N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification in eukaryotic RNA and is also present in various viral RNAs, where it plays a crucial role in regulating the viral life cycle. However, the molecular mechanisms through which viruses regulate host RNA m6A methylation are not fully understood. In this study, we reveal that SARS-CoV-2 and HCoV-OC43 infection enhance host m6A modification by activating the mTORC1 signalling pathway. Specifically, the viral non-structural protein nsp14 upregulates the expression of S-adenosylmethionine synthase MAT2A in an mTORC1-dependent manner. This mTORC1-MAT2A axis subsequently stimulates the synthesis of S-adenosylmethionine (SAM). The increase of SAM then enhances the m6A methylation of host RNA and facilitates viral replication. Our findings uncover a molecular mechanism by which viruses regulate host m6A methylation and provide insights into how SARS-CoV-2 hijacks host cellular epitranscriptomic modifications to promote its replication.

Therapeutic nucleic acids, including small interfering RNA (siRNA), and antisense oligonucleotides (ASOs), targeting RNA viruses such as influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV), play a crucial role in contemporary medicine. The primary goal of short oligonucleotide-based antivirals is to precisely inhibit viral mechanisms by interacting with viral RNA, thereby opening new avenues for infection treatment. RNA recently was also used to invent mRNA vaccine for different illness prevention. Therapeutic nucleic acids and mRNA vaccine attracted considerable attention during the COVID-19 pandemic due to the pressing necessity to develop an effective strategy to address this global threat. In addition to the advancement of therapeutic nucleic acids aimed at targeting respiratory viruses, the effective delivery of these molecules to infected cells is of paramount importance. Similarly, mRNA vaccine's effectiveness also depends on effective delivery. This article offers a comprehensive summary and analysis of various delivery strategies, along with the challenges encountered in their development. Representative studies conducted in cellular models, model organisms, and human are presented for examination. Furthermore, the article explores future perspectives regarding the delivery of therapeutic nucleic acids and mRNA vaccines aimed at combating IAV, SARS-CoV-2, and RSV.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiated a global pandemic resulting in an estimated 775 million infections with over 7 million deaths, it has become evident that COVID-19 is not solely a pulmonary disease. Emerging evidence has shown that, in a subset of patients, certain symptoms - including chest pain, stroke, anosmia, dysgeusia, diarrhea and abdominal pain - all indicate a role of vascular, neurological and gastrointestinal (GI) pathology in the disease process. Many of these disease processes persist long after the acute disease has been resolved, resulting in 'long COVID' or post-acute sequelae of COVID-19 (PASC). The molecular mechanisms underlying the acute and systemic conditions associated with COVID-19 remain incompletely defined. Appropriate animal models provide a method of understanding underlying disease mechanisms at the system level through the study of disease progression, tissue pathology, immune system response to the pathogen and behavioral responses. However, very few studies have addressed PASC and whether existing models hold promise for studying this challenging problem. Here, we review the current literature on cardiovascular, neurological and GI pathobiology caused by COVID-19 in patients, along with established animal models of the acute disease manifestations and their prospects for use in PASC studies. Our aim is to provide guidance for the selection of appropriate models in order to recapitulate certain aspects of the disease to enhance the translatability of mechanistic studies.

Early and accurate identification of SARS-CoV-2 infection is crucial for epidemic prevention and control. Lateral flow immunoassay (LFIA) has become the mainstream method for screening SARS-CoV-2 infection due to its rapid, simple and amenable for point-of-care detection (POCT), but still suffered from the poor sensitivity and accuracy. In this study, Au nanoparticles (NPs) with controllable Ag shell (Au@Ag) were manufactured via a seed-mediated growth method. The Au@Ag-based LFIA exhibited superb colorimetric (CM) signal and intense surface-enhanced Raman scattering (SERS) signal for dual-mode sensing of nucleocapsid protein (N protein), a naturally protein expression in vivo during SARS-CoV-2 infection. The limit of detection (LOD) of the SERS-LFIA mode was 2.16 pg/mL, which was around 150-time more sensitive than conventional visual CM-LFIA mode (300 pg/mL). More importantly, the proposed LFIA is capable of quantitatively detecting N protein-spiked real samples with satisfactory recoveries from 83 % to 91.4 %. Clinical pharyngeal swab samples of the infected patients (n = 20) and healthy subjects (n = 20) were effectively discriminated in the developed SERS-LFIA, where the negative accuracy rate was 100 % and the positive accuracy rate was 85 %, among which samples from P1, P18, and P19 were false-negative results. The results obtained from the LFIA immunoassay were in good agreement with the standard PCR method in clinic, and superior to those of the commercially colloidal gold strip by using the same antibodies. In conclusion, the LFIA proposed here can perform specific, rapid, and ultrasensitive analysis of N protein toward early warning of SARS-CoV-2 infection.

Acute respiratory diseases in humans can be caused by various viral pathogens such as respiratory syncytial virus (RSV), human coronavirus 229E (hCoV-229E), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To prevent severe cases by an early treatment, one effective strategy is to inhibit viral infection at the entry stage of the replication cycle. However, there is a lack of efficient, FDA-approved small-molecule drugs targeting these pathogens. Previously, we identified two dual RSV/hCoV-229E small-molecule inhibitors with activity in the single-digit micromolar range. In this study, we focused on a structure-guided optimization approach of the more promising prototype addressing activity, cell viability, selectivity, solubility and metabolic stability. We present valuable insights into the structure-activity relationship (SAR), and report the discovery of a sub-micromolar RSV entry inhibitor, a dual RSV/CoV-229E inhibitor and a highly potent compound against hCoV-229E.

Reverse genetics (rg) systems are indispensable tools for investigating the pathogenesis of RNA viruses, facilitating vaccine design, and advancing antiviral therapeutic strategies. In this study, we optimized the Infectious Subgenomic Amplicons (ISA) method for generating synthetic r-wt SARS-CoV-2 Wuhan-Hu-1. This system was validated by demonstrating the successful rescue of infectious viral particles from overlapping DNA fragments and their propagation in vitro. Sequencing confirmed 100 % identity of the recovered virus with the Wuhan-Hu-1 reference genome. Importantly, in vivo experiments using K18-hACE2 mice revealed that the r-wt SARS-CoV-2 Wuhan-Hu-1 strain caused clinical symptoms, weight loss, and mortality comparable to those induced by a virulent SARS-CoV-2 field variant. This ISA rg method offers a rapid and reproducible approach to generating synthetic coronaviruses, with potential applications in pathogenesis studies, antiviral testing, and vaccine development.

The coronavirus disease 2019 (COVID-19) global health crisis, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented unprecedented challenges to global healthcare systems, leading to rapid advances in treatment development. This review comprehensively examines the current therapeutic approaches for managing COVID-19, including direct-acting antivirals, immunomodulators, anticoagulants, and adjuvant therapies, as well as emerging and experimental approaches. Direct-acting antivirals target various stages of the viral life cycle, offering specific intervention points, while immunomodulators aim to modulate the host's immune response, reducing disease severity. Anticoagulant therapies address the coagulopathy frequently observed in severe cases, and adjuvant treatments provide supportive care to improve overall outcomes. We also explore the challenges and limitations of implementing these treatments, such as drug resistance, variable patient responses, and access to therapies, especially in resource-limited settings. The review also discusses future perspectives, including the potential of next-generation vaccines, personalized medicine, and global collaboration in shaping future COVID-19 treatment paradigms. Continuous innovation, combined with an integrated and adaptable approach, will be crucial to effectively managing COVID-19 and mitigating the impact of future pandemics.

Through screening of a nucleos(t)ide-focused chemical library, we identified 4'-thiouridine (1) as a potent antiviral agent against SARS-CoV-2 in Vero cells, with an EC50 value of 1.71 μM and a CC50 value exceeding 100 μM. Its triphosphate metabolite, compound 8, inhibited the RNA-dependent RNA polymerase activity of the SARS-CoV-2 Nsp12-Nsp7-Nsp82 complex, terminating nascent RNA synthesis through misincorporation. Additionally, compound 8 suppressed the function of the NiRAN domain of Nsp12, effectively blocking both RNAylation and NMPylation of Nsp9. Pharmacokinetic analysis in mice showed excellent oral bioavailability of compound 1. Oral administration at 100 mg/kg/day, twice daily for 5 days, protected mice from lethal SARS-CoV-2 infection, resulting in 40% survival and near-complete recovery of body weight by day 14 postinfection. Compound 1 also exhibited broad-spectrum activity against various coronaviruses and other RNA viruses. These findings highlight that compound 1 is a promising orally available antiviral candidate.

During infection, the coronavirus Nsp15, a uridine-specific endoribonuclease, suppresses the host cell's antiviral response. Recently, researchers have paid more attention to this relatively underexplored yet potentially viable drug target. In this study, we employed fluorescence resonance energy transfer-based screening assays to identify potent Nsp15 inhibitors. Subsequently, we used active-site in silico docking methods to design new molecules with enhanced inhibitory properties. Solution assays were used to measure the potency and determine the mechanism of these inhibitors. We identified a novel class of thiazolidinedione and rhodanine analogs that inhibit SARS-CoV-2 Nsp15. Docking these compounds into the uridine-binding site shows that most analogs form two hydrogen bonds with Ser294. The most potent inhibitors are compounds KCO237 and KCO251 (half-maximal inhibitory concentration: 0.304 μM, 0.931 μM respectively). The inhibition kinetics of KCO237 and KCO251 best align with a reversible mixed inhibition model. Mutating Ser294 did not completely abolish Nsp15 activity or the inhibitory effect of KCO237 or KCO251. These findings suggest that thiazolidinedione and rhodanine analogs likely inhibit Nsp15 by binding to the uridine active site while also implicating a possible secondary allosteric binding site. The ability of these compounds to inhibit VERO 6 cell infection with SARS-CoV-2 at subtoxic levels highlights their potential for development as novel antiviral treatments for SARS-CoV-2 and other coronavirus-related diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogenic agent responsible for the coronavirus disease 2019 (COVID-19) pandemic, uses the trimeric spike protein to gain entry into the host cell. Structural studies have revealed that the spike protein is comprised of the S1 and S2 subunits. The S1 subunit of the spike protein contains the receptor-binding domain (RBD), which binds to the human angiotensin-converting enzyme 2 (ACE2) receptor. The interaction between the RBD and ACE2 facilitates membrane fusion and host cell infection. The SARS-CoV-2 spike protein also contains a unique insertion of four amino acids that results in the 682-RRAR↓S-686 polybasic furin cleavage motif at the boundary of the S1 and S2 subunits. The furin cleavage motif contributes to the high infectivity and transmissibility of SARS-CoV-2. This review provides a comprehensive analysis of the molecular interactions of the spike protein, with a specific focus on the RBD and furin cleavage site. In addition to examining the binding characteristics with ACE2, the interactions with alternative receptors, such as neuropilin-1 (NRP1) and the nicotinic acetylcholine receptors (nAChRs) are highlighted. The ability of the spike protein to bind alternative receptors and host factors has been linked to the pathophysiology of COVID-19 and the persistence of symptoms in the post COVID-19 condition. Furthermore, we examine the impact of spike protein mutations on receptor affinity and disease severity. SARS-CoV-2 continues to evolve, with variants remaining an ongoing threat to public health. Understanding these molecular interactions is critical for the development of novel therapeutic interventions.

The COVID-19 pandemic is caused by the enveloped virus SARS-CoV-2. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Blocking endoplasmic reticulum (ER) to Golgi trafficking dramatically inhibits SARS-CoV-2 assembly and secretion without reducing viral genome replication. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion without affecting viral entry, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to modulate viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19) and has persistently caused infections since its emergence in late 2019. The main protease (Mpro) of SARS-CoV-2 plays a crucial role in its life-cycle; thus, it is an important target for drug development. One of the first virus-specific drugs that has been approved for the treatment of COVID-19 patients is Paxlovid, which contains nirmatrelvir, a covalent inhibitor of Mpro. Screening of inhibitor candidates and specificity studies also rely on efficient substrates and activity assays. Casein is one of the most commonly applied universal substrates that can be used to study a wide range of proteases, including SARS-CoV-2 Mpro. Casein is a known substrate for Mpro in vitro, but the specific casein isoform cleaved by Mpro remained unidentified, and the cleavage sites have yet to be determined. This work studied cleavage of α-, β- and κ-isoforms of bovine casein by SARS-CoV-2 Mpro, using in vitro and in silico approaches. The candidate cleavage sites were predicted in silico based on the protein sequences, and the cleavage positions were identified based on mass spectrometric analysis of cleavage fragments. Based on our results, only β-casein contains cleavage sites for Mpro and thus can be used as its substrate in vitro. The newly identified cleavage site sequences further widen the knowledge about the specificity of SARS-CoV-2 Mpro.

The study evaluated the effects of violet-blue light (VBL) on cell viability and replication, carbonylation of three structural proteins (S, E, and N) and one non-structural protein (NSP13), and direct damage to the RNA of SARS-CoV-2. The virus was exposed to increasing doses of VBL along with influenza A and B viruses to compare their susceptibility. At the highest dose (21.6 J/cm2), SARS-CoV-2 was significantly more susceptible to VBL than the influenza viruses, with a reduction in viral titer of 2.33 log10. Viral RNA did not show significant changes after exposure to VBL, as demonstrated by next-generation sequencing and real-time PCR quantification, suggesting that the inactivation process does not involve direct nucleic acid damage. To exclude the role of the culture suspension in the inactivation process, virus viability experiments were performed using different dilutions of Dulbecco's modified Eagle's medium (DMEM) in phosphate-buffered saline (PBS). The results indicated that the suspension medium played a secondary role in virus inactivation, as viability did not increase with increasing DMEM dilution. Subsequent tests with three different antioxidants (NAC, AsA, and SOD) at different concentrations prevented viral inactivation, from 99.99% to 85.43% (with SOD 0.003 mM). Carbonylation of S and E proteins was more pronounced when viruses were suspended in DMEM rather than PBS, although the tests demonstrated that the intrinsic properties of the viral membrane were a crucial element to consider in relation to its susceptibility to VBL.IMPORTANCELight-based disinfection methods are often used in combination with other cleaning methods due to their non-invasive nature, versatility, and environmental benefits. VBL is an effective approach as it induces the production of reactive oxygen species that reduce microbial viability. In this study, lipid peroxidation was identified as an important factor affecting the structural integrity and function of the viral envelope, reducing its ability to interact with host cells and consequently its ability to be infectious. The lipid envelope of SARS-CoV-2, composed mainly of glycerophospholipids and lacking cholesterol and sphingolipids, appears to be the critical factor in its susceptibility, distinguishing it from influenza viruses, which have a lipid profile richer in components that protect against oxidative stress.

Vaccination is a key control measure of coronavirus disease 2019 by preventing severe effects of disease outcomes, reducing hospitalization rates and death, and increasing immunity. However, vaccination can affect the evolution and adaptation of SARS-CoV-2 largely through vaccine-induced immune pressure. Here, we investigated intrahost recombination and single nucleotide variations (iSNVs) on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome in non-vaccinated and vaccinated sequences from the Kenyan population to profile intrahost viral genetic evolution and adaptations driven by vaccine-induced immune pressure. We identified recombination hotspots in the S, N, and ORF1a/b genes and showed the genetic evolution landscape of SARS-CoV-2 by comparing within- and inter-wave recombination events from the beginning of the pandemic (June 2020 to December 2022) in Kenya. We further reveal differential expression of recombinant RNA species between vaccinated and non-vaccinated individuals and perform an in-depth analysis of iSNVs to identify and characterize the functional properties of non-synonymous mutations found in ORF-1 a/b, S, and N genes. Lastly, we detected a minority variant in non-vaccinated patients in Kenya, with an immune escape mutation S255F of the spike gene, and showed differential recombinant RNA species. Overall, this work identified unique in vivo mutations and intrahost recombination patterns in SARS-CoV-2, which could have significant implications for virus evolution, virulence, and immune escape.IMPORTANCEThe impact of vaccination on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity in Kenya and much of Africa remains unknown. This can be attributed to lower sequencing rates; however, this information is relevant to improvement in vaccine and antiviral research. In this study, we investigated how vaccination and SARS-CoV-2 transmission waves affect intrahost non-homologous recombination and single nucleotide variations (iSNVs). We identified unique in vivo mutations and intrahost recombination patterns in SARS-CoV-2, which could have significant implications for virus evolution, virulence, and immune escape. We also demonstrate a methodology for studying genetic changes in a pathogen by a simultaneous analysis of both intrahost single nucleotide variations and recombination events. The study reveals the diversity of SARS-CoV-2 in Kenya and highlights the need for sustained genomic surveillance in Kenya and Africa to better understand how the virus evolves. Such surveillance ensures detection of drifts in evolution, allowing information for updates in vaccines, policy making, and containment of future variants of SARS-CoV-2.

Stem-loop 5 (SL5) is a structural element that is conserved across coronavirus genomic RNAs. It spans the start codon from which the long ORF1 is translated in full-length viral RNA. Phylogenetic conservation indicates that it is comprised of four paired elements, but the specific 3D arrangement of these helices has remained unknown. Now, we have solved the crystal structure of SL5 from SARS-CoV-2 at 3.3 Å resolution, finding that the RNA adopts a T-shaped four-way junction fold in which two coaxial stacks of two helices each pack orthogonally. This arrangement results in deep pockets at the helical junction, where cations bind. Except for limited interactions in this region, the structure is remarkable for the paucity of tertiary contacts. We confirmed the stability of this fold in solution by FRET and carried out single-particle cryogenic-sample electron microscopy (cryoEM). The resulting ∼5 Å resolution cryoEM map, and 3D variability analysis, suggest conformational flexibility at the junction. In vitro translation of structure-guided mutants demonstrated that SL5 inhibits protein synthesis. Thus, it is likely that SL5 recruits additional factors in vivo. This, and its characteristic clefts at the four-way junction, make SL5 an attractive target for the discovery of RNA-targeted antiviral small molecules.

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, most of these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased toward the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published functional screens and proteomics data to reveal (i) common pathways that were identified in all OMICs datasets-including regulation of Wnt signaling and gap junctions, (ii) pathways uniquely identified in this screen-including NADH oxidation, or (iii) pathways supported by this screen and proteomics data but not published functional screens-including arachionate production and MAPK signaling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 32 proteins that were determined to impact viral replication and 27 impacting late stages of infection, respectively. Additionally, a subset of proteins was tested across other coronaviruses revealing a subset of proviral factors that were conserved across pandemic SARS-CoV-2, epidemic SARS-CoV-1 and MERS-CoV, and the seasonal coronavirus OC43-CoV. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

Virus survival inside the host cell depends on the intricate mechanisms that recruit proteins involved in the arms race. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) proteome exhibits important levels of structural order. However, some of the SARS-CoV-2 proteins, such as the Nucleocapsid (N) and Non-structural protein 1 (Nsp1), contain a considerably significant amount of intrinsically disordered regions (IDRs) that play indispensable roles in the intra-viral and virus-host interaction. Here, focusing on proteins that contain a relevant percentage of IDRs, we discuss experimental and computational studies sought to support IDRs as a key player in the interplay with ordered domains, the biological role as potential origin for variants of SARS-CoV-2, and their association with virus transmissibility. Furthermore, we also highlight the potential involvement of IDRs in the viral-host protein interaction and host cellular machinery. Thus, shading lights on the dark proteome of the virus and looking for therapeutic approaches beyond the classic structure-function paradigm may contribute to the efforts sparking the quest for therapeutics.

The advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent coronavirus disease 2019 (COVID-19) pandemic have posed a serious threat to human health and society [...].

The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multisubunit replication complex consisting of nonstructural proteins (nsps) 12, 7, and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or coexpressed replication complex and RNA. Increasing binding times were observed, in this order, with nsp7 (none), nsp8, and nsp12, in nsp8 nsp12 mixtures and in reconstituted mixtures bearing all three proteins. Unstable, unstable→stable, and stable binding modes were recorded in the latter case, indicating that complexation is dynamic and the correct conformation must be achieved before stable RNA binding can occur. Notably, the coexpressed protein yields mostly stable binding even at low concentrations, while the reconstituted proteins exhibit unstable binding indicating inefficient complexation with reduced protein. The SM-PIFE assay distinguishes inhibitors that impact protein binding from those that prevent replication, as demonstrated with suramin and remdesivir, respectively. The data reveals a correlation between binding lifetime/affinity and protein activity and underscores differences between coexpressed vs reconstituted mixtures, suggesting the existence of trapped conformations that may not evolve to productive binding.

Myocardial injury is a critical determinant of prognosis in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, its underlying mechanisms remain incompletely understood. In this study, we examined the effects of SARS-CoV-2-derived RNA fragments on human cardiomyocytes. We identified a 19-nucleotide sequence within the viral genome that shares complete sequence homology with the human F1F0 ATP synthase subunit alpha gene (ATP5A). This sequence was found to associate with Argonaute 2 (AGO2) and downregulate ATP5A expression via a mechanism analogous to RNA interference. Consequently, oxidative phosphorylation was suppressed in cardiomyocytes, leading to impaired myocardial maturation and the emergence of heart failure-like phenotypes. Notably, exosome-mimetic liposomal delivery of this RNA fragment to cardiomyocytes reproduced the ATP5A-suppressive effect. These findings suggest that SARS-CoV-2-derived RNA fragments may contribute to myocardial injury through the siRNA-like modulation of mitochondrial gene expression. Further validation in animal models and patient-derived materials is warranted.

SARS-CoV-2 infections directly or indirectly cause unconscionable vascular events, significantly increasing the morbidity and mortality of COVID-19. Biomarkers associated with cardiac injury often elevate in individuals with COVID-19. Cytokine storm is a mechanism underlying myocardial cell injury caused by SARS-CoV-2 infections. Cell apoptosis in AC16 cells overexpressing structural and helper proteins of SARS-CoV-2 was detected by Western blot, MTT and TUNEL assay. The M protein was determined to play the most pronounced role in inducing apoptosis. Transcriptome sequencing on AC16 cells overexpressing the M protein was performed to screen differentially expressed genes (DEGs), which were further subjected to the gene set enrichment analysis. The regulatory effect of CXCL10 on cell apoptosis of AC16 cells overexpressing M protein was finally explored. Overexpression of M protein significantly increased the Bax/Bcl-2 and cleaved caspase-3/caspase-3(CC3/C3) ratios and the percentage of TUNEL-positive cells in AC16 cells, while markedly reducing cell viability. CXCL10 was the most prominent DEG in AC16 cells overexpressing M protein. Knockdown of CXCL10 partially reversed the increases in the Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, the percentage of TUNEL-positive cells, as well as the release of pro-inflammatory cytokines in AC16 cells overexpressing M protein. The M protein of SARS-CoV-2 triggers the production of CXCL10 and apoptosis of myocardial cells.

Current vaccines rely on the sequence of Spike (S) protein to induce immunity against the severe acute respiratory coronavirus-2 (SARS-CoV-2) virus. Because of the high mutation rate of the viral S protein, new mutant strains are developed to generate new infectivity profiles. Bioactive lipid mediators (LMs) derived from docosahexaenoic acid (DHA) are synthesized on demand to sustain homeostasis. The purpose of this study was to determine the action of selected LMs in the viral replication of SARS-CoV-2 Omicron BA.5 variant in human lung and nasal epithelial cells. Cells from healthy donors were infected with Omicron BA.5 for one hour and treated with 500 nM Elovanoid (ELV)-N32, ELV-N34, Resolvin D6 isomer (RvD6i), Neuroprotection D1 (NPD1), or vehicle before and after infection. Impedance was recorded to determine cell death by infectivity. Cells were then immunostained for nucleocapsid (N) protein, microtubule-associated protein 1B-light chain 3 (LC3B), and autophagic proteins. N and S RNA were measured to assess the synthesis of viral components. The addition of ELV-N34 or RvD6i decreased the synthesis of N RNA by 76.7% and 96.9%, respectively, in lung primary culture, while NPD1 exerted the same effect in nasal epithelial cells (61.7% reduction). In lung cells, transcription of autophagy-related gene-3 (ATG3) and Sequestosome 1 (SQSTM1/p62), components of the autophagy initiation process, decreased compared to the non-treated infected cells. The results suggest that specific LMs prevent viral autophagy machinery hijacking, leading to a decrease in BA.5 replication. This novel effect of the bioactive LMs as antivirals, regardless of the protein sequence, would potentially complement vaccination and other prevention and treatment therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) prolonged the coronavirus disease 2019 (COVID-19) pandemic. The continued development of novel pan-variant therapeutics to treat currently circulating and future VOCs is critically important. Photomedicine may offer broadly applicable, pan-variant treatments. In this study, we show that visible light centered around 425 nm inactivates each of the five SARS-CoV-2 VOC lineages that have been identified by the World Health Organization (Alpha, Beta, Delta, Gamma, and Omicron) in cell-free suspensions in a dose-dependent manner, including bamlanivimab-resistant variants. Specifically, 60 J/cm2 of 425 nm light reduced SARS-CoV-2 titers by >4 log10 relative to unilluminated controls. We observed that 425 nm light inactivates SARS-CoV-2 through restricted entry to host cells. In addition, a non-cytotoxic dosing regimen of 32 J/cm2 of 425 nm light reduced infectious virus titers in well-differentiated air-liquid interface (ALI) human airway epithelial (HAE) cells infected with the Beta, Delta, and Omicron variants that incorporate mutations associated with immune evasion and/or increased transmissibility. Infectious SARS-CoV-2 titers were reduced when dosing began during the early stages of infection or in more established infections. Finally, we translated these findings to the RD-X19, a novel medical device that emits 425 nm light; our results showed that the RD-X19 restricted spike binding to ACE-2 and reduced SARS-CoV-2 titers in cell-free suspensions (by >2 log10) and in the ALI HAE model (by >1 log10). These findings indicate that photomedicine utilizing 425 nm visible light may serve as a novel, pan-variant treatment modality for COVID-19.IMPORTANCEThe continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of variants that can evade public health measures, including vaccines and therapeutics. Thus, the continued development of broadly applicable measures to supplement current public health measures and standards of care remains critical. Photomedicine is one such approach. In this study, we show that non-ultraviolet visible light can inactivate each SARS-CoV-2 variant of concern (VOC) by preventing entry to host cells. Furthermore, visible light reduced the amount of virus produced in an infection model of the human airway at multiple stages of infection, demonstrating the antiviral capability of visible light. This study provides preclinical support for the development of visible light to serve as a SARS-CoV-2 countermeasure and warrants further investigation.

The COVID-19 pandemic caused immense mortality and morbidity reporting 704,753,890 cases worldwide. The repercussions of this pandemic are still being felt in the form of newly evolving variants and infections. The pandemic has pointed towards the need for the development of new and effective agents against SARS-CoV-2 infection. Sulfonohydrazides are a class of compounds with a wide range of therapeutic potential. The present study aims to identify the anti-SARS-CoV-2 potential of Sulfonohydrazide compounds. Twenty-five Sulfonohydrazides derivatives were evaluated for anti-viral potential via plaque reduction assay (PRA) and cytopathic effect (CPE) analysis in-vitro. Treatment point assay was employed for the strategic evaluation of antiviral compound at the particular stages of the SARS-CoV-2 life cycle. Gene expression analysis was also carried out, which was supported by immunofluorescence assays targeting the N and S proteins of SARS-CoV-2, alongside fold-change analysis, to identify a robust and multifaceted approach for the understanding of viral dynamics. Moreover, ligand-inhibitor interactions were assessed by in- silico studies. Compound 24 (4(E)-4-methyl-N'-(2,3,4-trihydroxybenzylidene)benzenesulfonohydrazide) was identified as the most potent molecule that inhibited SARS-CoV-2 infection (92.85 ± 3.57%) via PRA. The time point assay revealed that the effect of the compound might be at the entry point, which might be due to the down-regulation of the Spike (S) and Angiotensin-converting enzyme 2 (ACE-2) genes by the compound. The gene expression analysis of ORF1a/b by qRT-PCR indicated reduction in viral load after compound treatment, as indicated by a higher cycle threshold (Ct) value. Moreover, the compound 24 also downregulated the expression of S, RdRp, and ACE-2. Furthermore, the interaction of compound 24 with S, RdRp, and ACE-2 was predicted via molecular docking, which validated the interaction and possible anti-SARS-CoV-2 effect. Additionally, immunofluorescence staining analysis of spike and nucleocapsid proteins also showed downregulation in SARS-CoV-2 infected cells. Overall, the acquired data suggested that Sulfonohydrazide derivative 24 inhibits SARS-CoV-2 entry and replication.

SARS-CoV-2's continued global health impact underscores the importance of ongoing pathogenesis research. Insights into the host's first line of defense against severe COVID-19 identify actionable biomarkers, informing disease management or therapeutics. Yet, the innate immune response, including cytokines, chemokines, adenosine deaminases (ADAs) and Toll-like receptors (TLRs), relevant to COVID-19 remain incompletely characterized.

Peripheral blood was longitudinally collected between May 2020 and March 2021 from COVID-19 hospitalized adults (N = 79) and healthy controls (HCs) (N = 14; not tested, assumed COVID-negative, no viral exposure or symptoms). Heparinized blood was fractionated for plasma cryopreservation and in vitro whole blood TLR-stimulation employing TLR-3, -4, and -7/8 agonists. Post-stimulation culture supernatants were analyzed using multiplex and enzymatic assays.

Upon hospitalization, plasma concentrations of IFNγ, IL-6, CXCL10, and ADAs were significantly upregulated compared to convalescent time points and HCs. Participants with fatal COVID-19 exhibited higher IL-27, CXCL10, and ADAs concentrations upon admission. Plasma cytokines, chemokines, and ADAs were positively correlated and associated with distinct temporal patterns. TLR-stimulated cell cultures from patients produced reduced IFNα2, IFNγ, IL-12p40, and IL-12p70 compared to HCs or later time points.

Higher plasma concentrations of IL-27, CXCL10, and ADAs at admission were associated with severe COVID-19 and mortality. Reduced TLR-mediated IFNα2, IFNγ, and IL-12p70 production suggests COVID dampens Th1-polarizing innate immune responses, providing insight into immunological sequelae of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a highly transmissible acute respiratory infection that can result in severe pneumonia and death. Many details of SARS-CoV-2 infection are not fully understood, including the cell biology and host-virus interactions involved in coronavirus assembly and release, in which the Envelope (E) structural protein is instrumental. Deletion of E in other coronaviruses has been shown previously to either attenuate or abrogate infection. To determine the role of E on SARS-CoV-2 virus production and infectivity, we produced reporter SARS-CoV-2 with or without the E gene deleted using a bacterial artificial chromosome. Replication of ΔE SARS-CoV-2 was attenuated in Vero E6 cells expressing human ACE2 and TMPRSS2 and in human epithelial cell lines. Electron and immunofluorescence microscopy and virology assays showed that ΔE SARS-CoV-2 increased cell surface expression of Spike (S) glycoprotein, leading to reduced S incorporation into ΔE SARS-CoV-2 particles and promotion of increased cell-to-cell transmission that evades neutralizing antibody inhibition. Trans-complementation of E partially rescued ΔE SARS-CoV-2 S incorporation and restored cell-free transmission. In addition to validating the role of E in retention of S in the ER-Golgi intermediate complex (ERGIC), our results showed that a lack of E led to reorganization of the ERGIC during SARS-CoV-2 infection. Improved understanding of E in SARS-CoV-2 replication and host pathogenesis may help development of novel therapeutics.

Non-S coronavirus structural proteins, including E, are conserved, making them potential pan-coronavirus therapeutic targets. Many details about these proteins and their roles in viral replication and host pathogenesis are unknown. In this study, we showed that SARS-CoV-2 replicates without E but is attenuated and impaired for virus particle formation, with less S incorporated into virions and more S expressed on the cell surface compared to wild-type virus. SARS-CoV-2 lacking E spread primarily via cell fusion and evaded neutralizing antibodies. In addition, the absence of E resulted in the reorganization of the ERGIC cell secretory compartment during SARS-CoV-2 infection. A better understanding of how E influences SARS-CoV-2 replication could guide directed design of novel therapeutics for treatment of COVID-19 patients, as well as the potential for pan-coronavirus protection against future coronavirus outbreaks.

Severe acute respiratory syndrome coronavirus 2 non-structural protein 8 (SARS-CoV-2 nsp8) is a multifunctional protein essential for viral replication and immune evasion. However, the host factors that regulate nsp8 stability and function remain unclear. In this study, we identify HECT and RCC-like domain-containing protein 5 (HERC5) as an essential E3 ligase that regulates nsp8 stability through ISGylation, a ubiquitin-like post-translational modification that facilitates proteasome-dependent degradation. HERC5 overexpression significantly enhances nsp8 degradation in an enzymatic activity-dependent manner, whereas SARS-CoV-2 papain-like protease (PLpro) counteracts this process by deconjugating interferon-stimulated gene 15 (ISG15) from nsp8-thereby preventing its degradation and facilitating viral replication. Mass spectrometry and mutational analyses revealed that the N2 domain of nsp8 is indispensable for ISGylation, with multiple lysine residues acting as primary modification sites. Additionally, we demonstrated that the ISGylation system, including HERC5, ubiquitin-like modifier activating enzyme 7 (UBA7), and ISG15, effectively suppresses SARS-CoV-2 replication across multiple variants, including Omicron BA.5 and XBB.1.5.15. These findings provide novel insights into the role of ISGylation in host antiviral defense and highlight the interplay between HERC5 and PLpro in modulating viral replication. This study establishes a foundation for developing therapeutic strategies targeting HERC5 or PLpro to inhibit SARS-CoV-2 replication.

Structurally constrained cyclic β-amino acids are attractive building blocks for peptide drugs because they induce unique and stable conformations. Introduction of (1S,2S)-2-aminocyclopentanecarboxylic acid [(1S,2S)-2-ACPC] into peptides stabilizes helical conformations, so improving proteolytic stability and cell membrane permeability. We report on the ribosomal synthesis of a helical peptide library incorporating (1S,2S)-2-ACPC at every third position and its application for the discovery of SARS-CoV-2 main protease (Mpro) inhibitors. We identified two peptide sequences containing multiple (1S,2S)-2-ACPC residues, which exhibit helical conformations and superior proteolytic stability compared with their α-Ala or β-Ala counterparts. Studies using the chloroalkane cell-penetration assay showed that their cell permeability values (CP50) are comparable with or even slightly better than that of the cell-penetrating nona-arginine (R9) peptide. The new approach is thus a highly efficient method that combines a helical peptide library containing structurally constrained cyclic β-amino acids with the classical RaPID discovery method, enabling de novo discovery of proteolytically stable and cell-penetrating bioactive peptides that target intracellular proteins.

Among the identified targets for developing anti-coronavirus therapies, SARS-CoV-2 Mpro stands out as one of the most promising due to its crucial role in viral replication and its low mutability across various coronaviruses, making it a potential broad-spectrum target. Currently, although the approved drugs targeting Mpro are peptidomimetic inhibitors with an adequate efficacy, they exhibit relatively poor pharmacokinetic properties commonly associated with peptide-based compounds. On the contrary, using non-peptidic small-molecules Mpro inhibitors can offer many advantages, including reduced off-target toxicity, improved metabolic stability and drug-like properties more appropriate for oral administration. This topic has sparked interest in the scientific community, leading to the publication of numerous studies in recent years. In this review, we summarize the most recent progress over the past two years in the identification and development of synthetic small-molecule inhibitors of SARS-CoV-2 Mpro.

Endocytosis is a specialized transport mechanism in which the cell membrane folds inward to enclose large molecules, fluids, or particles, forming vesicles that are transported within the cell. It plays a crucial role in nutrient uptake, immune responses, and cellular communication. However, many pathogens exploit the endocytic pathway to invade and survive within host cells, allowing them to evade the immune system and establish infection. Endocytosis can be classified as clathrin-mediated (CME) or clathrin-independent (CIE), based on the mechanism of vesicle formation. Unlike CME, which involves the formation of clathrin-coated vesicles that bud from the plasma membrane, CIE does not rely on clathrin-coated vesicles. Instead, other mechanisms facilitate membrane invagination and vesicle formation. CIE encompasses a variety of pathways, including caveolin-mediated, Arf6-dependent, and flotillin-dependent pathways. In this review, we discuss key features of CIE pathways, including cargo selection, vesicle formation, routes taken by internalized cargo, and the regulatory mechanisms governing CIE. Many viruses and bacteria hijack host cell CIE mechanisms to facilitate intracellular trafficking and persistence. We also revisit the exploitation of CIE by bacterial and viral pathogens, highlighting recent discoveries in entry mechanisms, intracellular fate, and host-pathogen interactions. Understanding how pathogens manipulate CIE in host cells can inform the development of novel antimicrobial and immunomodulatory interventions, offering new avenues for disease prevention and treatment.

Human coronaviruses (CoV) cause respiratory infections that range from mild to severe. CoVs are a large family of viruses with considerable genetic heterogeneity and a multitude of viral types, making preventing and treating these viruses difficult. Comprehensive treatments that inhibit CoV infections fulfill a pressing medical need and may be immensely valuable in managing emerging and endemic CoV infections. As the main protease (Mpro) is highly conserved across many CoVs, this protease has been identified as a route for broad CoV inhibition. We utilize the advanced generative chemistry platform Chemistry42 for de novo molecular design and obtained novel small-molecule, non-peptide-like inhibitors targeting the SARS-CoV-2 Mpro. ISM3312 is identified as an irreversible, covalent Mpro inhibitor from extensive virtual screening and structure-based optimization efforts. ISM3312 exhibits low off-target risk and outstanding antiviral activity against multiple human coronaviruses, including SARS-CoV-2, MERS-CoV, 229E, OC43, NL63, and HKU1 independent of P-glycoprotein (P-gp) inhibition. Furthermore, ISM3312 shows significant inhibitory effects against Nirmatrelvir-resistant Mpro mutants, suggesting ISM3312 may contribute to reduced viral escape in these settings. Incorporating ISM3312 and Nirmatrelvir into antiviral strategy could improve preparedness and reinforce defenses against future coronavirus threats.

COVID-19 is a human respiratory syndrome caused by the infection of the SARS-CoV-2 virus that has a high rate of infection and mortality. Viruses modulate the host machinery by altering cellular mechanisms that favor their replication. One of the mechanisms that viruses exploit is the protein folding and processing of post-translational modifications that occur in the endoplasmic reticulum (ER). When ER function is impaired, there is an accumulation of misfolded proteins leading to endoplasmic reticulum stress (ER stress). To maintain homeostasis, cells trigger an adaptive signaling mechanism called the Unfolded Protein Response (UPR) which helps cells deal with stress, but under severe conditions, can activate the apoptotic cell death mechanism. This study elucidated an activation of a diversity of molecular mechanisms by Brazilian variants of SARS-CoV-2 by a time-resolved and large-scale characterization of SARS-CoV-2-infected cells proteomics and immunoblotting. Furthermore, it was shown that pharmacological UPR modulation could reduce viral release by counteracting the different viral activations of its cellular response. Analysis of human clinical specimens and disease outcomes focusing on ER stress reinforces the importance of UPR modulation as a host regulatory mechanism during viral infection and could point to novel therapeutic targets. SIGNIFICANCE: Since the emergence of SARS-CoV-2 and the consequent COVID-19 pandemic, the rapid emergence of variants of this new coronavirus has been a cause for concern since many of them have significantly higher rates of transmissibility and virulence, being called Variants of Concern (VOC). In this work, we studied the VOCs Gamma (P.1) and Zeta (P.2), also known as Brazilian variants. Constant evidence has reported that there are particularities related to each variant of SARS-CoV-2, with different rates of transmissibility, replication and modulation of host biological processes being observed, in addition to the mutations present in the variants. For this reason, this work focused on infections caused by the Brazilian variants of SARS-CoV-2 in different cell lines, in which we were able to observe that the infections caused by the variants induced endoplasmic reticulum stress in the infected cells and activated the UPR pathways, presenting specific modulations of each variant in this pathway. Furthermore, transcriptome analysis of patients revealed a correlation between ER-related genes and COVID-19 progression. Finally, we observed that the use of UPR modulators in host cells decreased viral release of all variants without affecting cell viability. The data presented in this work complement the observations of other studies that aim to understand the pathogenicity of SARS-CoV-2 VOCs and possible new therapeutic strategies, mainly targeting biological processes related to the endoplasmic reticulum.

The SARS-CoV-2 nucleocapsid protein, or N-protein, is a structural protein that plays an important role in the SARS-CoV-2 life cycle. The N-protein takes part in the regulation of viral RNA replication and drives highly specific packaging of full-length genomic RNA prior to virion formation. One regulatory mechanism that is proposed to drive the switch between these two operating modes is the phosphorylation state of the N-protein. Here, we assess the dynamic behavior of non-phosphorylated and phosphorylated versions of the N-protein homodimer through atomistic molecular dynamics simulations. We show that the introduction of phosphorylation yields a more dynamic protein structure and decreases the binding affinity between the N-protein and RNA. Furthermore, we find that secondary structure is essential for the preferential binding of particular RNA elements from the 5' UTR of the viral genome to the N-terminal domain of the N-protein. Altogether, we provide detailed molecular insights into N-protein dynamics, N-protein:RNA interactions, and phosphorylation. Our results corroborate the hypothesis that phosphorylation of the N-protein serves as a regulatory mechanism that determines N-protein function.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a threat due to the emergence of variants with increased transmissibility and enhanced escape from immune responses. Like other coronaviruses before, SARS-CoV-2 likely emerged after its transmission from bats. The successful propagation of SARS-CoV-2 in humans might have been facilitated by usurping evolutionarily conserved cellular factors to execute crucial steps in its life cycle, such as the generation of replication organelles-membrane structures where coronaviruses assemble their replication-transcription complex. In this study, we found that RAB5, which is highly conserved across mammals, is a critical host dependency factor for the replication of the SARS-CoV-2 genome. Our results also suggest that SARS-CoV-2 uses RAB5+ membranes to build replication organelles with the aid of COPB1, a component of the COP-I complex, and that the virus protein NSP6 participates in this process. Hence, targeting NSP6 represents a promising approach to interfere with SARS-CoV-2 RNA synthesis and halt its propagation.IMPORTANCEIn this study, we sought to identify the host dependency factors that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for the generation of replication organelles: cellular membranous structures that SARS-CoV-2 builds in order to support the replication and transcription of its genome. We uncovered that RAB5 is an important dependency factor for SARS-CoV-2 replication and the generation of replication organelles, and that the viral protein NSP6 participates in this process. Hence, NSP6 represents a promising target to halt SARS-CoV-2 replication.