SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis.

Viral infection-induced cell death has long been considered as a double-edged sword in the inhibition or exacerbation of viral infections. Patients with severe Coronavirus Disease 2019 (COVID-19) are characterized by multiple organ dysfunction syndrome and cytokine storm, which may result from SARS-CoV-2-induced cell death. Previous studies have observed enhanced ROS level and signs of ferroptosis in SARS-CoV-2 infected cells or specimens of patients with COVID-19, but the exact mechanism is not clear yet. Here, we find SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis. SARS-CoV-2 ORF3a promotes the degradation of NRF2 through recruiting Keap1, thereby attenuating cellular resistance to oxidative stress and facilitated cells to ferroptotic cell death. Our study uncovers that SARS-CoV-2 ORF3a functions as a positive regulator of ferroptosis, which might explain SARS-CoV-2-induced damage in multiple organs in COVID-19 patients and imply the potential of ferroptosis inhibition in COVID-19 treatment.

Degradation of natural organic matter and disinfection byproducts formation by solar photolysis of free available chlorine.

Environment disinfection effectively curbs transmission of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). However, elevated concentration of free available chlorine (FAC) in disinfectants can be discharged into surface water, generating toxic disinfection byproducts (DBPs). The impact of solar photolysis of FAC on natural organic matter (NOM) to form DBPs has not been well studied. In this work, solar photolysis of FAC was found to result in higher formation of DBPs, DBPs formation potential (DBPsFP), total organic chlorine (TOCl) and lower specific ultraviolet absorbance at 254 nm (SUVA254), compared to dark chlorination. In solar photolysis of FAC, formation of total DBPs was promoted by pH=8, but hindered by the addition of HCO3-, radical scavenger or deoxygenation, while addition of NO3-and NH4+both enhanced the formation of nitrogenous DBPs. Differences in the formation of DBPs in solar photolysis of FAC under various conditions were influenced by reactive species. The formation of trichloromethane (TCM) and haloacetic acids (HAAs) in solar photolysis of FAC positively correlated with the steady-state concentrations of ClO• and O3. The steady-state concentrations of •NO and •NH2 positively correlated with the formation of halonitromethanes (HNMs). HAAs and haloacetonitriles (HANs) mainly contributed to calculated cytotoxicity of DBPs. This study demonstrates that solar photolysis of FAC may significantly impact the formation of DBPs in surface water due to extensive use of disinfectants containing FAC during SARS-CoV-2 pandemic.

Synthesis of deuterated S-217622 (Ensitrelvir) with antiviral activity against coronaviruses including SARS-CoV-2.

S-217622 (Ensitrelvir) is a reversible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro) inhibitor which obtained emergency regulatory approval in Japan for the treatment of SARS-CoV-2 infection on Nov 22, 2022. Herein, analogs of S-271622 with deuterium-for-hydrogen replacement were synthesized for comparison of the antiviral activities and pharmacokinetic (PK) profiles. Compared to the parent compound, C11-d2-S-217622 compound YY-278 retained in vitro activity against 3CLpro and SARS-CoV-2. X-ray crystal structural studies showed similar interactions of SARS-CoV-2 3CLpro with YY-278 and S-271622. The PK profiling revealed the relatively favorable bioavailability and plasma exposure of YY-278. In addition, YY-278, as well as S-217622, displayed broadly anti-coronaviral activities against 6 other coronaviruses that infect humans and animals. These results laid the foundation for further research on the therapeutic potential of YY-278 against COVID-19 and other coronaviral diseases.

Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models.

The outbreak of coronavirus disease 2019 (COVID-19) has resulted in a global pandemic due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of this manuscript's publication, remdesivir is the only COVID-19 treatment approved by the United States Food and Drug Administration. However, its effectiveness is still under question due to the results of the large Solidarity Trial conducted by the World Health Organization. Herein, we report that the parent nucleoside of remdesivir, GS-441524, potently inhibits the replication of SARS-CoV-2 in Vero E6 and other cell lines. Challenge studies in both an AAV-hACE2 mouse model of SARS-CoV-2 and in mice infected with murine hepatitis virus, a closely related coronavirus, showed that GS-441524 was highly efficacious in reducing the viral titers in CoV-infected organs without notable toxicity. Our results support that GS-441524 is a promising and inexpensive drug candidate for treating of COVID-19 and other CoV diseases.

A soluble DR5-Fc chimeric protein attenuates inflammatory responses induced by coronavirus MHV-A59 and SARS-CoV-2.

Mortality in coronavirus disease 2019 (COVID-19) patients has been linked to the presence of a "cytokine storm" induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which involves elevated levels of circulating cytokines and immune-cell hyperactivation. Targeting cytokines during the management of COVID-19 patients has the potential to improve survival rates and reduce mortality. Although cytokine blockers and immune-host modulators are currently being tested in severely ill COVID-19 patients to cope with the overwhelming systemic inflammation, there is not too many successful cases, thus finding new cytokine blockers to attenuate the cytokine storm syndrome is meaningful. In this paper, we significantly attenuated the inflammatory responses induced by mouse hepatitis viruses A59 and SARS-CoV-2 through a soluble DR5-Fc (sDR5-Fc) chimeric protein that blocked the TNF-related apoptosis-inducing ligand-death receptor 5 (TRAIL-DR5) interaction. Our findings indicates that blocking the TRAIL-DR5 pathway through the sDR5-Fc chimeric protein is a promising strategy to treat COVID-19 severe patients requiring intensive care unit  admission or with chronic metabolic diseases.

The Relationship Between Adjustment and Mental Health of Chinese Freshmen: The Mediating Effect of Security and the Moderating Effect of Gender.

During the COVID-19 pandemic, many universities are confronted with campus lockdown or even school closures to reduce the risk of infection. However, these measures pose a threat to the mental health of adolescents. In particular, freshmen who have just entered the university campus may suffer from more serious mental health risks. In this study, 1,818 freshmen were analyzed by using the Chinese College Student Adjustment Scale (CCSAS), Sense of Security Questionnaire (SQ), and Symptom Check List 90 (SCL-90) of the qualitative method. The results showed that adjustment had an impact on mental health. Firstly, there was a significant negative association between adjustment and mental health. Secondly, adjustment had a significant predictive effect on mental health. Finally, a sense of security and gender affected the relationship between adjustment and mental health. Collectively, adjustment, sense of security, and gender exerted impacts on the mental health of freshmen, suggesting that we should create a warm and harmonious campus environment for students and conduct targeted education for male and female students.

Evidence of Infection of Human Embryonic Stem Cells by SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially described to target the respiratory system and now has been reported to infect a variety of cell types, including cardiomyocytes, neurons, hepatocytes, and gut enterocytes. However, it remains unclear whether the virus can directly infect human embryonic stem cells (hESCs) or early embryos. Herein, we sought to investigate this question in a cell-culture system of hESCs. Both the RNA and S protein of SARS-CoV-2 were detected in the infected hESCs and the formation of syncytium was observed. The increased level of subgenomic viral RNA and the presence of dsRNA indicate active replication of SARS-CoV-2 in hESCs. The increase of viral titers in the supernatants revealed virion release, further indicating the successful life cycle of SARS-CoV-2 in hESCs. Remarkably, immunofluorescence microscopy showed that only a small portion of hESCs were infected, which may reflect low expression of SARS-CoV-2 receptors. By setting |log2 (fold change)| > 0.5 as the threshold, a total of 1,566 genes were differentially expressed in SARS-CoV-2-infected hESCs, among which 17 interferon-stimulated genes (ISGs) were significantly upregulated. Altogether, our results provide novel evidence to support the ability of SARS-CoV-2 to infect and replicate in hESCs.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

Comparison of model-specific histopathology in mouse models of COVID-19.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the causative agent of the current coronavirus disease 2019 pandemic. Development of animal models that parallel the clinical and pathologic features of disease are highly essential to understanding the pathogenesis of SARS-CoV-2 infection and the development of therapeutics and prophylactics. Several mouse models that express the human angiotensin converting enzyme 2 (hACE2) have been created, including transgenic and knock-in strains, and viral vector-mediated delivery of hACE2. However, the comparative pathology of these mouse models infected with SARS-CoV-2 are unknown. Here, we perform systematic comparisons of the mouse models including K18-hACE2 mice, KI-hACE2 mice, Ad5-hACE2 mice and CAG-hACE2 mice, which revealed differences in the distribution of lesions and the characteristics of pneumonia induced. Based on these observations, the hACE2 mouse models meet different needs of SARS-CoV-2 researches. The similarities or differences among the model-specific pathologies may help in better understanding the pathogenic process of SARS-CoV-2 infection and aiding in the development of effective medications and prophylactic treatments for SARS-CoV-2.

A comprehensive evolutionary and epidemiological characterization of insertion and deletion mutations in SARS-CoV-2 genomes.

SARS-CoV-2, which causes the current pandemic of respiratory illness, is evolving continuously and generating new variants. Nevertheless, most of the sequence analyses thus far focused on nucleotide substitutions despite the fact that insertions and deletions (indels) are equally important in the evolution of SARS-CoV-2. In this study, we analyzed 1,099,664 high-quality sequences of SARS-CoV-2 genomes to re-construct the evolutionary and epidemiological histories of indels. Our analysis revealed 289 circulating indel types (237 deletion and 52 insertion types, each represented by more than ten genomic sequences), among which eighteen were recurrent indel types, each represented by more than 500 genome sequences. Although indels were identified across the entire genome, most of them were identified in nsp6, S, ORF8, and N genes, among which ORF8 indel types had the highest frequencies of frameshift. Geographical and temporal analyses of these variants revealed a few alterations of dominant indel types, each accompanied by geographic expansion to different countries and continents, which resulted in the fixation of several types of indels in the field, including the current variants of concern. Evolutionary and structural analyses revealed that indels involving S N-terminal domain regions were linked to the 3/4 variants of concern, resulting in significantly altered S protein that might contribute to the selective advantage of the corresponding variant. In sum, our study highlights the important role of insertions and deletions in the evolution and spread of SARS-CoV-2.

N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition.

The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2'-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5' cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5' cap in vitro. Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHVnsp14-Y414A) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHVnsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHVnsp14-Y414A mutant was restored in Ifnar-/- mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2nsp14-Y420A) also significantly decreased N7-methyltransferase activity in vitro, and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2nsp14-Y420A conferred complete protection against subsequent and otherwise lethal SARS-CoV-2 infection in mice, indicating the vaccine potential of this mutant. IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development.

The Impact of Accumulated Mutations in SARS-CoV-2 Variants on the qPCR Detection Efficiency.

SARS-CoV-2 is evolving with mutations throughout the genome all the time and a number of major variants emerged, including several variants of concern (VOC), such as Delta and Omicron variants. In this study, we demonstrated that mutations in the regions corresponding to the sequences of the probes and 3'-end of primers have a significant impact on qPCR detection efficiency. We also found that the G28916T mutation of the N gene accounts for 78.78% sequenced genomes of Delta variant. It was found that detection sensitivity of G28916T mutant was 2.35 and 1.74 times less than that of the wt sequence and detection limit was reduced from 1 copy/µl to 10 copies/µl for the commercially available CP3 and CP4 primer/probe sets. These results indicate that the detection probes and primers should be optimized to keep maximal detection efficiency in response to the emergence of new variants.

A comprehensive evolutionary and epidemiological characterization of insertion and deletion mutations in SARS-CoV-2 genomes

SARS-CoV-2, which causes the current pandemic of respiratory illness, is evolving continuously and generating new variants. Nevertheless, most of the sequence analyses thus far focused on nucleotide substitutions despite the fact that insertions and deletions (indels) are equally important in the evolution of SARS-CoV-2. In this study, we analyzed 1,099,664 high-quality sequences of SARS-CoV-2 genomes to re-construct the evolutionary and epidemiological histories of indels. Our analysis revealed 289 circulating indel types (237 deletion and 52 insertion types, each represented by more than ten genomic sequences), among which eighteen were recurrent indel types, each represented by more than 500 genome sequences. Although indels were identified across the entire genome, most of them were identified in nsp6, S, ORF8, and N genes, among which ORF8 indel types had the highest frequencies of frameshift. Geographical and temporal analyses of these variants revealed a few alterations of dominant indel types, each accompanied by geographic expansion to different countries and continents, which resulted in the fixation of several types of indels in the field, including the current variants of concern. Evolutionary and structural analyses revealed that indels involving S N-terminal domain regions were linked to the 3/4 variants of concern, resulting in significantly altered S protein that might contribute to the selective advantage of the corresponding variant. In sum, our study highlights the important role of insertions and deletions in the evolution and spread of SARS-CoV-2.

Oxidative stress transforms 3CLpro into an insoluble and more active form to promote SARS-CoV-2 replication.

3CLpro is a key proteinase for SARS-CoV-2 replication and serves as an important target for antiviral drug development. However, how its activity is regulated intracellularly is still obscure. In this study, we developed a 3CLpro protease activity reporter system to examine the impact of various factors, including nutrient supplements, ions, pHs, or oxidative stress inducers, on 3CLpro protease activity. We found that oxidative stress could increase the overall activity of 3CLpro. Not altering the expression, oxidative stress decreased the solubility of 3CLpro in the lysis buffer containing 1% Triton-X-100. The Triton-X-100-insoluble 3CLpro was correlated with aggregates' formation and responsible for the increased enzymatic activity. The disulfide bonds formed between Cys85 sites of 3CLpro protomers account for the insolubility and the aggregation of 3CLpro. Besides being regulated by oxidative stress, 3CLpro impaired the cellular antioxidant capacity by regulating the cleavage of GPx1 at its N-terminus. This cleavage could further elevate the 3CLpro-proximate oxidative activity, favor aggregation and activation of 3CLpro, and thus lead to a positive feedback loop. In summary, we reported that oxidative stress transforms 3CLpro into a detergent-insoluble form that is more enzymatically active, leading to increased viral replication/transcription. Our study provided mechanistic evidence that suggests the therapeutic potential of antioxidants in the clinical treatment of COVID-19 patients.

Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication.

BACKGROUND: Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. RESULTS: Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. CONCLUSIONS: Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

Live attenuated coronavirus vaccines deficient in N7-Methyltransferase activity induce both humoral and cellular immune responses in mice.

Coronaviruses (CoVs) can infect a variety of hosts, including humans, livestock and companion animals, and pose a serious threat to human health and the economy. The current COVID-19 pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed millions of people. Unfortunately, effective treatments for CoVs infection are still lacking, suggesting the importance of coronavirus vaccines. Our previous work showed that CoV nonstuctural protein 14 (nsp14) functions as (guanine-N7)-methyltransferase (N7-MTase), which is involved in RNA cap formation. Moreover, we found that N7-MTase is well conserved among different CoVs and is a universal target for developing antivirals against CoVs. Here, we show that N7-MTase of CoVs can be an ideal target for designing live attenuated vaccines. Using murine hepatitis virus strain A59 (MHV-A59), a representative and well-studied model of coronaviruses, we constructed N7-MTase-deficient recombinant MHV D330A and Y414A. These two mutants are highly attenuated in mice and exhibit similar replication efficiency to the wild-type (WT) virus in the cell culture. Furthermore, a single dose immunization of D330A or Y414A can induce long-term humoral immune responses and robust CD4+ and CD8+ T cell responses, which can provide full protection against the challenge of a lethal-dose of MHV-A59. Collectively, this study provides an ideal strategy to design live attenuated vaccines for coronavirus by abolishing viral RNA N7-MTase activity. This approach may apply to other RNA viruses that encode their own conservative viral N7-methyltransferase.

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery.

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far. Due to the high infectivity and pathogenicity of this virus, all studies on the live virus are strictly confined in the biosafety level 3 (BSL3) laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious. In this study, we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome (BAC) vector with deletion of the spike (S) gene. Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein. Therefore, such a replicon system is not infectious and can be used in ordinary biological laboratories. We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus. By mutational analysis of nsp12 and nsp14, we showed that the RNA polymerase, exonuclease, and cap N7 methyltransferase play essential roles in genome replication and sgRNA production. We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors. Thus, such a one-plasmid system is biosafe and convenient to use, which will benefit both fundamental research and development of antiviral drugs.

Understanding building-occupant-microbiome interactions toward healthy built environments: A review.

Built environments, occupants, and microbiomes constitute a system of ecosystems with extensive interactions that impact one another. Understanding the interactions between these systems is essential to develop strategies for effective management of the built environment and its inhabitants to enhance public health and well-being. Numerous studies have been conducted to characterize the microbiomes of the built environment. This review summarizes current progress in understanding the interactions between attributes of built environments and occupant behaviors that shape the structure and dynamics of indoor microbial communities. In addition, this review also discusses the challenges and future research needs in the field of microbiomes of the built environment that necessitate research beyond the basic characterization of microbiomes in order to gain an understanding of the causal mechanisms between the built environment, occupants, and microbiomes, which will provide a knowledge base for the development of transformative intervention strategies toward healthy built environments. The pressing need to control the transmission of SARS-CoV-2 in indoor environments highlights the urgency and significance of understanding the complex interactions between the built environment, occupants, and microbiomes, which is the focus of this review.