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1.
ACS Chem Biol ; 15(9): 2331-2337, 2020 09 18.
Article in English | MEDLINE | ID: covidwho-860112

ABSTRACT

We report on using the synthetic aminoadamantane-CH2-aryl derivatives 1-6 as sensitive probes for blocking M2 S31N and influenza A virus (IAV) M2 wild-type (WT) channels as well as virus replication in cell culture. The binding kinetics measured using electrophysiology (EP) for M2 S31N channel are very dependent on the length between the adamantane moiety and the first ring of the aryl headgroup realized in 2 and 3 and the girth and length of the adamantane adduct realized in 4 and 5. Study of 1-6 shows that, according to molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations, all bind in the M2 S31N channel with the adamantyl group positioned between V27 and G34 and the aryl group projecting out of the channel with the phenyl (or isoxazole in 6) embedded in the V27 cluster. In this outward binding configuration, an elongation of the ligand by only one methylene in rimantadine 2 or using diamantane or triamantane instead of adamantane in 4 and 5, respectively, causes incomplete entry and facilitates exit, abolishing effective block compared to the amantadine derivatives 1 and 6. In the active M2 S31N blockers 1 and 6, the phenyl and isoxazolyl head groups achieve a deeper binding position and high kon/low koff and high kon/high koff rate constants, compared to inactive 2-5, which have much lower kon and higher koff. Compounds 1-5 block the M2 WT channel by binding in the longer area from V27-H37, in the inward orientation, with high kon and low koff rate constants. Infection of cell cultures by influenza virus containing M2 WT or M2 S31N is inhibited by 1-5 or 1-4 and 6, respectively. While 1 and 6 block infection through the M2 block mechanism in the S31N variant, 2-4 may block M2 S31N virus replication in cell culture through the lysosomotropic effect, just as chloroquine is thought to inhibit SARS-CoV-2 infection.


Subject(s)
Adamantane/pharmacology , Influenza A virus/drug effects , Influenza, Human/prevention & control , Ion Channels/antagonists & inhibitors , Molecular Probes/chemistry , Viral Matrix Proteins/antagonists & inhibitors , Adamantane/analogs & derivatives , Adamantane/chemistry , Adamantane/metabolism , Betacoronavirus/drug effects , Binding Sites , Cells, Cultured , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Genetic Variation , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza, Human/drug therapy , Kinetics , Molecular Probes/metabolism , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Protein Binding , Structure-Activity Relationship , Virus Replication/drug effects
2.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-840682

ABSTRACT

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.


Subject(s)
Pestivirus Infections/virology , Pestivirus/physiology , RNA, Viral , Viral Envelope Proteins/metabolism , Virus Replication , Gene Deletion , Gene Expression , Genes, Reporter , Genetic Engineering , Host-Pathogen Interactions , Pestivirus Infections/immunology , Replicon , Viral Envelope Proteins/genetics , Virion , Virus Assembly
3.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: covidwho-840609

ABSTRACT

Alpha/beta interferon (IFN-α/ß) signaling through the IFN-α/ß receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression patterns of factors associated with the IFN-α/ß pathway in different CNS resident cell populations implicate complex cooperative pathways in IFN-α/ß induction and responsiveness. Here we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) expressing neurons (CaMKIIcre:IFNARfl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (mouse hepatitis virus A59 strain [MHV-A59]) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNARfl/fl mice compared to IFNARfl/fl mice affected neurons not only in the forebrain but also in the mid-hind brain and spinal cords but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNARfl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs but a decrease in mRNA levels of IFNα/ß pathway genes as well as Il6, Tnf, and Il1ß between days 4 and 6 postinfection (p.i.). T cell accumulation and IFN-γ production, an essential component of virus control, were not altered. However, IFN-γ responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFN-α/ß signaling in neurons can worsen disease course and disrupt complex interactions between the IFN-α/ß and IFN-γ pathways in achieving optimal antiviral responses.IMPORTANCE IFN-α/ß induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/ß functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/ß receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/ß pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/ß and IFN-γ signaling pathways to optimize antiviral IFN-γ activity.


Subject(s)
Central Nervous System/virology , Interferon Type I/metabolism , Interferon-gamma/metabolism , Macrophages/metabolism , Microglia/metabolism , Neurons/metabolism , Signal Transduction , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Central Nervous System/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Models, Animal , Encephalomyelitis/immunology , Encephalomyelitis/virology , Macrophages/virology , Mice , Mice, Mutant Strains , Microglia/virology , Murine hepatitis virus/physiology , Neurons/virology , Neutrophil Infiltration , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Virus Replication
4.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-833255

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.


Subject(s)
CD13 Antigens/genetics , Coronavirus Infections/veterinary , Coronavirus/physiology , Gene Expression Regulation, Viral , Host-Pathogen Interactions , Swine Diseases/metabolism , Swine Diseases/virology , Viral Tropism , Animals , Enterocolitis/metabolism , Enterocolitis/pathology , Enterocolitis/virology , Interferons/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , Swine , Swine Diseases/pathology , Virus Replication
5.
Viruses ; 12(2)2020 02 21.
Article in English | MEDLINE | ID: covidwho-833229

ABSTRACT

Porcine epidemic diarrhea virus (PEDV), being highly virulent and contagious in piglets, has caused significant damage to the pork industries of many countries worldwide. There are no commercial drugs targeting coronaviruses (CoVs), and few studies on anti-PEDV inhibitors. The coronavirus 3C-like protease (3CLpro) has a conserved structure and catalytic mechanism and plays a key role during viral polyprotein processing, thus serving as an appealing antiviral drug target. Here, we report the anti-PEDV effect of the broad-spectrum inhibitor GC376 (targeting 3Cpro or 3CLpro of viruses in the picornavirus-like supercluster). GC376 was highly effective against the PEDV 3CLpro and exerted similar inhibitory effects on two PEDV strains. Furthermore, the structure of the PEDV 3CLpro in complex with GC376 was determined at 1.65 Å. We elucidated structural details and analyzed the differences between GC376 binding with the PEDV 3CLpro and GC376 binding with the transmissible gastroenteritis virus (TGEV) 3CLpro. Finally, we explored the substrate specificity of PEDV 3CLpro at the P2 site and analyzed the effects of Leu group modification in GC376 on inhibiting PEDV infection. This study helps us to understand better the PEDV 3CLpro substrate specificity, providing information on the optimization of GC376 for development as an antiviral therapeutic against coronaviruses.


Subject(s)
Antiviral Agents/pharmacology , Peptide Hydrolases/chemistry , Porcine epidemic diarrhea virus/drug effects , Protease Inhibitors/pharmacology , Pyrrolidines/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Catalytic Domain , Chlorocebus aethiops , Crystallography, X-Ray , Models, Molecular , Peptide Hydrolases/metabolism , Porcine epidemic diarrhea virus/enzymology , Porcine epidemic diarrhea virus/physiology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Pyrrolidines/chemistry , Pyrrolidines/metabolism , Substrate Specificity , Transmissible gastroenteritis virus/enzymology , Vero Cells , Virus Replication/drug effects
6.
Nephron ; 144(5): 213-221, 2020.
Article in English | MEDLINE | ID: covidwho-829878

ABSTRACT

Here, we review the most recent findings on the effects of SARS-CoV-2 infection on kidney diseases, including acute kidney injury, and examine the potential effects of ARBs on the outcomes of patients with COVID-19. Lastly, we discuss the clinical management of COVID-19 patients with existing chronic renal disorders, particularly those in dialysis and with kidney transplants.


Subject(s)
Angiotensins/antagonists & inhibitors , Coronavirus Infections/complications , Kidney Diseases/complications , Kidney Diseases/drug therapy , Pneumonia, Viral/complications , Angiotensin Receptor Antagonists , Angiotensin-Converting Enzyme Inhibitors , Betacoronavirus/physiology , Humans , Kidney/virology , Kidney Transplantation , Nephrologists , Pandemics , Peptidyl-Dipeptidase A , Renal Dialysis , Virus Replication
7.
Antiviral Res ; 173: 104646, 2020 01.
Article in English | MEDLINE | ID: covidwho-829317

ABSTRACT

Human coronaviruses (HCoVs) are important pathogens that cause upper respiratory tract infections and have neuroinvasive abilities; however, little is known about the dynamic infection process of CoVs in vivo, and there are currently no specific antiviral drugs to prevent or treat HCoV infection. Here, we verified the replication ability and pathogenicity of a reporter HCoV-OC43 strain expressing Renilla luciferase (Rluc; rOC43-ns2DelRluc) in mice with different genetic backgrounds (C57BL/6 and BALB/c). Additionally, we monitored the spatial and temporal progression of HCoV-OC43 through the central nervous system (CNS) of live BALB/c mice after intranasal or intracerebral inoculation with rOC43-ns2DelRluc. We found that rOC43-ns2DelRluc was fatal to suckling mice after intranasal inoculation, and that viral titers and Rluc expression were detected in the brains and spinal cords of mice infected with rOC43-ns2DelRluc. Moreover, viral replication was initially observed in the brain by non-invasive bioluminescence imaging before the infection spread to the spinal cord of BALB/c mice, consistent with its tropism in the CNS. Furthermore, the Rluc readout correlated with the HCoV replication ability and protein expression, which allowed quantification of antiviral activity in live mice. Additionally, we validated that chloroquine strongly inhibited rOC43-ns2DelRluc replication in vivo. These results provide new insights into the temporal and spatial dissemination of HCoV-OC43 in the CNS, and our methods provide an extremely sensitive platform for evaluating the efficacy of antiviral therapies to treat neuroinvasive HCoVs in live mice.


Subject(s)
Central Nervous System/virology , Coronavirus Infections/virology , Coronavirus OC43, Human/physiology , Animals , Antiviral Agents/administration & dosage , Brain/diagnostic imaging , Brain/virology , Central Nervous System/diagnostic imaging , Chloroquine/administration & dosage , Coronavirus Infections/diagnostic imaging , Coronavirus Infections/drug therapy , Coronavirus OC43, Human/genetics , Genes, Reporter , Humans , Luciferases, Renilla/genetics , Luciferases, Renilla/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Imaging , Virus Replication/drug effects
8.
J Virol ; 94(6)2020 02 28.
Article in English | MEDLINE | ID: covidwho-827743

ABSTRACT

TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.


Subject(s)
Adenosine Triphosphatases/metabolism , Nucleocapsid/metabolism , Nucleopolyhedroviruses/physiology , Virus Replication , Animals , Cell Nucleus/virology , Cytoplasm/virology , DNA Helicases/metabolism , DNA, Viral/biosynthesis , DNA-Binding Proteins/metabolism , Host-Pathogen Interactions , Sf9 Cells/virology , Vacuoles/virology , Viral Proteins/metabolism , Virion
9.
Biochem J ; 477(5): 1009-1019, 2020 03 13.
Article in English | MEDLINE | ID: covidwho-827308

ABSTRACT

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.


Subject(s)
RNA-Binding Proteins/genetics , Sequence Alignment/methods , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics , Coronavirus Infections/genetics , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Fluorescence Resonance Energy Transfer , Protein Structure, Secondary , RNA-Binding Proteins/chemistry , Viral Nonstructural Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry , Virus Replication/physiology
10.
Antiviral Res ; 173: 104651, 2020 01.
Article in English | MEDLINE | ID: covidwho-824493

ABSTRACT

Emerging coronaviruses (CoVs) primarily cause severe gastroenteric or respiratory diseases in humans and animals, and no approved therapeutics are currently available. Here, A9, a receptor tyrosine kinase inhibitor (RTKI) of the tyrphostin class, is identified as a robust inhibitor of transmissible gastroenteritis virus (TGEV) infection in cell-based assays. Moreover, A9 exhibited potent antiviral activity against the replication of various CoVs, including murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV). We further performed a comparative phosphoproteomic analysis to investigate the mechanism of action of A9 against TGEV infection in vitro. We specifically identified p38 and JNK1, which are the downstream molecules of receptor tyrosine kinases (RTKs) required for efficient TGEV replication, as A9 targets through plaque assays, qRT-PCR and Western blotting assays. p38 and JNK1 inhibitors and RNA interference further showed that the inhibitory activity of A9 against TGEV infection was mainly mediated by the p38 mitogen-activated protein kinase (MAPK) signaling pathway. All these findings indicated that the RTKI A9 directly inhibits TGEV replication and that its inhibitory activity against TGEV replication mainly occurs by targeting p38, which provides vital clues to the design of novel drugs against CoVs.


Subject(s)
Antiviral Agents/pharmacology , Host-Pathogen Interactions , MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Transmissible gastroenteritis virus/drug effects , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Cats , Cell Line , Cells, Cultured , Chlorocebus aethiops , Chromatography, Liquid , Gastroenteritis, Transmissible, of Swine/drug therapy , Gastroenteritis, Transmissible, of Swine/metabolism , Gastroenteritis, Transmissible, of Swine/virology , High-Throughput Screening Assays , Life Cycle Stages , Phosphoproteins/metabolism , Protein Kinase Inhibitors/chemistry , Proteomics/methods , Small Molecule Libraries , Swine , Tandem Mass Spectrometry , Vero Cells
11.
Virology ; 539: 38-48, 2020 01 02.
Article in English | MEDLINE | ID: covidwho-822398

ABSTRACT

Ionic calcium (Ca2+) is a versatile intracellular second messenger that plays important roles in cellular physiological and pathological processes. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes serious vomiting and diarrhea in suckling piglets. In this study, the role of Ca2+ to PDCoV infection was investigated. PDCoV infection was found to upregulate intracellular Ca2+ concentrations of IPI-2I cells. Chelating extracellular Ca2+ by EGTA inhibited PDCoV replication, and this inhibitory effect was overcome by replenishment with CaCl2. Treatment with Ca2+ channel blockers, particularly the L-type Ca2+ channel blocker diltiazem hydrochloride, inhibited PDCoV infection significantly. Mechanistically, diltiazem hydrochloride reduces PDCoV infection by inhibiting the replication step of the viral replication cycle. Additionally, knockdown of CACNA1S, the L-type Ca2+ voltage-gated channel subunit, inhibited PDCoV replication. The combined results demonstrate that PDCoV modulates calcium influx to favor its replication.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Coronavirus/physiology , Swine Diseases/metabolism , Swine Diseases/virology , Virus Replication , Animals , Calcium Signaling , Swine , Swine, Miniature
12.
Signal Transduct Target Ther ; 5(1): 218, 2020 10 03.
Article in English | MEDLINE | ID: covidwho-813983

Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Cardiac Glycosides/pharmacology , Gene Expression Regulation/drug effects , Host-Pathogen Interactions/drug effects , Animals , Antiviral Agents/chemistry , Betacoronavirus/pathogenicity , Biological Products/chemistry , Biological Products/pharmacology , Bufanolides/chemistry , Bufanolides/pharmacology , Cardiac Glycosides/chemistry , Cell Survival/drug effects , Chlorocebus aethiops , Chloroquine/chemistry , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Digoxin/chemistry , Digoxin/pharmacology , High-Throughput Screening Assays , Host-Pathogen Interactions/genetics , Humans , Janus Kinases/antagonists & inhibitors , Janus Kinases/genetics , Janus Kinases/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Pandemics , Phenanthrenes/chemistry , Phenanthrenes/pharmacology , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Signal Transduction , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Vero Cells , Virus Replication/drug effects
13.
Nat Commun ; 11(1): 4938, 2020 10 02.
Article in English | MEDLINE | ID: covidwho-811573

ABSTRACT

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Dimethyl Fumarate/agonists , NF-E2-Related Factor 2/metabolism , Pneumonia, Viral/drug therapy , Succinates/agonists , Adult , Antioxidants/pharmacology , Betacoronavirus/metabolism , Coronavirus Infections/virology , Dimethyl Fumarate/pharmacology , Female , Gene Expression , Gene Knockdown Techniques , Humans , Interferon Type I , Lung/pathology , Male , NF-E2-Related Factor 2/genetics , Pandemics , Pneumonia, Viral/virology , Signal Transduction/drug effects , Succinates/pharmacology , Virus Replication/drug effects
14.
Sci Rep ; 10(1): 16200, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-811545

ABSTRACT

The current coronavirus (COVID-19) pandemic is exacerbated by the absence of effective therapeutic agents. Notably, patients with COVID-19 and comorbidities such as hypertension and cardiac diseases have a higher mortality rate. An efficient strategy in response to this issue is repurposing drugs with antiviral activity for therapeutic effect. Digoxin (DIG) and ouabain (OUA) are FDA drugs for heart diseases that have antiviral activity against several coronaviruses. Thus, we aimed to assess antiviral activity of DIG and OUA against SARS-CoV-2 infection. The half-maximal inhibitory concentrations (IC50) of DIG and OUA were determined at a nanomolar concentration. Progeny virus titers of single-dose treatment of DIG, OUA and remdesivir were approximately 103-, 104- and 103-fold lower (> 99% inhibition), respectively, than that of non-treated control or chloroquine at 48 h post-infection (hpi). Furthermore, therapeutic treatment with DIG and OUA inhibited over 99% of SARS-CoV-2 replication, leading to viral inhibition at the post entry stage of the viral life cycle. Collectively, these results suggest that DIG and OUA may be an alternative treatment for COVID-19, with potential additional therapeutic effects for patients with cardiovascular disease.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Digoxin/pharmacology , Ouabain/pharmacology , Virus Replication , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Betacoronavirus/physiology , Chlorocebus aethiops , Chloroquine/pharmacology , Inhibitory Concentration 50 , Vero Cells
15.
Antimicrob Agents Chemother ; 64(10)2020 09 21.
Article in English | MEDLINE | ID: covidwho-810756

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Atazanavir Sulfate/pharmacology , Betacoronavirus/drug effects , Cytokines/metabolism , Ritonavir/pharmacology , Animals , Atazanavir Sulfate/chemistry , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , Cell Death/drug effects , Chlorocebus aethiops , Coronavirus Infections/drug therapy , Coronavirus Infections/metabolism , Coronavirus Infections/pathology , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Drug Therapy, Combination , Humans , Inflammation/metabolism , Inflammation/virology , Lopinavir/pharmacology , Molecular Docking Simulation , Monocytes/virology , Pandemics , Pneumonia, Viral/drug therapy , Pneumonia, Viral/metabolism , Pneumonia, Viral/pathology , Protease Inhibitors/pharmacology , Vero Cells , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
17.
Eur Respir J ; 56(3)2020 09.
Article in English | MEDLINE | ID: covidwho-810458

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has elicited a swift response by the scientific community to elucidate the pathogenesis of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)-induced lung injury and develop effective therapeutics. Clinical data indicate that severe COVID-19 most commonly manifests as viral pneumonia-induced acute respiratory distress syndrome (ARDS), a clinical entity mechanistically understood best in the context of influenza A virus-induced pneumonia. Similar to influenza, advanced age has emerged as the leading host risk factor for developing severe COVID-19. In this review we connect the current understanding of the SARS-CoV-2 replication cycle and host response to the clinical presentation of COVID-19, borrowing concepts from influenza A virus-induced ARDS pathogenesis and discussing how these ideas inform our evolving understanding of COVID-19-induced ARDS. We also consider important differences between COVID-19 and influenza, mainly the protean clinical presentation and associated lymphopenia of COVID-19, the contrasting role of interferon-γ in mediating the host immune response to these viruses, and the tropism for vascular endothelial cells of SARS-CoV-2, commenting on the potential limitations of influenza as a model for COVID-19. Finally, we explore hallmarks of ageing that could explain the association between advanced age and susceptibility to severe COVID-19.


Subject(s)
Aging/physiology , Betacoronavirus/physiology , Coronavirus Infections/complications , Pneumonia, Viral/complications , Respiratory Distress Syndrome, Adult/virology , Disease Susceptibility , Humans , Pandemics , Virus Replication
18.
mBio ; 11(5)2020 09 25.
Article in English | MEDLINE | ID: covidwho-796616

ABSTRACT

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.


Subject(s)
Betacoronavirus/genetics , Chromosomes, Artificial, Bacterial/genetics , Animals , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , Chlorocebus aethiops , Coronavirus Infections/virology , Cricetinae , DNA, Complementary/genetics , Genome, Viral/genetics , Pandemics , Pneumonia, Viral/virology , RNA, Viral/genetics , Reverse Genetics , Vero Cells , Virus Replication
20.
Eur Rev Med Pharmacol Sci ; 24(17): 9182-9187, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-790180

ABSTRACT

COVID-19 pandemic has underlined that unknown viral infections, which jump from animals to humans, can be extremely dangerous. In case of new viruses as SARS-CoV2, available drugs can fail to contrast the virus aggressiveness leading patients to death. Long time is necessary to create a vaccine, but immediate solutions are necessary to stop the mortality COVID-19 related. We have learned that the immune-system is the key to reduce the severity of COVID-19 and, through its modulation, it has been possible saving people's life. In this short communication, we discuss the use of nutraceuticals to modulate and stimulate the immune answer for reducing the severity of COVID-19 symptoms. The nutraceuticals are safe and can be administered to all ages. In addition, combination of natural anti-viral elements and immune-stimulating molecules already successfully tested against others upper-respiratory tract infections-could be efficient against SARS-CoV2. We believe that these natural molecules could really be a valid ally against COVID-19, especially in this moment in which a SARS-CoV2 vaccine is still not available.


Subject(s)
Coronavirus Infections/therapy , Dietary Supplements , Pneumonia, Viral/therapy , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Betacoronavirus/isolation & purification , Coronavirus Infections/pathology , Coronavirus Infections/virology , Humans , Lactobacillus/physiology , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Selenium/pharmacology , Selenium/therapeutic use , Severity of Illness Index , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Virus Replication/drug effects
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