Structure of the 5' untranslated region in SARS-CoV-2 genome and its specific recognition by innate immune system via the human oligoadenylate synthase 1

The 2'-5'-oligoadenylate synthetase 1 (OAS1) have been identified as one of the key enzymes driving the innate immune system response to SARS-CoV-2 infection and has been related to COVID-19 severity. OAS1 is a sensor of endogenous RNA that triggers the 2'-5' oligoadenylate/RNase L pathway in response to viral infections, ultimately activating the RNA-Lyase which cleaves endogenous and exogenous RNA hence impeding the viral maturation. Upon SARS-CoV-2 infection, OAS1 is responsible for the recognition of viral RNA and has been shown to possess a particularly high sensitivity for the 5'-untranslated (5'-UTR) RNA region, which is organized in a double-strand stem loop motif (SL1). Yet the structure of the nucleic acid/protein complex has not been resolved. Here, we report the structure of the OAS1/SL1 complex generated by molecular modeling, including enhanced sampling approaches. We also pinpoint how SL1 region enhances the interaction network with the enzyme, promoting specific hydrogen bonds which are absent in normal double strand RNA fragments, hence rationalizing the high affinity shown by OAS1.

A highly potent and safe pyrrolopyridine-based allosteric HIV-1 integrase inhibitor targeting host LEDGF/p75-integrase interaction site

Allosteric integrase inhibitors (ALLINIs) are a class of experimental anti-HIV agents that target the noncatalytic sites of the viral integrase (IN) and interfere with the IN-viral RNA interaction during viral maturation. Here, we report a highly potent and safe pyrrolopyridine-based ALLINI, STP0404, displaying picomolar IC50 in human PBMCs with a >24,000 therapeutic index against HIV-1. X-ray structural and biochemical analyses revealed that STP0404 binds to the host LEDGF/p75 protein binding pocket of the IN dimer, which induces aberrant IN oligomerization and blocks the IN-RNA interaction. Consequently, STP0404 inhibits proper localization of HIV-1 RNA genomes in viral particles during viral maturation. Y99H and A128T mutations at the LEDGF/p75 binding pocket render resistance to STP0404. Extensive in vivo pharmacological and toxicity investigations demonstrate that STP0404 harbors outstanding therapeutic and safety properties. Overall, STP0404 is a potent and first-in-class ALLINI that targets LEDGF/p75 binding site and has advanced to a human trial.

A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.

Conformational Plasticity of Hepatitis B Core Protein Spikes Promotes Peptide Binding Independent of the Secretion Phenotype

Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.

Cleavage of TANK-Binding Kinase 1 by HIV-1 Protease Triggers Viral Innate Immune Evasion

Type-I interferons (IFN-I) are the innate immune system’s principal defense against viral infections. Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host’s innate immunity in order to survive and replicate to sustain infection. Suppression of IFN-I is one among the multiple escape strategies used by HIV-1 to prevent its clearance. HIV-1 protease which helps in viral maturation has also been observed to cleave host cellular protein kinases. In this study we performed a comprehensive screening of a human kinase library using AlphaScreen assay and identified that TANK binding kinase-1 (TBK1) was cleaved by HIV-1 protease (PR). We demonstrate that PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity and further reveal that the PR mediated suppression of IFN-I could be counteracted by protease inhibitors (PI) in vitro. We have also revealed that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme’s ability to cleave TBK1. The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.

The E3 Ubiquitin Ligase RNF5 Facilitates SARS-CoV-2 Membrane Protein-Mediated Virion Release

As enveloped virus, SARS-CoV-2 membrane protein (M) mediates viral release from cellular membranes, but the molecular mechanisms of SARS-CoV-2 virions release remain poorly understood. Here, we performed RNAi screening and identified the E3 ligase RNF5 which mediates ubiquitination of SARS-CoV-2 M at residue K15 to enhance the interaction of viral envelope (E) with M. M-E complex ensures the uniform size of viral particles for viral maturation and mediates viral release. Moreover, overexpression of M induces complete autophagy which is dependent on RNF5-mediated ubiquitin modification. M inhibits the activity of lysosome protease, and uses autolysosomes for virion release. Consequently, all these results demonstrate that RNF5 mediates ubiquitin modification of SARS-CoV-2 M to stabilize the M-E complex and induce autophagy for virion release.

In Silico Study of Coumarins and Quinolines Derivatives as Potent Inhibitors of SARS-CoV-2 Main Protease

The pandemic that started in Wuhan (China) in 2019 has caused a large number of deaths, and infected people around the world due to the absence of effective therapy against coronavirus 2 of the severe acute respiratory syndrome (SARS-CoV-2). Viral maturation requires the activity of the main viral protease (Mpro), so its inhibition stops the progress of the disease. To evaluate possible inhibitors, a computational model of the SARS-CoV-2 enzyme Mpro was constructed in complex with 26 synthetic ligands derived from coumarins and quinolines. Analysis of simulations of molecular dynamics and molecular docking of the models show a high affinity for the enzyme (∆Ebinding between −5.1 and 7.1 kcal mol−1). The six compounds with the highest affinity show Kd between 6.26 × 10–6 and 17.2 × 10–6, with binding affinity between −20 and −25 kcal mol−1, with ligand efficiency less than 0.3 associated with possible inhibitory candidates. In addition to the high affinity of these compounds for SARS-CoV-2 Mpro, low toxicity is expected considering the Lipinski, Veber and Pfizer rules. Therefore, this novel study provides candidate inhibitors that would allow experimental studies which can lead to the development of new treatments for SARS-CoV-2.

In silico Exploration of Inhibitors for SARS-CoV-2's Papain-Like Protease

Coronavirus disease 2019 (COVID-19) is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with very limited treatments so far. Demonstrated with good druggability, two major proteases of SARS-CoV-2, namely main protease (Mpro) and papain-like protease (PLpro) that are essential for viral maturation, have become the targets for many newly designed inhibitors. Unlike Mpro that has been heavily investigated, PLpro is not well-studied so far. Here, we carried out the in silico high-throughput screening of all FDA-approved drugs via the flexible docking simulation for potential inhibitors of PLpro and explored the molecular mechanism of binding between a known inhibitor rac5c and PLpro. Our results, from molecular dynamics simulation, show that the chances of drug repurposing for PLpro might be low. On the other hand, our long (about 450 ns) MD simulation confirms that rac5c can be bound stably inside the substrate-binding site of PLpro and unveils the molecular mechanism of binding for the rac5c-PLpro complex. The latter may help perform further structural optimization and design potent leads for inhibiting PLpro.

Search for new therapeutics against HIV-1 via dual inhibition of RNase H and integrase: current status and future challenges

Reverse transcriptase and integrase are key enzymes that play a pivotal role in HIV-1 viral maturation and replication. Reverse transcriptase consists of two active sites: RNA-dependent DNA polymerase and RNase H. The catalytic domains of integrase and RNase H share striking similarity, comprising two aspartates and one glutamate residue, also known as the catalytic DDE triad, and a Mg2+ pair. The simultaneous inhibition of reverse transcriptase and integrase can be a rational drug discovery approach for combating the emerging drug resistance problem. In the present review, the dual inhibition of RNase H and integrase is systematically discussed, including rationality of design, journey of development, advancement and future perspective.

Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its...