RNA-dependent RNA Polymerase Research Articles

Antiviral effects and mechanism of Qi pi pill against influenza viruses.

Qi pi pill (QPP), which contains Renshen, Baizhu, Fuling, Gancao, Chenpi, Shanyao, Lianzi, Shanzha, Liushenqu, Maiya, and Zexie, was recommended for preventing and treating COVID-19 in Shandong Province (China). However, the mechanism by which QPP treats infectious diseases remains unclear. This study aims to investigate the therapeutic effect of QPP invitro and on acute influenza infection in mice, exploring its mechanism of action against influenza A virus (IAV). The invitro activity of QPP was assessed using dose-response curve analysis and titer reduction assay, and its antiviral mechanism was identified invitro by real-time polymerase chain reaction (PCR), time-of-addition, and enzymatic assays. The antiviral efficacy of QPP was further evaluated invivo using BALB/c mice infected with IAV. At the same time, each single Chinese herbal medicine in QPP was evaluated to preliminarily identify those with antiviral effects. In vitro results showed that QPP exhibited a higher potency antiviral effect against both influenza A and B viruses, inhibiting viral RNA replication and release by targeting RNA-dependent RNA polymerase and neuraminidase. Additionally, QPP significantly decreased the expression of inflammatory cytokines in A549 cells. Invivo study revealed that QPP significantly reduced the lung index and viral load in lung tissue of mice infected with IAV. Renshen, Gancao, Zexie, and Lianzi were the Chinese herbal medicines from QPP that showed anti-IAV activity. The antiviral activity of QPP targets IAV replication and release, cytokine modulation in host cells, and provides protection in mice with acute influenza infection.

Efficacy and safety of SHEN26, a novel oral small molecular RdRp inhibitor for COVID-19 treatment: a multicenter, randomized, double-blinded, placebo-controlled, phase II clinical trial

BackgroundSHEN26 (ATV014) is an oral RNA-dependent RNA polymerase (RdRp) inhibitor with potential anti-SARS-CoV-2 activity. Safety, tolerability, and pharmacokinetic characteristics were verified in a Phase I study. This phase II study aimed to verify the efficacy and safety of SHEN26 in COVID-19 patients.MethodsThis was a multicenter randomized double-blind placebo-controlled study. Mild-to-moderate adult patients with COVID-19 were recruited and randomly assigned to the high-dose (400 mg), low-dose (200 mg), or placebo groups (1:1:1). The primary outcome measure was “changes in RNA levels on Day seven (D7)”. The second outcome measures were “changes of RNA levels on D3, D5, D10, D28,” “Time of clearance of virus.”ResultsA total of 91 patients were recruited in this study between December 08, 2022, and January 27, 2023. Twelve patients dropped out due to a lack of examination results. Finally, the data of 79 patients (24 in the placebo group, 31 in the 200 mg group, and 24 in the 400 mg group) were analyzed. No significant differences in the baseline data were observed between the groups. The changes of viral load were significantly higher on D3 (P = 0.0119), and D5 (P = 0.0120) in 400 mg group (vs. placebo group), and the difference value achieved 1.06 log10 copies/mL on D3 and 1.21 log10 copies/mL on D5. No significant difference was found in the viral clearance time between SHEN26 administrating groups and placebo groups. Administration of SHEN26 did not enhance drug-related ADEs and did not induce ADEs, and ADE inducing drug withdrawal, dose reduction, or death. Moreover, SHEN26 did not worsen the renal function.ConclusionsOur findings indicate a better efficacy of a high dose (400 mg) for COVID-19 treatment. These preliminary data on the efficacy and safety provide useful information and a working basis for further verification and development of SHEN26 as a novel oral small-molecule antiviral drug for treating COVID-19.

Remdesivir and Obeldesivir Retain Potent Antiviral Activity Against SARS-CoV-2 Omicron Variants

As new SARS-CoV-2 variants continue to emerge, it is important to evaluate the potency of antiviral drugs to support their continued use. Remdesivir (RDV; VEKLURY®) an approved antiviral treatment for COVID-19, and obeldesivir (ODV) are inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase Nsp12. Here we show these two compounds retain antiviral activity against the Omicron variants BA.2.86, BF.7, BQ.1, CH.1.1, EG.1.2, EG.5.1, EG.5.1.4, FL.22, HK.3, HV.1, JN.1, JN.1.7, JN.1.18, KP.2, KP.3, LB.1, XBB.1.5, XBB.1.5.72, XBB.1.16, XBB.2.3.2, XBC.1.6, and XBF when compared with reference strains. Genomic analysis identified 29 Nsp12 polymorphisms in these and previous Omicron variants. Phenotypic analysis of these polymorphisms confirmed no impact on the antiviral activity of RDV or ODV and suggests Omicron variants containing these Nsp12 polymorphisms remain susceptible to both compounds. These data support the continued use of RDV in the context of circulating SARS-CoV-2 variants and the development of ODV as an antiviral therapeutic.

Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch.

The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza A virus catalyzes viral RNA transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) by adopting different conformations. A switch from transcription to replication occurs at a relatively late stage of infection. We recently reported that the viral NS2 protein, expressed at later stages from a spliced transcript of the NS segment messenger RNA (mRNA), inhibits transcription, promotes replication and plays a key role in the transcription-to-replication switch. In this study, we performed comprehensive functional analyses to elucidate how NS2 promotes viral genome replication. Using a cell-based single-step RNP reconstitution assay, we found that NS2 specifically promotes the first-step vRNA-to-cRNA synthesis. Further investigation revealed that this promotion is tightly associated with the intrinsic properties of the 3'-vRNA promoter. Employing a highly sensitive complementation reporter assay, we demonstrated that NS2 associates more strongly with the vRNA-resident RdRp than the cRNA-resident RdRp. These findings were further validated through in vitro replication analyses. We, therefore, propose that influenza A virus NS2 protein targets vRNA-resident RdRp to drive the transcription-to-replication switch during infection.

Identification of Benzothiophene-Derived Inhibitors of Flaviviruses by Targeting RNA-Dependent RNA Polymerase

Flaviviruses such as Dengue, West Nile, and Zika viruses are mosquito-borne RNA viruses that can cause serious diseases in humans. To develop effective drugs for treating these viruses’ infections, we create a new approach for developing common or shared drugs that may work for several different viral species of flaviviruses. It is based on the conserved RNA-dependent RNA polymerase (RdRp), which is the key enzyme for viral replication. We built up a common structure of RdRps (POLcon) from their consensus sequence. A conserved Triple-D structural motif was identified at the active site of POLcon that has been used for virtual compound screening. We have identified three inhibitors that have potent activities against Dengue, West Nile, and Zika viruses. All these three inhibitors are Benzothiophene derivatives. This is the first report of Benzothiophene-derived compounds as inhibitors for flaviviruses. Furthermore, our approach has provided a proof-of-concept that it is feasible to identify shared drugs for several different viral species of flaviviruses.

The role of M2 proteins of pneumoviruses in transcription regulation, prevention of hypermutation, and activation of the type I interferon pathway.

Human metapneumovirus (HMPV) is an important causative agent of respiratory tract disease. Fundamental knowledge of the interaction between HMPV and the innate immune system could lead to the design of novel antiviral therapies. Previously, we demonstrated that HMPV M2-2 deletion mutants had hypermutated genomes and contained defective interfering particles (DIs), which are potent inducers of the IFN response. Here, we investigated the role of the HMPV M2-2 protein as IFN antagonist using chimeric HMPV expressing M2 proteins of other pneumoviruses: respiratory syncytial virus (RSV) and avian metapneumovirus type C (AMPV/C). Chimeric HMPVs expressing the M2 proteins of RSV or AMPV/C were attenuated in HEp-2 cells but did not activate the IFN response, and their genomes were not hypermutated. In contrast, chimeric HMPVs expressing the M2-2 proteins of RSV and AMPV/C, in combination with HMPV M2-1, did activate the IFN response, and their genomes were hypermutated. Investigation of the role of the pneumovirus M2 proteins in transcription regulation demonstrated that the M2-2 protein, only in concerted action with autologous M2-1 protein, acted as a transcription elongation factor. As a second approach, chimeric RSV in which the IFN antagonists NS1 and NS2 were replaced by the HMPV M2-2 gene failed to suppress an IFN response, indicating that the HMPV M2-2 protein is not a potent IFN antagonist. These data indicate that expression of autologous M2-1 and M2-2 proteins is important for the fidelity of the RNA-dependent RNA polymerase, necessary to prevent the accumulation of mutations, and possibly DIs, thereby preventing activation of the IFN responses.IMPORTANCEThe M2-2 protein of human metapneumovirus is suggested to function as a type I interferon antagonist, a function so far not assigned to the M2 proteins of other pneumoviruses. Although M2-2 deletion mutants of HMPV activate the type I interferon pathway, these mutants have hypermutated genomes and contain defective interfering RNAs, known to activate the interferon pathway. Here, we show that the M2-2 protein, in concerted action with autologous M2-1 protein, acts as a transcription elongation factor, which could explain the accumulation of DIs in M2-2 deletion mutants. Additionally, chimeric RSV in which the IFN antagonists NS1 and NS2 were replaced by the HMPV M2-2 gene failed to suppress an IFN response. These data indicate that expression of autologous M2-1 and M2-2 proteins is required for the fidelity of the RNA-dependent RNA polymerase to prevent genome hypermutation and activation of the type I IFN pathway.

Sequence analysis and genome organization of a new marafivirus from Leptochloa chinensis.

High-throughput sequencing was used to identify and characterize a novel marafivirus from the weed Leptochloa chinensis, which was tentatively named "Leptochloa chinensis marafivirus" (LcMV). The complete genome of the virus consists of 6,178 base pairs, and its nucleotide sequence is 73.82% identical to that of Sorghum almum marafivirus, which is a member of the genus Marafivirus within the family Tymoviridae. The LcMV genome contains a relatively large open reading frame (ORF) encoding a single polyprotein (220.6 kDa) with five functional domains (methyltransferase, papain-like protease, helicase, RNA-dependent RNA polymerase, and coat proteins), which is a characteristic of members of this genus. Furthermore, a 16-nucleotide conserved marafibox sequence was identified at nucleotide positions 5341-5356. The coat protein of LcMV is 68.02% identical to that of Sorghum almum marafivirus. Phylogenetic analysis based on nucleotide and polyprotein sequences showed that LcMV is closely related to members of the genus Marafivirus. Our findings support the classification of LcMV as a member of a new species within this genus. This is the first report of a marafivirus infecting Leptochloa chinensis, a very important weed of rice.

Discovery of novel 'sugarcane totivirus 1' from Saccharum officinarum by high-throughput sequencing.

Sugarcane is cultivated globally and affected by more than 125 pathogens, which lead to various plant diseases. In recent years, high-throughput sequencing (HTS)-based genome analyses have been broadly adopted for the discovery of both characterized and un-characterized viruses from plant samples. In this study, the HTS data of sugarcane pooled sample retrieved from sequence read archive (SRA) were de novo re-assembled using CLC Genomic Workbench. The genomic sequence of a novel dsRNA totivirus, 5,384 nucleotides (nt) long, excluding the 5' untranslated region (UTR) and 3' UTR, was discovered and named sugarcane totivirus 1 (STV 1). The genome contains two open reading frames (ORFs): a putative coat protein (CP) encoding 866 amino acids (aa) and RNA-dependent RNA polymerase (RdRp) encoding 824 aa. Phylogenetic studies based on the genomic sequences (nt), and the aa sequences of CP as well as RdRp regions revealed that STV 1 is closely related to other members of the genus Totivirus. Pairwise sequence identity of CP and RdRp aa sequences showed 30.0-51.5% and 26.3-47.2% similarity, respectively with other members of the family Totiviridae. The HTS results were further validated and confirmed through OneStep RT-PCR assay and Sanger sequencing. A novel totivirus (STV 1) in the genus Totivirus, family Totiviridae has been identified. This is the first report of dsRNA totivirus STV 1 associated with sugarcane from India.

Peri-centrosomal localization of small interfering RNAs in C. elegans.

The centrosome is the microtubule-organizing center and a crucial part of cell division. Centrosomal RNAs (cnRNAs) have been reported to enable precise spatiotemporal control of gene expression during cell division in many species. Whether and how cnRNAs exist in C. elegans are unclear. Here, using the nuclear RNAi Argonaute protein NRDE-3 as a reporter, we observed potential peri-centrosome localized small interfering (si)RNAs in C. elegans. NRDE-3 was previously shown to associate with pre-mRNAs and pre-rRNAs via a process involving the presence of complementary siRNAs. We generated a GFP-NRDE-3 knock-in transgene through CRISPR/Cas9 technology and observed that NRDE-3 formed peri-centrosomal foci neighboring the tubulin protein TBB-2, other centriole proteins and pericentriolar material (PCM) components in C. elegans embryos. The peri-centrosomal accumulation of NRDE-3 depends on RNA-dependent RNA polymerase (RdRP)-synthesized 22G siRNAs and the PAZ domain of NRDE-3, which is essential for siRNA binding. Mutation of eri-1, ergo-1, or drh-3 significantly increased the percentage of pericentrosome-enriched NRDE-3. At the metaphase of the cell cycle, NRDE-3 was enriched in both the peri-centrosomal region and the spindle. Moreover, the integrity of centriole proteins and pericentriolar material (PCM) components is also required for the peri-centrosomal accumulation of NRDE-3. Therefore, we concluded that siRNAs could accumulate in the pericentrosomal region in C. elegans and suggested that the peri-centrosomal region may also be a platform for RNAi-mediated gene regulation.

Nuclear Argonaute protein NRDE-3 switches small RNA partners during embryogenesis to mediate temporal-specific gene regulatory activity.

RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. We also observe that many other RNAi factors form foci in embryonic cells distinct from "SIMR granules", including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Prior work has shown that NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.

First Detection of Alphacoronavirus in Bats from the World's Largest Wetland, the Pantanal, Brazil.

Coronaviruses (CoV) infect a wide variety of hosts, causing epidemics in humans, birds, and mammals over the years. Bats (order Chiroptera) are one of the natural hosts of the Coronaviridae family. They represent 40% of the total number of mammal species in the Pantanal, a biodiversity hotspot in South America. Given the recent SARS-CoV-2 pandemic, we investigated the presence of CoV in bats captured in the Brazilian Pantanal. Oral and rectal swabs collected in 2021 from 419 bats were analyzed using Pancoronavirus-nested PCR targeting the RNA-dependent RNA-polymerase (RdRp) gene. Orthocoronavirinae was detected in 16.7% (70/419) of the bats; nine samples were sequenced, confirming that Carollia perspicillata (4), Phyllostomus hastatus (2), Desmodus rotundus (1), Molossus rufus (1), and Myotis cf. nigricans (1) collected in buildings formally used by humans were infected by Alphacoronavirus genera. This is the first description of Alphacoronavirus in bats from the Pantanal. As they are natural reservoirs of CoVs, constant monitoring of bats is important to comprehend the epidemiology of emerging viruses, especially in the Pantanal biome.

In silico Evaluation of H1-Antihistamine as Potential Inhibitors of SARS-CoV-2 RNA-dependent RNA Polymerase: Repurposing Study of COVID-19 Therapy.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), from the family Coronaviridae, is the seventh known coronavirus to infect humans and cause acute respiratory syndrome. Although vaccination efforts have been conducted against this virus, which emerged in Wuhan, China, in December 2019 and has spread rapidly around the world, the lack of an Food and Drug Administration-approved antiviral agent has made drug repurposing an important approach for emergency response during the COVID-19 pandemic. The aim of this study was to investigate the potential of H1-antihistamines as antiviral agents against SARS-CoV-2 RNA-dependent RNA polymerase enzyme. Using molecular docking techniques, we explored the interactions between H1-antihistamines and RNA-dependent RNA polymerase (RdRp), a key enzyme involved in viral replication. The three-dimensional structure of 37 H1-antihistamine molecules was drawn and their energies were minimized using Spartan 0.4. Subsequently, we conducted a docking study with Autodock Vina to assess the binding affinity of these molecules to the target site. The docking scores and conformations were then visualized using Discovery Studio. The results examined showed that the docking scores of the H1-antihistamines were between 5.0 and 8.3 kcal/mol. These findings suggested that among all the analyzed drugs, bilastine, fexofenadine, montelukast, zafirlukast, mizolastine, and rupatadine might bind with the best binding energy (< -7.0 kcal/mol) and inhibit RdRp, potentially halting the replication of the virus. This study highlights the potential of H1-antihistamines in combating COVID-19 and underscores the value of computational approaches in rapid drug discovery and repurposing efforts. Finally, experimental studies are required to measure the potency of H1-antihistamines before their clinical use against COVID-19 as RdRp inhibitors.

A post-assembly conformational change makes the SARS-CoV-2 polymerase elongation-competent.

Coronaviruses (CoV) encode sixteen non-structural proteins (nsps), most of which form the replication-transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles into a processive polymerase remains poorly understood. We show that the core RTC preferentially assembles by first having nsp12-polymerase bind to the RNA template, followed by the subsequent association of nsp7 and nsp8. Once assembled on the RNA template, the core RTC requires hundreds of seconds to undergo a conformational change that enables processive elongation. In the absence of RNA, the (apo-)RTC requires several hours to adopt its elongation-competent conformation. We propose that this obligatory activation step facilitates the recruitment of additional nsp's essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.

Genomic Characterization of Laodelphax striatellus Permutotetra-like Virus and Self-Cleavage Function of Viral Capsid Protein

Laodelphax striatellus permutotetra-like virus (LsPLV) is a novel insect virus identified via small RNA deep sequencing. At present, there is a lack of awareness of LsPLV, restricting research on its utilization in biocontrol. In this paper, the full-length genome of LsPLV was cloned and analyzed, then viral capsid protein (CP) was expressed and prepared as an antibody, and CP property was tested. It was found that the LsPLV genome was 4667 nt in length, encoding two proteins, RNA-dependent RNA polymerase (RdRP) and CP, and the palm subdomain conserved region in RdRp was arranged in a “C–A–B” permutation pattern, exhibiting the typical characteristics of permutotetra-like viruses. Phylogenetic analysis suggested that LsPLV shared the highest homology (excluding LsPLV1) with a Nodaviridae virus (QLI47702.1), and their nucleotide identities of RdRP and CP were 55.4% and 59.2%, respectively. After expression, purified CP exhibited two bands of 60 kDa and 47 kDa, suggesting a potential cleavage in the protein. LsPLV CP in L. striatellus was detected by Western blot, and except for the complete CP band, the specific bands with molecular weights lower than CP were also detected, indicating that CP underwent cleavage. Detection of purified CP in vitro showed that the cleavage could occur independent of any protease, confirming that CP has self-cleavage characteristics.

Synthesis, Characterization, and Molecular Modeling Studies of Novel Indenopyridazine-thiazole Molecular Hybrids

Background: Previous studies have reported various biological activities of indeno-pyridazine and thiazole derivatives, including antiviral activity and CoV-19 inhibition. In this paper, the authors aimed to design, synthesize, and characterize a novel series of indenopyridazinethiazoles, starting with 2-(4-cyano-3-oxo-2,3-dihydro-9H-indeno[2,1-c]pyridazin-9-ylidene)-hydrazine-1-car-bothioamide and available laboratory reagents. Methods: The strategy involved the synthesis of indeno[2,1-c]pyridazincarbothioamide, followed by its reaction with various hydrazonoyl chlorides and α-halocompounds (phenacyl bromides and α-chloroketones) to obtain the desired indenopyridazinethiazole derivatives. The synthesized structures were confirmed using IR, NMR, mass spectra, elemental analysis, and alternative synthesis when possible. Docking scores and poses of thirteen synthesized compounds were examined using Auto-Dock4.2.6 software against multiple targets of SARS-CoV-2, including 3C-like protease (3CLpro), helicase, receptor binding domain (RBD), papain-like protease (PLpro), neuropilin-1 (NRP-1), RNA-dependent RNA polymerase (RdRp), and human angiotensin‐converting enzyme 2 (ACE2). Results: Docking predictions revealed that compound 13d exhibited high potency against 3CLpro and helicase, with docking scores of -10.9 and -10.5 kcal/mol, respectively. Compound 10c showed su-perior docking scores against RBD and ACE2, with values of -8.7 and -11.8 kcal/mol, respectively. Compounds 10a, 13c, and 7b demonstrated excellent docking scores against RdRp, PLpro, and NRP-1, with values of -10.3, -10.4, and -8.6 kcal/mol, respectively. Conclusion: The authors recommend further experimental assessments of compounds 13d, 10c, 10a, 13c, and 7b against SARS-CoV-2 multi-targets, considering their promising docking scores.

In silico evaluation of bisphosphonates identifies leading candidates for SARS-CoV-2 RdRp inhibition.

The novel coronavirus disease (COVID-19) pandemic has resulted in 777 million confirmed cases and over 7 million deaths worldwide, with insufficient treatment options. Innumerable efforts are being made around the world for faster identification of therapeutic agents to treat the deadly disease. Post Acute Sequelae of SARS-CoV-2 infection or COVID-19 (PASC), also called Long COVID, is still being understood and lacks treatment options as well. A growing list of drugs are being suggested by various in silico, in vitro and ex vivo models, however currently only two treatment options are widely used: the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir, and the main protease inhibitor nirmatrelvir in combination with ritonavir. Computational drug development tools and in silico studies involving molecular docking, molecular dynamics, entropy calculations and pharmacokinetics can be useful to identify new targets to treat COVID-19 and PASC, as shown in this work and our recent paper that identified alendronate as a promising candidate. In this study, we have investigated all bisphosphonates (BPs) on the ChEMBL database which can bind competitively to nidovirus RdRp-associated nucleotidyl (NiRAN) transferase domain, and systematically down selected seven candidates (CHEMBL608526, CHEMBL196676, CHEMBL164344, CHEMBL4291724, CHEMBL4569308, CHEMBL387132, CHEMBL98211), two of which closely resemble the approved drugs minodronate and zoledronate. This work and our recent paper together provide an in silico mechanistic explanation for alendronate and zoledronate users having dramatically reduced odds of SARS-CoV-2 testing, COVID-19 diagnosis, and COVID-19-related hospitalizations, and indicate that similar observational studies in Japan with minodronate could be valuable.

Investigating Pb2 CAP-binding domain inhibitors from marine bacteria for targeting the influenza A H5N1.

The ongoing increase in the prevalence and mutation rate of the influenza virus remains a critical global health issue. A promising strategy for antiviral drug development involves targeting the RNA-dependent RNA polymerase, specifically the PB2-cap binding domain of Influenza A H5N1. This study employs an in-silico approach to inhibit this domain, crucial for viral replication, using potential inhibitors derived from marine bacterial compounds. Utilizing the MTi-OpenScreen web server, we screened a library of compounds to assess their molecular interactions with the target. This process identified four potential inhibitors: CMNPD25830, CMNPD18675, CMNPD18676, and CMNPD27216. Subsequent molecular dynamics simulations, conducted using the Amber software suite, evaluated their binding affinities and dynamic interactions with the PB2 protein. Notably, CMNPD25830 and CMNPD27216 emerged as the most promising candidates, exhibiting higher binding affinities and more favourable interaction profiles compared to the control molecule. Additional analyses, including post-simulation free energy calculations and free energy landscape analysis, strengthened the potential of these compounds as effective PB2-cap binding domain inhibitors. This comprehensive computational investigation identifies CMNPD27216 and CMNPD25830 as standout candidates due to their superior binding energies and dynamic stability, suggesting their strong potential as therapeutic agents against influenza. This research sets the stage for further in vitro validation and optimization of these lead compounds, potentially supporting the development of more effective influenza treatments.

Marburg Virus: A comprehensive examination of a critical pathogen

Marburg virus infection is a rare, severe zoonotic illness caused by the Marburg virus (MARV), with the primary reservoir being Egyptian fruit bats. Marburgviruses have reemerged on multiple occasions, resulting in a very high fatality ratio of up to 88% during the most significant outbreaks. MVD exhibits prominent clinical signs and symptoms such as haemorrhagic fever, coagulopathies, multi-organ failure in the affected individuals. This review provides insight into the MVD structure, genomic organisation, replication, clinical manifestations, and special emphasis on the available treatment strategies. Although no specific therapeutic interventions exist for management, supportive care like fluid administration and treatment of specific symptoms can improve survival rates and clinical outcomes. T-705 Favipiravir blocks the influenza virus's RNA-dependent RNA polymerase (RdRp) and suggests potential effectiveness in patients exhibiting lower viral loads. Remdesivir has shown efficacy in a study of MARV-infected Cynomolgus macaques; treatment improved clinical ratings, decreased plasma viral RNA, and boosted kidney and liver functions. BCX4430 Galidesivir showed 24-hour and 48-hour survival in animal groups after receiving the drug with no apparent toxicity and improved liver enzyme values.MARV can induce significantly more severe epidemics than previously thought. These widespread outbreaks had a remarkably high mortality rate throughout a large geographical region, presenting substantial challenges in the field of medicine. High fever, haemorrhagic manifestation, organ failure, and coma are the prominent clinical signs of MVD. Supportive care and volume resuscitation are fervently recommended. Monoclonal antibodies and antivirals like Remdesivir and Favipiravir are potential treatment options; however, Galidesivir and Favipiravir can also be used.

Structure of the Nipah virus polymerase complex

Nipah virus is a highly virulent zoonotic paramyxovirus causing severe respiratory and neurological disease. Despite its lethality, there is no approved treatment for Nipah virus infection. The viral polymerase complex, composed of the polymerase (L) and phosphoprotein (P), replicates and transcribes the viral RNA genome. Here, we describe structures of the Nipah virus L-P polymerase complex and the L-protein’s Connecting Domain (CD). The cryo-electron microscopy L-P complex structure reveals the organization of the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyl transferase (PRNTase) domains of the L-protein, and shows how the P-protein, which forms a tetramer, interacts with the RdRp-domain of the L-protein. The crystal structure of the CD-domain alone reveals binding of three Mg ions. Modelling of this domain onto an AlphaFold 3 model of an RNA-L-P complex suggests a catalytic role for one Mg ion in mRNA capping. These findings offer insights into the structural details of the L-P polymerase complex and the molecular interactions between L-protein and P-protein, shedding light on the mechanisms of the replication machinery. This work will underpin efforts to develop antiviral drugs that target the polymerase complex of Nipah virus.

Synthesis and Antiviral Evaluation of 5-(4-Aryl-1,3-butadiyn-1-yl)-uridines and Their Phosphoramidate Pronucleotides.

The emergence of RNA viruses driven by global population growth and international trade highlights the urgent need for effective antiviral agents that can inhibit viral replication. Nucleoside analogs, which mimic natural nucleotides, have shown promise in targeting RNA-dependent RNA polymerases (RdRps). Starting from protected 5-iodouridine, we report the synthesis of hitherto unknown C5-substituted-(1,3-diyne)-uridines nucleosides and their phosphoramidate prodrugs. The modifications at C5 include 4-(trifluoromethyl)benzene (a), 4-pentyl-benzene (b), 3,5-dimethoxy-benzene (c), 4-(trifluoromethoxy)benzene (d), 3-aniline (e), 4-pyridine (f), 3-thiophene (g), C6H13 (h), 2-pyrimidine (i), cyclopropyl (j), and phenyl (k) groups. These compounds were synthesized using Sonogashira palladium-catalyzed reactions and nickel-copper-catalyzed C-H activation between various alkynes, yielding between 25% and 67%. The antiviral activities of obtained compounds were measured through HTS against RNA viruses including influenza H1N1 and H3N2, human respiratory syncytial virus (RSV), SARS-CoV-2, Zika, hepatitis C virus (HCV), Hepatitis E virus (HEV), as well as against coronavirus (HCoV-229E). Unfortunately, none of them showed promising antiviral activity, with less than 85% inhibition observed in the cell viability screening of infected cells.