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3.
J Mol Biol ; 434(6): 167277, 2022 03 30.
Article in English | MEDLINE | ID: covidwho-2061566

ABSTRACT

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.


Subject(s)
Flavivirus , Interferons , Virus Replication , Apolipoproteins L/genetics , Apolipoproteins L/metabolism , Flavivirus/physiology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Interferons/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , SARS-CoV-2/physiology , Zika Virus/physiology
4.
EMBO Rep ; 23(11): e54061, 2022 Nov 07.
Article in English | MEDLINE | ID: covidwho-2056517

ABSTRACT

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.


Subject(s)
DEAD-box RNA Helicases , HIV-1 , Positive-Strand RNA Viruses , Virus Replication , Humans , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , HIV-1/physiology , Positive-Strand RNA Viruses/physiology , SARS-CoV-2/physiology
5.
JCO Glob Oncol ; 7: 464-473, 2021 04.
Article in English | MEDLINE | ID: covidwho-2054022

ABSTRACT

PURPOSE: To evaluate stress levels among the health care workers (HCWs) of the radiation oncology community in Asian countries. METHODS: HCWs of the radiation oncology departments from 29 tertiary cancer care centers of Bangladesh, India, Indonesia and Nepal were studied from May 2020 to July 2020. A total of 758 eligible HCWs were identified. The 7-Item Generalized Anxiety Disorder, 9-Item Patient Health Questionnaire, and 22-Item Impact of Events Scale-Revised were used for assessing anxiety, depression, and post-traumatic stress disorder. Univariate and multivariate analysis was done to identify the causative factors affecting mental health. RESULTS: A total of 758 participants from 794 HCWs were analyzed. The median age was 31 years (IQR, 27-28). The incidence of moderate to severe levels of anxiety, depression, and stress was 34.8%, 31.2%, and 18.2%, respectively. Severe personal concerns were noticed by 60.9% of the staff. On multivariate analysis, the presence of commonly reported symptoms of COVID-19 during the previous 2 weeks, contact history (harzard ratio [HR], 2.04; CI, 1.15 to 3.63), and compliance with precautionary measures (HR, 1.69; CI, 1.19 to 2.45) for COVID-19 significantly predicted for increasing anxiety (HR, 2.67; CI, 1.93 to 3.70), depression (HR, 3.38; CI 2.36 to 4.84), and stress (HR, 2.89; CI, 1.88 to 4.43) (P < .001). A significant regional variation was also noticed for anxiety, stress, and personal concerns. CONCLUSION: This survey conducted during the COVID-19 pandemic revealed that a significant proportion of HCWs in the radiation oncology community experiences moderate to severe levels of anxiety, depression, and stress. This trend is alarming and it is important to identify and intervene at the right time to improve the mental health of HCWs to avoid any long-term impacts.


Subject(s)
COVID-19/prevention & control , Health Personnel/statistics & numerical data , Radiation Oncology/statistics & numerical data , Stress, Psychological/prevention & control , Surveys and Questionnaires , Adult , Anxiety Disorders/epidemiology , Anxiety Disorders/prevention & control , Anxiety Disorders/psychology , Bangladesh/epidemiology , COVID-19/epidemiology , COVID-19/virology , Cross-Sectional Studies , Depression/epidemiology , Depression/prevention & control , Depression/psychology , Female , Health Personnel/psychology , Humans , India/epidemiology , Indonesia/epidemiology , Male , Middle Aged , Nepal/epidemiology , Pandemics , Radiation Oncology/methods , SARS-CoV-2/physiology , Stress Disorders, Post-Traumatic/epidemiology , Stress Disorders, Post-Traumatic/prevention & control , Stress Disorders, Post-Traumatic/psychology , Stress, Psychological/epidemiology , Stress, Psychological/psychology
7.
Proc Natl Acad Sci U S A ; 119(38): e2209514119, 2022 09 20.
Article in English | MEDLINE | ID: covidwho-2017036

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.


Subject(s)
COVID-19 , Nasal Cavity , SARS-CoV-2 , Serine Endopeptidases , Virus Internalization , COVID-19/virology , Furin/genetics , Furin/metabolism , Humans , Hydrogen-Ion Concentration , Nasal Cavity/chemistry , Nasal Cavity/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
8.
Proc Natl Acad Sci U S A ; 119(39): e2204624119, 2022 09 27.
Article in English | MEDLINE | ID: covidwho-2017031

ABSTRACT

The high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, postexposure TIP dose lowers SARS-CoV-2 nasal shedding, and at 5 days postinfection, infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Cohoused "contact" animals exposed to infected, TIP-treated animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals cohoused with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission.


Subject(s)
COVID-19 , RNA, Messenger , RNA, Small Interfering , SARS-CoV-2 , Virus Shedding , Animals , COVID-19/therapy , COVID-19/transmission , Cricetinae , RNA, Messenger/administration & dosage , RNA, Small Interfering/administration & dosage , SARS-CoV-2/genetics , SARS-CoV-2/physiology
9.
Nat Commun ; 13(1): 5196, 2022 09 03.
Article in English | MEDLINE | ID: covidwho-2008279

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, produces polyproteins 1a and 1ab that contain, respectively, 11 or 16 non-structural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for viral assembly and maturation. Using C-terminally substituted Mpro chimeras, we have determined X-ray crystallographic structures of Mpro in complex with 10 of its 11 viral cleavage sites, bound at full occupancy intermolecularly in trans, within the active site of either the native enzyme and/or a catalytic mutant (C145A). Capture of both acyl-enzyme intermediate and product-like complex forms of a P2(Leu) substrate in the native active site provides direct comparative characterization of these mechanistic steps as well as further informs the basis for enhanced product release of Mpro's own unique C-terminal P2(Phe) cleavage site to prevent autoinhibition. We characterize the underlying noncovalent interactions governing binding and specificity for this diverse set of substrates, showing remarkable plasticity for subsites beyond the anchoring P1(Gln)-P2(Leu/Val/Phe), representing together a near complete analysis of a multiprocessing viral protease. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for antiviral therapeutic development.


Subject(s)
COVID-19 , Coronavirus 3C Proteases/metabolism , Polyproteins , SARS-CoV-2/physiology , Cysteine Endopeptidases/metabolism , Humans , Peptide Hydrolases , Polyproteins/chemistry , Viral Proteins/chemistry , X-Rays
10.
Sci Rep ; 12(1): 3794, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-2004784

ABSTRACT

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.


Subject(s)
Microscopy/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/transmission , COVID-19/virology , Chlorocebus aethiops , Cytoplasm/chemistry , Cytoplasm/ultrastructure , Cytoplasm/virology , Extracellular Vesicles/chemistry , Extracellular Vesicles/ultrastructure , Giant Cells/chemistry , Giant Cells/physiology , Helium/chemistry , Humans , Ions/chemistry , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
11.
Emerg Microbes Infect ; 11(1): 2160-2175, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1997031

ABSTRACT

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.


Subject(s)
COVID-19 , Lung , Orthomyxoviridae Infections , Animals , Antiviral Agents/pharmacology , COVID-19/pathology , Fluoxetine/pharmacology , Humans , Influenza A virus/physiology , Influenza, Human/pathology , Interferons , Lung/virology , Mice , Orthomyxoviridae Infections/pathology , SARS-CoV-2/physiology , Tissue Culture Techniques , Virus Replication
12.
mBio ; 13(4): e0194422, 2022 08 30.
Article in English | MEDLINE | ID: covidwho-1986333

ABSTRACT

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.


Subject(s)
COVID-19 , Nasal Mucosa , Organoids , SARS-CoV-2 , COVID-19/virology , Humans , Nasal Mucosa/virology , Organoids/virology , SARS-CoV-2/classification , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Tissue Culture Techniques
13.
J Virol ; 96(15): e0075322, 2022 08 10.
Article in English | MEDLINE | ID: covidwho-1962094

ABSTRACT

Circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population leads to further viral evolution. The new variants that arise during this evolution are more infectious. Our data suggest that newer variants have shifted from utilizing both cathepsin/endosome- and TMPRSS2-mediated entry mechanisms to rely on a TMPRSS2-dependent entry pathway. Accordingly, only the early lineages of SARS-CoV-2 are capable of infecting and forming syncytia in Vero/ACE2 cells which lack TMPRSS2 expression. The presence of an intact multibasic furin cleavage site (FCS) in the S protein was a key requirement for cell-to-cell fusion. Deletion of FCS makes SARS-CoV-2 more infectious in vitro but renders it incapable of syncytium formation. Cell-to-cell fusion likely represents an alternative means of virus spread and is resistant to the presence of high levels of neutralizing monoclonal antibodies (MAbs) and immune sera in the media. In this study, we also noted that cells infected with SARS-CoV-2 with an intact FCS or alphavirus replicon expressing S protein (VEErep/S) released high levels of free S1 subunit. The released S1 is capable of activating the TLR4 receptor and inducing a pro-inflammatory response. Thus, S1 activation of TLR4 may be an important contributor to SARS-CoV-2-induced COVID-19 disease and needs to be considered in the design of COVID mRNA vaccines. Lastly, a VEErep/S-replicon was shown to produce large amounts of infectious, syncytium-forming pseudoviruses and thus could represent alternative experimental system for screening inhibitors of virus entry and syncytium formation. IMPORTANCE The results of this study demonstrate that the late lineages of SARS-CoV-2 evolved to more efficient use of the TMPRSS2-mediated entry pathway and gradually lost an ability to employ the cathepsins/endosome-mediated entry. The acquisition of a furin cleavage site (FCS) by SARS-CoV-2-specific S protein made the virus a potent producer of syncytia. Their formation is also determined by expression of ACE2 and TMPRSS2 and is resistant to neutralizing human MAbs and immune sera. Syncytium formation appears to be an alternative means of infection spread following the development of an adaptive immune response. Cells infected with SARS-CoV-2 with an intact FCS secrete high levels of the S1 subunit. The released S1 demonstrates an ability to activate the TLR4 receptor and induce pro-inflammatory cytokines, which represent a hallmark of SARS-CoV-2 pathogenesis. Alphavirus replicons encoding SARS-CoV-2 S protein cause spreading, syncytium-forming infection, and they can be applied as an experimental tool for studying the mechanism of syncytium formation.


Subject(s)
COVID-19 , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/metabolism , Evolution, Molecular , Furin/metabolism , Humans , Immune Sera , SARS-CoV-2/genetics , Signal Transduction , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Toll-Like Receptor 4 , Virus Internalization
14.
J Cell Biol ; 221(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1960887

ABSTRACT

ß-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.


Subject(s)
COVID-19 , Membrane Proteins , SARS-CoV-2 , Viral Replication Compartments , Autophagy/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Organelles/metabolism , Phosphatidylserines , SARS-CoV-2/physiology , Viral Nonstructural Proteins/genetics , Virus Replication
15.
Proc Natl Acad Sci U S A ; 119(32): e2205690119, 2022 08 09.
Article in English | MEDLINE | ID: covidwho-1960628

ABSTRACT

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.


Subject(s)
COVID-19 , Furin , Proteolysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Amino Acid Motifs/genetics , Animals , COVID-19/virology , Chlorocebus aethiops , Furin/chemistry , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Deletion , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication/genetics
16.
J Immunotoxicol ; 18(1): 93-104, 2021 07 24.
Article in English | MEDLINE | ID: covidwho-1947806

ABSTRACT

The aging immune system is characterized by a low-grade chronic systemic inflammatory state ("inflammaging") marked by elevated serum levels of inflammatory molecules such as interleukin (IL)-6 and C-reactive protein (CRP). These inflammatory markers were also reported to be strong predictors for the development/severity of Type 2 diabetes, obesity, and COVID-19. The levels of these markers have been positively associated with those of advanced glycation end-products (AGEs) generated via non-enzymatic glycation and oxidation of proteins and lipids during normal aging and metabolism. Based on the above observations, it is clinically important to elucidate how dietary AGEs modulate inflammation and might thus increase the risk for aging-exacerbated diseases. The present narrative review discusses the potential pro-inflammatory properties of dietary AGEs with a focus on the inflammatory mediators CRP, IL-6 and ferritin, and their relations to aging in general and Type 2 diabetes in particular. In addition, underlying mechanisms - including those related to gut microbiota and the receptors for AGEs, and the roles AGEs might play in affecting physiologies of the healthy elderly, obese individuals, and diabetics are discussed in regard to any greater susceptibility to COVID-19.


Subject(s)
COVID-19/metabolism , Diabetes Mellitus, Type 2/metabolism , Glycation End Products, Advanced/metabolism , Inflammation Mediators/metabolism , SARS-CoV-2/physiology , Aging , Animals , Diet , Dysbiosis , Gastrointestinal Microbiome , Glycation End Products, Advanced/immunology , Homeostasis , Humans , Immunity , Lipid Metabolism
17.
Proc Natl Acad Sci U S A ; 119(30): e2123065119, 2022 07 26.
Article in English | MEDLINE | ID: covidwho-1947760

ABSTRACT

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.


Subject(s)
Antiviral Agents , COVID-19 , Host-Pathogen Interactions , Molecular Targeted Therapy , Protein Processing, Post-Translational , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Caco-2 Cells , Exoribonucleases/metabolism , Host-Pathogen Interactions/drug effects , Humans , Protein Processing, Post-Translational/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sirtuins/metabolism , Succinates/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
18.
Proc Natl Acad Sci U S A ; 119(30): e2122236119, 2022 07 26.
Article in English | MEDLINE | ID: covidwho-1947759

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.


Subject(s)
Astrocytes , Cerebral Cortex , SARS-CoV-2 , Viral Tropism , Angiotensin-Converting Enzyme 2/metabolism , Astrocytes/enzymology , Astrocytes/virology , Cerebral Cortex/virology , Humans , Organoids/virology , Primary Cell Culture , SARS-CoV-2/physiology
19.
ACS Infect Dis ; 8(8): 1533-1542, 2022 08 12.
Article in English | MEDLINE | ID: covidwho-1931304

ABSTRACT

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5'-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 µM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.


Subject(s)
Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases , COVID-19/virology , DNA, Single-Stranded , Humans , Methyltransferases/antagonists & inhibitors , Nucleic Acids/metabolism , RNA Helicases/antagonists & inhibitors , SARS-CoV-2/physiology , Viral Nonstructural Proteins/antagonists & inhibitors
20.
PLoS One ; 17(6): e0270609, 2022.
Article in English | MEDLINE | ID: covidwho-1910688

ABSTRACT

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.


Subject(s)
ADAM17 Protein , Angiotensin-Converting Enzyme 2 , COVID-19 , Colon , Estradiol , Lung , Receptors, Estradiol , ADAM17 Protein/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/drug therapy , COVID-19/virology , Colon/drug effects , Colon/metabolism , Estradiol/pharmacology , Female , Lung/metabolism , Male , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Wistar , Receptors, Estradiol/genetics , Receptors, Estradiol/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Transcription, Genetic/drug effects , Virus Internalization
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