SARS-CoV-2 N protein enhances the anti-apoptotic activity of MCL-1 to promote viral replication.

Viral infection in respiratory tract usually leads to cell death, impairing respiratory function to cause severe disease. However, the diversity of clinical manifestations of SARS-CoV-2 infection increases the complexity and difficulty of viral infection prevention, and especially the high-frequency asymptomatic infection increases the risk of virus transmission. Studying how SARS-CoV-2 affects apoptotic pathway may help to understand the pathological process of its infection. Here, we uncovered SARS-CoV-2 imployed a distinct anti-apoptotic mechanism via its N protein. We found SARS-CoV-2 virus-like particles (trVLP) suppressed cell apoptosis, but the trVLP lacking N protein didn't. Further study verified that N protein repressed cell apoptosis in cultured cells, human lung organoids and mice. Mechanistically, N protein specifically interacted with anti-apoptotic protein MCL-1, and recruited a deubiquitinating enzyme USP15 to remove the K63-linked ubiquitination of MCL-1, which stabilized this protein and promoted it to hijack Bak in mitochondria. Importantly, N protein promoted the replications of IAV, DENV and ZIKV, and exacerbated death of IAV-infected mice, all of which could be blocked by a MCL-1 specific inhibitor, S63845. Altogether, we identifed a distinct anti-apoptotic function of the N protein, through which it promoted viral replication. These may explain how SARS-CoV-2 effectively replicates in asymptomatic individuals without cuasing respiratory dysfunction, and indicate a risk of enhanced coinfection with other viruses. We anticipate that abrogating the N/MCL-1-dominated apoptosis repression is conducive to the treatments of SARS-CoV-2 infection as well as coinfections with other viruses.

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.

TUT4/7-mediated uridylation of a coronavirus subgenomic RNAs delays viral replication.

Coronaviruses are positive-strand RNA viruses with 3' polyadenylated genomes and subgenomic transcripts. The lengths of the viral poly(A) tails change during infection by mechanisms that remain poorly understood. Here, we use a splint-ligation method to measure the poly(A) tail length and poly(A) terminal uridylation and guanylation of the mouse hepatitis virus (MHV) RNAs. Upon infection of 17-CL1 cells with MHV, a member of the Betacoronavirus genus, we observe two populations of terminally uridylated viral transcripts, one with poly(A) tails ~44 nucleotides long and the other with poly(A) tails shorter than ~22 nucleotides. The mammalian terminal uridylyl-transferase 4 (TUT4) and terminal uridylyl-transferase 7 (TUT7), referred to as TUT4/7, add non-templated uracils to the 3'-end of endogenous transcripts with poly(A) tails shorter than ~30 nucleotides to trigger transcript decay. Here we find that depletion of the host TUT4/7 results in an increased replication capacity of the MHV virus. At late stages of infection, the population of uridylated subgenomic RNAs with tails shorter than ~22 nucleotides is reduced in the absence of TUT4/7 while the viral RNA load increases. Our findings indicate that TUT4/7 uridylation marks the MHV subgenomic RNAs for decay and delays viral replication.

All Domains of SARS-CoV-2 nsp1 Determine Translational Shutoff and Cytotoxicity of the Protein.

Replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable ß5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity. IMPORTANCE The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes.

ZFP36 ring finger protein like 1 significantly suppresses human coronavirus OC43 replication.

CCCH-type zinc figure proteins (ZFP) are small cellular proteins that are structurally maintained by zinc ions. Zinc ions coordinate the protein structure in a tetrahedral geometry by binding to cystine-cystine or cysteines-histidine amino acids. ZFP's unique structure enables it to interact with a wide variety of molecules including RNA; thus, ZFP modulates several cellular processes including the host immune response and virus replication. CCCH-type ZFPs have shown their antiviral efficacy against several DNA and RNA viruses. However, their role in the human coronavirus is little explored. We hypothesized that ZFP36L1 also suppresses the human coronavirus. To test our hypothesis, we used OC43 human coronavirus (HCoV) strain in our study. We overexpressed and knockdown ZFP36L1 in HCT-8 cells using lentivirus transduction. Wild type, ZFP36L1 overexpressed, and ZFP36L1 knockdown cells were each infected with HCoV-OC43, and the virus titer in each cell line was measured over 96 hours post-infection (p.i.). Our results show that HCoV-OC43 replication was significantly reduced with ZFP36L1 overexpression while ZFP36L1 knockdown significantly enhanced virus replication. ZFP36L1 knockdown HCT-8 cells started producing infectious virus at 48 hours p.i. which was an earlier timepoint as compared to wild -type and ZFP36L1 overexpressed cells. Wild-type and ZFP36L1 overexpressed HCT-8 cells started producing infectious virus at 72 hours p.i. Overall, the current study showed that overexpression of ZFP36L1 suppressed human coronavirus (OC43) production.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

PRPF19 Limits Porcine Epidemic Diarrhea Virus Replication through Targeting and Degrading Viral Capsid Protein.

Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.

Analysis of critical protein-protein interactions of SARS-CoV-2 capping and proofreading molecular machineries towards designing dual target inhibitory peptides.

In recent years, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as the cause of the coronavirus disease (COVID-19) global pandemic, and its variants, especially those with higher transmissibility and substantial immune evasion, have highlighted the imperative for developing novel therapeutics as sustainable solutions other than vaccination to combat coronaviruses (CoVs). Beside receptor recognition and virus entry, members of the SARS-CoV-2 replication/transcription complex are promising targets for designing antivirals. Here, the interacting residues that mediate protein-protein interactions (PPIs) of nsp10 with nsp16 and nsp14 were comprehensively analyzed, and the key residues' interaction maps, interaction energies, structural networks, and dynamics were investigated. Nsp10 stimulates both nsp14's exoribonuclease (ExoN) and nsp16's 2'O-methyltransferase (2'O-MTase). Nsp14 ExoN is an RNA proofreading enzyme that supports replication fidelity. Nsp16 2'O-MTase is responsible for the completion of RNA capping to ensure efficient replication and translation and escape from the host cell's innate immune system. The results of the PPIs analysis proposed crucial information with implications for designing SARS-CoV-2 antiviral drugs. Based on the predicted shared protein-protein interfaces of the nsp16-nsp10 and nsp14-nsp10 interactions, a set of dual-target peptide inhibitors was designed. The designed peptides were evaluated by molecular docking, peptide-protein interaction analysis, and free energy calculations, and then further optimized by in silico saturation mutagenesis. Based on the predicted evolutionary conservation of the interacted target residues among CoVs, the designed peptides have the potential to be developed as dual target pan-coronavirus inhibitors.

Fidelity of Ribonucleotide Incorporation by the SARS-CoV-2 Replication Complex.

The SARS-CoV-2 coronavirus has caused a global pandemic. Despite the initial success of vaccines at preventing infection, genomic variation has led to the proliferation of variants capable of higher infectivity. Mutations in the SARS-CoV-2 genome are the consequence of replication errors, highlighting the importance of understanding the determinants of SARS-CoV-2 replication fidelity. The RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit for SARS-CoV-2 RNA replication and genome transcription. Here, we report the fidelity of ribonucleotide incorporation by SARS-CoV-2 RdRp (nsp12), along with its co-factors nsp7/nsp8, using steady-state kinetic analysis. Our analysis suggests that in the absence of the proofreading subunit (nsp14), the nsp12/7/8 complex has a surprisingly low base substitution fidelity (10-1-10-3). This is orders of magnitude lower than the fidelity reported for other coronaviruses (10-6-10-7), highlighting the importance of proofreading for faithful SARS-CoV-2 replication. We performed a mutational analysis of all reported SARS-CoV-2 genomes and identified mutations in both nsp12 and nsp14 that appear likely to lower viral replication fidelity through mechanisms that include impairing the nsp14 exonuclease activity or its association with the RdRp. Our observations provide novel insight into the mechanistic basis of replication fidelity in SARS-CoV-2 and the potential effect of nsp12 and nsp14 mutations on replication fidelity, informing the development of future antiviral agents and SARS-CoV-2 vaccines.

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

Defective Interfering Particles of Influenza Virus and Their Characteristics, Impacts, and Use in Vaccines and Antiviral Strategies: A Systematic Review.

Defective interfering particles (DIPs) are particles containing defective viral genomes (DVGs) generated during viral replication. DIPs have been found in various RNA viruses, especially in influenza viruses. Evidence indicates that DIPs interfere with the replication and encapsulation of wild-type viruses, namely standard viruses (STVs) that contain full-length viral genomes. DIPs may also activate the innate immune response by stimulating interferon synthesis. In this review, the underlying generation mechanisms and characteristics of influenza virus DIPs are summarized. We also discuss the potential impact of DIPs on the immunogenicity of live attenuated influenza vaccines (LAIVs) and development of influenza vaccines based on NS1 gene-defective DIPs. Finally, we review the antiviral strategies based on influenza virus DIPs that have been used against both influenza virus and SARS-CoV-2. This review provides systematic insights into the theory and application of influenza virus DIPs.

Examples of Structural Motifs in Viral Genomes and Approaches for RNA Structure Characterization.

The relationship between conserved structural motifs and their biological function in the virus replication cycle is the interest of many researchers around the world. RNA structure is closely related to RNA function. Therefore, technological progress in high-throughput approaches for RNA structure analysis and the development of new ones are very important. In this mini review, we discuss a few perspectives on the structural elements of viral genomes and some methods used for RNA structure prediction and characterization. Based on the recent literature, we describe several examples of studies concerning the viral genomes, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV). Herein, we emphasize that a better understanding of viral genome architecture allows for the discovery of the structure-function relationship, and as a result, the discovery of new potential antiviral therapeutics.

Anticipating the Next Chess Move: Blocking SARS-CoV-2 Replication and Simultaneously Disarming Viral Escape Mechanisms.

The COVID-19 pandemic initiated a race to determine the best measures to control the disease and to save as many people as possible. Efforts to implement social distancing, the use of masks, and massive vaccination programs turned out to be essential in reducing the devastating effects of the pandemic. Nevertheless, the high mutation rates of SARS-CoV-2 challenge the vaccination strategy and maintain the threat of new outbreaks due to the risk of infection surges and even lethal variations able to resist the effects of vaccines and upset the balance. Most of the new therapies tested against SARS-CoV-2 came from already available formulations developed to treat other diseases, so they were not specifically developed for SARS-CoV-2. In parallel, the knowledge produced regarding the molecular mechanisms involved in this disease was vast due to massive efforts worldwide. Taking advantage of such a vast molecular understanding of virus genomes and disease mechanisms, a targeted molecular therapy based on siRNA specifically developed to reach exclusive SARS-CoV-2 genomic sequences was tested in a non-transformed human cell model. Since coronavirus can escape from siRNA by producing siRNA inhibitors, a complex strategy to simultaneously strike both the viral infectious mechanism and the capability of evading siRNA therapy was developed. The combined administration of the chosen produced siRNA proved to be highly effective in successfully reducing viral load and keeping virus replication under control, even after many days of treatment, unlike the combinations of siRNAs lacking this anti-anti-siRNA capability. Additionally, the developed therapy did not harm the normal cells, which was demonstrated because, instead of testing the siRNA in nonhuman cells or in transformed human cells, a non-transformed human thyroid cell was specifically chosen for the experiment. The proposed siRNA combination could reduce the viral load and allow the cellular recovery, presenting a potential innovation for consideration as an additional strategy to counter or cope COVID-19.

Subgenomic RNAs and Their Encoded Proteins Contribute to the Rapid Duplication of SARS-CoV-2 and COVID-19 Progression.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently widespread throughout the world, accompanied by a rising number of people infected and breakthrough infection of variants, which make the virus highly transmissible and replicable. A comprehensive understanding of the molecular virological events and induced immunological features during SARS-CoV-2 replication can provide reliable targets for vaccine and drug development. Among the potential targets, subgenomic RNAs and their encoded proteins involved in the life cycle of SARS-CoV-2 are extremely important in viral duplication and pathogenesis. Subgenomic RNAs employ a range of coping strategies to evade immune surveillance from replication to translation, which allows RNAs to synthesize quickly, encode structural proteins efficiently and complete the entire process of virus replication and assembly successfully. This review focuses on the characteristics and functions of SARS-CoV-2 subgenomic RNAs and their encoded proteins and explores in depth the role of subgenomic RNAs in the replication and infection of host cells to provide important clues to the mechanism of COVID-19 pathogenesis.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.

SARS-CoV-2 variant spike and accessory gene mutations alter pathogenesis.

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.

Characterization of SARS-CoV-2 replication complex elongation and proofreading activity.

The replication complex (RC) of SARS-CoV-2 was recently shown to be one of the fastest RNA-dependent RNA polymerases of any known coronavirus. With this rapid elongation, the RC is more prone to incorporate mismatches during elongation, resulting in a highly variable genomic sequence. Such mutations render the design of viral protein targets difficult, as drugs optimized for a given viral protein sequence can quickly become inefficient as the genomic sequence evolves. Here, we use biochemical experiments to characterize features of RNA template recognition and elongation fidelity of the SARS-CoV-2 RdRp, and the role of the exonuclease, nsp14. Our study highlights the 2'OH group of the RNA ribose as a critical component for RdRp template recognition and elongation. We show that RdRp fidelity is reduced in the presence of the 3' deoxy-terminator nucleotide 3'dATP, which promotes the incorporation of mismatched nucleotides (leading to U:C, U:G, U:U, C:U, and A:C base pairs). We find that the nsp10-nsp14 heterodimer is unable to degrade RNA products lacking free 2'OH or 3'OH ribose groups. Our results suggest the potential use of 3' deoxy-terminator nucleotides in RNA-derived oligonucleotide inhibitors as antivirals against SARS-CoV-2.

The emerging role of miRNAs in the pathogenesis of COVID-19: Protective effects of nutraceutical polyphenolic compounds against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can cause immunosuppression and cytokine storm, leading to lung damage and death. The clinical efficacy of anti-SARS-CoV-2 drugs in preventing viral entry into host cells and suppressing viral replication remains inadequate. MicroRNAs (miRNAs) are crucial to the immune response to and pathogenesis of coronaviruses, such as SARS-CoV-2. However, the specific roles of miRNAs in the life cycle of SARS-CoV-2 remain unclear. miRNAs can participate in SARS-CoV-2 infection and pathogenesis through at least four possible mechanisms: 1. host cell miRNA expression interfering with SARS-CoV-2 cell entry, 2. SARS-CoV-2-derived RNA transcripts acting as competitive endogenous RNAs (ceRNAs) that may attenuate host cell miRNA expression, 3. host cell miRNA expression modulating SARS-CoV-2 replication, and 4. SARS-CoV-2-encoded miRNAs silencing the expression of host protein-coding genes. SARS-CoV-2-related miRNAs may be used as diagnostic or prognostic biomarkers for predicting outcomes among patients with SARS-CoV-2 infection. Furthermore, accumulating evidence suggests that dietary polyphenolic compounds may protect against SARS-CoV-2 infection by modulating host cell miRNA expression. These findings have major implications for the future diagnosis and treatment of COVID-19.

Targeting ACLY efficiently inhibits SARS-CoV-2 replication.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the biggest public health challenge the world has witnessed in the past decades. SARS-CoV-2 undergoes constant mutations and new variants of concerns (VOCs) with altered transmissibility, virulence, and/or susceptibility to vaccines and therapeutics continue to emerge. Detailed analysis of host factors involved in virus replication may help to identify novel treatment targets. In this study, we dissected the metabolome derived from COVID-19 patients to identify key host factors that are required for efficient SARS-CoV-2 replication. Through a series of metabolomic analyses, in vitro, and in vivo investigations, we identified ATP citrate lyase (ACLY) as a novel host factor required for efficient replication of SARS-CoV-2 wild-type and variants, including Omicron. ACLY should be further explored as a novel intervention target for COVID-19.