Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development. © 2022 by the authors.

Rapid quantitative monitoring of SARS-CoV-2 spike protein-mediated syncytia formation using split NanoLuc.

SARS-CoV-2 infection causes syncytial pneumocyte in patients and this has been considered as a defining feature of severe COVID-19 cases. Traditional methods of syncytia quantification require the morphology characterization of fused cells either with light microscope or fluorescent microscope, which is time-consuming and not accurate. Here we developed a rapid and sensitive coculture system measuring spike-induced syncytia by using NanoLuc complementation system. We found the formation of syncytia occurred rapidly after ACE2-expressing cells exposure to spike protein. In addition, we found furin cleavage as well as the cell surface protease TMPRSS2 enhanced syncytia formation. Finally, we showed that this coculture system can be used to test the ability of different compound to inhibit syncytia formation, thus providing a useful tool to screen anti-syncytial drugs.

SYSTEMATIC ANALYSIS of INNATE IMMUNE ANTAGONISM of SARS-CoV-2 and ITS EVOLUTION

Background: The innate immune system is a powerful anti-viral defense mechanism, which includes the interferon (IFN) system and autophagy. Thus, successful pathogens like SARS-CoV-2 need to counteract or evade these defenses to establish an infection. However, due to its ongoing, worldwide spread in the human population SARS-CoV-2 is evolving and in the meantime four variants with selection advantages (variants of concern) emerged. Methods: Using expression constructs for 29 SARS-CoV-2 proteins we evaluated the impact of individual viral proteins on induction of cytokines (IFNA4, IFNB1, IRF3-signalling, NF-κB-signaling) and cytokine signaling (IFNα2, IFNβ, IFNγ, IFNa;1, IL-1α, TNFα) in luciferase reporter assays, validated by endogenous transcription factor phosphorylation analysis. We assessed the influence of SARS-CoV-2 proteins on autophagy using a flow cytometry-based system. Underlying molecular mechanisms were investigated on an endogenous level using Western blot, confocal fluorescence microscopy, and flow cytometry. In addition, we examined the susceptibility of SARS-CoV-2 including all variants of concern towards type-I,-II, and-III interferons. Results: To understand how SARS-CoV-2 efficiently manipulates the host's innate immune defenses, we systematically analyzed the impact of SARS-CoV-2 encoded proteins on induction of various IFNs and pro-inflammatory cytokines, IFN signaling, and autophagy. Our results reveal the range of innate immune antagonists encoded by SARS-CoV-2 and we characterized selected molecular mechanisms employed by Nsp1 and Nsp14 to downregulate the IFN system or ORF3a and ORF7a to prevent autophagic degradation. Interestingly, our assays show that variants of concern of SARS-CoV-2 remain sensitive to type-II interferon signaling but show increased resistance towards type-I and/or type-III interferons. Conclusion: SARS-CoV-2 has evolved to counteract innate immunity using several synergistic approaches but remains relatively sensitive to type-II and-III interferons. However, emerged variants of concern remain sensitive overall but are less susceptible towards IFNα2/β and IFNa;1 than early SARS-CoV-2 isolates.

ORF1 REGION of SARS-C V-2 GENOMIC RNA AS A PROMISING TARGET for siRNA-BASED THERAPY

Background: A promising approach to tackle the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. However, it is unclear so far, which viral replication steps can be efficiently inhibited with siRNAs. Here we report the first-ever in-depth analysis of RNAi-accessible SARS-CoV-2 replication steps. Methods: siRNAs were designed against four genomic regions of SARS-CoV-2. Initial screening of siRNA activity was performed with a dual luciferase reporter assay. Efficacy of siRNAs to terminate various viral replication steps was analyzed by infecting VeroE6 cells with wildtype SARS-CoV-2 or a GFP expressing recombinant SARS-CoV-2 and monitoring viral spread in real-time by time-lapse fluorescence microscopy. Positive and negative sense viral RNA transcripts were distinctly quantified via sense specific cDNA synthesis and reverse-transcriptase quantitative PCR. Finally, the antiviral activity of the siRNAs was primarily evaluated in a highly relevant model, SARS-CoV-2 infected human lung explants. Results: When applied in a prophylactic fashion, siRNAs were able to target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, terminating replication before start of transcription, thereby preventing cytopathic effects. Surprisingly, siRNAs were not active against intermediate negative sense transcripts formed during replication. Targeting sequences that are commonly shared by all viral transcripts indeed allowed a simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. However, siRNAs that targeted ORF1 which is solely part of gRNA, presented an enhanced antiviral activity. We show that the reason for this was that siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs. Based on these findings, we developed a chemically stabilized siRNA, which targets a highly conserved region of ORF1, and which inhibited SARS-CoV-2 replication by >90% ex vivo in explants of the human lung. Conclusion: Our work strongly encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with targeting ORF1, might be key for high antiviral efficacy.

Combined Network Pharmacology, Molecular Docking, and Experimental Verification Approach to Investigate the Potential Mechanisms of Polydatin Against COVID-19

Background: Coronavirus disease 2019 (COVID-19) has posed a serious threat to human health and there is an urgent need for drug development. In this study, we explored the potential mechanisms underlying the efficacy of polydatin against COVID-19. Methods: A combined approach of network pharmacology, molecular docking, and experimental verification were employed in this study. Potential targets of polydatin for treating COVID-19 were obtained from multiple drug and disease databases. Protein–protein interaction and enrichment analyses were performed to predict the potential mechanism of action of polydatin against COVID-19. The binding potential of polydatin and key targets was evaluated through molecular docking. Furthermore, experimental methods including flow cytometry and luciferase assay were used to validate the results of computational analyses. Results: The main diseases identified as polydatin targets included metabolic diseases, lung diseases, inflammation, infectious diseases, and tumors. Polydatin may be used to treat COVID-19 through interventions that alter the immune and inflammatory responses, including IL-17 signaling pathway, T-cell activation, cytokines and inflammatory response, lipopolysaccharide-mediated signaling pathway, as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) innate immunity evasion and cell-specific immune response. Polydatin can potentially bind to the target proteins related to COVID-19, such as SARS-CoV-2 Mpro, RdRp, and human angiotensin-converting enzyme 2 (ACE2), while directly exerting its regulatory or therapeutic functions. The experimental results showed that polydatin decreased the infectivity of the SARS-CoV-2 spike pseudovirus in HEK293T-ACE2 cells. Accordingly, polydatin may retard the entry of SARS-CoV-2 into cells by competitively binding to human ACE2. Conclusion: The potential targets and signaling pathways of polydatin against COVID-19 were preliminarily identified. The findings may benefit the development and application of polydatin as a treatment for COVID-19.

Semi-Automated High-Throughput Substrate Screening Assay for Nucleoside Kinases.

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and nucleotide analogue production in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening method for NKs is of great importance. Here, we report on the validation of a well-known luciferase-based assay for the detection of NK activity in a 96-well plate format. The assay was semi-automated using a liquid handling robot. Good linearity was demonstrated (r² > 0.98) in the range of 0-500 µM ATP, and it was shown that alternative phosphate donors like dATP or CTP were also accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplarily used for the comparison of the substrate spectra of four NKs using 20 (8 natural, 12 modified) substrates. The screening results correlated well with literature data, and additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays.

SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.