-micromolar antiviral activity, and they inhibit integrase through DNA-binding inhibition. They probably bind to the C-terminal domain of integrase, in the cavity normally occupied by the noncleaved strand of the viral DNA substrate. Because of this original mode of action compared to active site strand transfer inhibitors, they do not exhibit cross-resistance to the three main resistance pathways to integrase inhibitors (G140S-Q148H, N155H, and Y143R). Further structure-activity optimization should enable the development of more active and less toxic derivatives with potential clinical relevance.

Author Correction: RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Modulation of the functional association between the HIV-1 intasome and the nucleosome by histone amino-terminal tails.

BACKGROUND: Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. RESULTS: We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. CONCLUSIONS: Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

BACKGROUND: Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. RESULTS: Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. CONCLUSIONS: Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

Exploring the impact of the side-chain length on peptide/RNA binding events.

The impact of the amino-acid side-chain length on peptide-RNA binding events has been investigated using HIV-1 Tat derived peptides as ligands and the HIV-1 TAR RNA element as an RNA model. Our studies demonstrate that increasing the length of all peptide side-chains improves unexpectedly the binding affinity (KD) but reduces the degree of compactness of the peptide-RNA complex. Overall, the side-chain length appears to modulate in an unpredictable way the ability of the peptide to compete with the cognate TAR RNA partner. Beyond the establishment of non-intuitive fundamental relationships, our results open up new perspectives in the design of effective RNA ligand competitors, since a large number of them have already been identified but few studies report on the modulation of the biological activity by modifying in the same way the length of all chains connecting RNA recognition motives to the central scaffold of a ligand.

De novo RNA synthesis catalyzed by the Zika Virus RNA polymerase domain.

Mosquito- and tick-borne pathogens including Chikungunya, Dengue, Japanese encephalitis, West Nile, Yellow fever and Zika virus, represent a new economic and public health challenge. In the absence of effective vaccines and specific therapies, only supportive regimens are administrated for most of these infections. Thus, the development of a targeted therapy is mandatory to stop the rapid progression of these pathogens and preoccupant associated burdens such as Guillain-Barre syndrome, microcephaly. For this, it is essential to develop biochemical tools to help study and target key viral enzymes involved in replication such as helicase complexes, methyl-transferases and RNA-dependent RNA polymerases. Here, we show that a highly purified ZIKV polymerase domain is active in vitro. Importantly, we show that this isolated domain is capable of de novo synthesis of the viral genome and efficient elongation without terminal nucleotide transferase activity. Altogether, this isolated polymerase domain will be a precious tool to screen and optimize specific nucleoside and non-nucleoside inhibitors to fight against Zika infections.

Hepatitis C virus intragenomic interactions are modulated by the SLVI RNA structure of the core coding sequence.

Several RNA interactions are thought to play a role in the regulation of the hepatitis C virus (HCV) life cycle. Most of these interactions involve the 5BSL3.2 domain and therefore occur at the 3' end of the viral genomic RNA. A long-range interaction has also been described between 5BSL3.2 and the 5' untranslated region (UTR). Another interaction involves the SLVI stem loop of the core coding region and the 5'UTR. We aimed to analyse the role of this SLVI domain, which likely interferes with others interactions. By evaluating RNA stability, translation and RNA synthesis, we showed that the SLVI stem loop extensively modulates the effect of the interactions mediated by the 5BSL3.2 domain and strongly affects the IIId/5BSL3.2 interaction. Numerous interactions in HCV genomic RNA have been described in the UTRs and the coding sequence but their roles are poorly understood. We showed that the SLVI domain located in the core coding sequence plays an important role in the translation of the polyprotein, but also in the modulation of long-range RNA interactions centred on the 5BSL3.2 domain. The SLVI domain has been absent from most studies, especially from the extensively used subgenomic replicon; our data highlight the importance of this domain in the studies of these long-range interactions in the HCV life cycle.

2-hydroxyisoquinoline-1,3(2H,4H)-diones (HIDs) as human immunodeficiency virus type 1 integrase inhibitors: Influence of the alkylcarboxamide substitution of position 4.

Herein, we report further insight into the biological activities displayed by the 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HID) scaffold. Previous studies have evidenced the marked fruitful effect of substitution of this two-metal binding pharmacophore at position 4 by phenyl and benzyl carboxamido chains. Strong human immunodeficiency virus type 1 integrase (HIV-1 IN) inhibitors in the low nanomolar range with micromolar (even down to low nanomolar) anti-HIV activities were obtained. Keeping this essential 4-carboxamido function, we investigated the influence of the replacement of phenyl and benzyl groups by various alkyl chains. This study shows that the recurrent halogenobenzyl pharmacophore found in the INSTIs can be efficiently replaced by an n-alkyl group. With an optimal length of six carbons, we observed a biological profile and a high barrier to resistance equivalent to those of a previously reported hit compound bearing a 4-fluorobenzyl group.

Anticancer molecule AS1411 exhibits low nanomolar antiviral activity against HIV-1.

During clinical trials, a number of fully characterized molecules are dropped along the way because they do not provide enough benefit for the patient. Some of them show limited side effects and might be of great use for other applications. AS1411 is a nucleolin-targeting aptamer that underwent phase II clinical trials as anticancer agent. Here, we show that AS1411 exhibits extremely potent antiviral activity and is therefore an attractive new lead as anti-HIV agent.

Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration.

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.