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The ongoing global pandemic of the coronavirus 2019 (COVID‐19) disease is caused by the virus SARS‐CoV‐2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9–nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS‐CoV‐2 and any newly emerging coronaviruses. [ABSTRACT FROM AUTHOR]

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients. [ABSTRACT FROM AUTHOR]

During the Covid-19 pandemic, the resurgence of SARS-CoV-2 was due to the development of novel variants of concern (VOC). Thus, genomic surveillance is essential to monitor continuing evolution of SARS-CoV-2 and to track the emergence of novel variants. In this study, we performed phylogenetic, mutation, and selection pressure analyses of the Spike, nsp12, nsp3, and nsp5 genes of SARS-CoV-2 isolates circulating in Yogyakarta and Central Java provinces, Indonesia from May 2021 to February 2022. Various bioinformatics tools were employed to investigate the evolutionary dynamics of distinct SARS-CoV-2 isolates. During the study period, 213 and 139 isolates of Omicron and Delta variants were identified, respectively. Particularly in the Spike gene, mutations were significantly more abundant in Omicron than in Delta variants. Consistently, in all of four genes studied, the substitution rates of Omicron were higher than that of Delta variants, especially in the Spike and nsp12 genes. In addition, selective pressure analysis revealed several sites that were positively selected in particular genes, implying that these sites were functionally essential for virus evolution. In conclusion, our study demonstrated a distinct evolutionary pattern of SARS-CoV-2 variants circulating in Yogyakarta and Central Java provinces, Indonesia. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19. [ABSTRACT FROM AUTHOR]

Abstract The disease coronavirus COVID-19 has been the cause of millions of deaths worldwide. Among the proteins of SARS-CoV-2, non-structural protein 12 (NSP12) plays a key role during COVID infection and is part of the RNA-dependent RNA polymerase complex. The monitoring of NSP12 polymorphisms is extremely important for the design of new antiviral drugs and monitoring of viral evolution. This study analyzed the NSP12 mutations detected in circulating SARS-CoV-2 during the years 2020 to 2022 in the population of the city of Manaus, Amazonas, Brazil. The most frequent mutations found were P323L and G671S. Reports in the literature indicate that these mutations are related to transmissibility efficiency, which may have contributed to the extremely high numbers of cases in this location. In addition, two mutations described here (E796D and R914K) are close and have RMSD that is similar to the mutations M794V and N911K, which have been described in the literature as influential on the performance of the NSP12 enzyme. These data demonstrate the need to monitor the emergence of new mutations in NSP12 in order to better understand their consequences for the treatments currently used and in the design of new drugs.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

• The siRNA strategy is competent for RdRp targeting therapy for SARS-CoV2. • RBD-11b efficiently targets the lungs of mice, which provides a reference for the lung-targeted delivery of drugs. • siRNAs against nsp12 restore host type I interferon responses. The RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), known as nsp12, is crucial to viral replication and spread. Being one of the most conserved proteins of SARS-CoV-2, nsp12 is also a promising therapeutic target for COVID-19. In addition, nsp12 has been reported to suppress the host type I interferon (IFN-I) responses. We used HEK293T cells exogenously expressing nsp12-luciferase to identify siRNAs against nsp12. A series of in vitro and in vivo experiments were employed to examine the effect of RBD-11b-modified cationic liposomes (CLs) on targeted delivery of siRNAs to the mouse lungs. Mice were airway-administered adenovirus to induce the exogenous expression of nsp12 in the mouse lungs, followed by nsp12 siRNAs@RBD-11b-CLs treatment. Nsp12 expression and the activity of the IRF3/IFN-I axis in the lungs were then examined. High-efficiency nsp12 siRNAs were identified and successfully delivered to the mouse lungs by the RBD-11b-modified CLs. The nsp12 siRNAs@RBD-11b-CLs efficiently inhibited the exogenous expression of nsp12 in the mouse lungs. Moreover, nsp12 siRNAs@ RBD-11b-CLs successfully restored the nucleus transport of IRF3, thereby increasing the expression of IFNβ and several IFN effector genes in alveolar cells. We establish a lung targeting system to efficiently deliver siRNAs against SARS-CoV-2 nsp12 and significantly restore host type I interferon responses. This study will provide fresh perspectives and forthcoming approaches for the treatment of COVID-19 and other RNA viruses. [ABSTRACT FROM AUTHOR]

• SARS-CoV-2 significantly impacts the UPS, a key cellular regulatory mechanism. • SARS-CoV-2 encoded proteinsinteract with host UPS, influencing immune signaling and apoptosis. • Stability of ORF3a and ORF8 may or may not be influenced by host proteasome. • Unlike previous studies, recent studies do not show ORF3a as an ion channel. • SARS-CoV-2 PLpro alters host immune responses via interfering withUPS. • Proteomic studies reveal changes in ubiquitination in SARS-CoV-2 infected cells. • Promising treatment include targeting PLpro with zinc-ejector drugs, targeting viral nsp12 via heat treatment. The Ubiquitin proteasome system (UPS), an essential eukaryotic/host/cellular post-translational modification (PTM), plays a critical role in the regulation of diverse cellular functions including regulation of protein stability, immune signaling, antiviral activity, as well as virus replication. Although UPS regulation of viral proteins may be utilized by the host as a defense mechanism to invade viruses, viruses may have adapted to take advantage of the host UPS. This system can be manipulated by viruses such as the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to stimulate various steps of the viral replication cycle and facilitate pathogenesis, thereby causing the respiratory disease COVID-19. Many SARS-CoV-2 encoded proteins including open reading frame 3a (ORF3a), ORF6, ORF7a, ORF9b, and ORF10 interact with the host's UPS machinery, influencing host immune signaling and apoptosis. Moreover, SARS-CoV-2 encoded papain-like protease (PLpro) interferes with the host UPS to facilitate viral replication and to evade the host's immune system. These alterations in SARS-CoV-2 infected cells have been revealed by various proteomic studies, suggesting potential targets for clinical treatment. To provide insight into the underlying causes of COVID-19 and suggest possible directions for therapeutic interventions, this paper reviews the intricate relationship between SARS-CoV-2 and UPS. Promising treatment strategies are also investigated in this paper including targeting PLpro with zinc-ejector drugs, as well as targeting viral non-structural protein (nsp12) via heat treatment associated ubiquitin-mediated proteasomal degradation to reduce viral pathogenesis. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 nsp12, the RNA-dependent RNA-polymerase plays a crucial role in virus replication. Monitoring the effect of its emerging mutants on viral replication and response to antiviral drugs is important. Nsp12 of two Egyptian isolates circulating in 2020 and 2021 were sequenced. Both isolates included P323L, one included the A529V. Tracking A529V mutant frequency, it relates to the transience peaked C.36.3 variant and its parent C.36, both peaked worldwide on February-August 2021, enlisted as high transmissible variants under investigation (VUI) on May 2021. Both Mutants were reported to originate from Egypt and showed an abrupt low frequency upon screening, we analyzed all 1104 nsp12 Egyptian sequences. A529V mutation was in 36 records with an abrupt low frequency on June 2021. As its possible reappearance might obligate actions for a candidate VUI, we analyzed the predicted co-effect of P323L and A529V mutations on protein stability and dynamics through protein structure simulations. Three available structures for drug-nsp12 interaction were used representing remdesivir, suramin and favipiravir drugs. Remdesivir and suramin showed an increase in structure stability and considerable change in flexibility while favipiravir showed an extreme interaction. Results predict a favored efficiency of the drugs except for favipiravir in case of the reported mutations., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)