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1.
Viruses ; 13(12)2021 12 17.
Article in English | MEDLINE | ID: covidwho-1580424

ABSTRACT

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.


Subject(s)
Coronavirus/metabolism , Intracellular Membranes/metabolism , RNA, Viral/biosynthesis , Animals , Cell Line , Coronavirus/classification , Coronavirus/growth & development , Cytoplasm/metabolism , Humans , Infectious bronchitis virus/growth & development , Infectious bronchitis virus/metabolism , RNA, Double-Stranded/metabolism , Viral Replication Compartments/metabolism
2.
Microbiol Spectr ; 9(2): e0090821, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1452921

ABSTRACT

Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.


Subject(s)
Autophagy/physiology , Blood Proteins/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Coronavirus/growth & development , Poly(A)-Binding Proteins/metabolism , Virus Replication/physiology , Animals , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Infectious bronchitis virus/growth & development , Murine hepatitis virus/growth & development , Nuclear Proteins/metabolism , Porcine epidemic diarrhea virus/growth & development , Proteolysis , Sp1 Transcription Factor/metabolism , Swine , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Vero Cells
3.
J Biol Chem ; 295(51): 17781-17801, 2020 12 18.
Article in English | MEDLINE | ID: covidwho-985572

ABSTRACT

Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of "self" in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess "nonself" cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA-binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus/growth & development , Coronavirus/genetics , Protein Biosynthesis , RNA Cap-Binding Proteins/metabolism , RNA Caps/metabolism , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Coronavirus/metabolism , Disease Models, Animal , HEK293 Cells , Humans , Mice , Mice, Knockout , Models, Molecular , Mutation , Protein Binding , RAW 264.7 Cells , RNA-Binding Proteins/genetics
4.
Methods Mol Biol ; 2203: 223-229, 2020.
Article in English | MEDLINE | ID: covidwho-729909

ABSTRACT

All viruses have to overcome the innate immune response in order to establish infection. Methods have been developed to assay if, and how, viruses overcome these responses, and many can be directly applied to coronaviruses. Here, in vitro methods to determine how coronaviruses overcome this response are described.


Subject(s)
Coronavirus/growth & development , Coronavirus/metabolism , Virus Cultivation/methods , Animals , Cell Line , Coronavirus/pathogenicity , Host-Pathogen Interactions , Humans , Immunity, Innate/immunology , Interferons , Middle East Respiratory Syndrome Coronavirus/growth & development , Middle East Respiratory Syndrome Coronavirus/pathogenicity , RNA, Viral , Viral Nonstructural Proteins , Virus Replication
5.
Nature ; 593(7859): 418-423, 2021 05.
Article in English | MEDLINE | ID: covidwho-1137788

ABSTRACT

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.


Subject(s)
Antiviral Agents/pharmacology , Clofazimine/pharmacology , Coronavirus/classification , Coronavirus/drug effects , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Animals , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antiviral Agents/pharmacokinetics , Antiviral Agents/therapeutic use , Biological Availability , Cell Fusion , Cell Line , Clofazimine/pharmacokinetics , Clofazimine/therapeutic use , Coronavirus/growth & development , Coronavirus/pathogenicity , Cricetinae , DNA Helicases/antagonists & inhibitors , Drug Synergism , Female , Humans , Life Cycle Stages/drug effects , Male , Mesocricetus , Pre-Exposure Prophylaxis , SARS-CoV-2/growth & development , Species Specificity , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Transcription, Genetic/drug effects , Transcription, Genetic/genetics
6.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 605-611, 2020 Apr 25.
Article in Chinese | MEDLINE | ID: covidwho-1024807

ABSTRACT

Cyclophilin A (CypA) is a widely distributed and highly conserved protein in organisms. It has peptidyl-prolyl cis/trans isomerase activity and is a receptor for cyclosporin A (CsA). Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses. Seven types of coronaviruses are currently known to infect humans, among which SARS-CoV, MERS-CoV, and SARS-CoV-2 are fatal for humans. It is well established that CypA is essential for the replication of various coronaviruses such as SARS-CoV, CoV-229E, CoV-NL63, and FCoV. Additionally, CsA and its derivatives (ALV, NIM811, etc.) have obvious inhibitory effects on a variety of coronaviruses. These results suggest that CypA is a potential antiviral target and the existing drug CsA might be used as an anti-coronavirus drug. At the end of 2019, SARS-CoV-2 raged in China, which seriously theatern human health and causes huge economic lases. In view of this, we describe the effects of CypA on the replication of coronaviruses and the antiviral activities of its inhibitors, which will provide the scientific basis and ideas for the development of antiviral drugs for SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Infections , Coronavirus/drug effects , Coronavirus/growth & development , Cyclophilin A/antagonists & inhibitors , Cyclosporine/pharmacology , Cyclosporine/therapeutic use , Pandemics , Pneumonia, Viral , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Betacoronavirus/growth & development , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Cyclosporine/chemistry , Humans , Pneumonia, Viral/drug therapy , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , SARS Virus/drug effects , SARS Virus/growth & development , SARS-CoV-2 , Virus Replication/drug effects
7.
Virology ; 554: 75-82, 2021 02.
Article in English | MEDLINE | ID: covidwho-989370

ABSTRACT

Human population growth, climate change, and globalization are accelerating the emergence of novel pathogenic viruses. In the past two decades alone, three such members of the coronavirus family have posed serious threats, spurring intense efforts to understand their biology as a way to identify targetable vulnerabilities. Coronaviruses use a programmed -1 ribosomal frameshift (-1 PRF) mechanism to direct synthesis of their replicase proteins. This is a critical switch in their replication program that can be therapeutically targeted. Here, we discuss how nearly half a century of research into -1 PRF have provided insight into the virological importance of -1 PRF, the molecular mechanisms that drive it, and approaches that can be used to manipulate it towards therapeutic outcomes with particular emphasis on SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Coronavirus/genetics , Frameshifting, Ribosomal/drug effects , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Coronavirus/growth & development , Coronavirus/physiology , Coronavirus Infections/drug therapy , Frameshifting, Ribosomal/genetics , Frameshifting, Ribosomal/physiology , Gene Expression Regulation, Viral , Humans , Mutation , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/growth & development , SARS-CoV-2/physiology , Virus Replication
8.
Methods Mol Biol ; 2203: 135-143, 2020.
Article in English | MEDLINE | ID: covidwho-761350

ABSTRACT

Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).


Subject(s)
Coronavirus/pathogenicity , Viral Plaque Assay/methods , Animals , Cells, Cultured , Coronavirus/growth & development , Infectious bronchitis virus/growth & development , Infectious bronchitis virus/pathogenicity , Trachea/cytology
9.
Pathog Dis ; 78(7)2020 10 07.
Article in English | MEDLINE | ID: covidwho-733382

ABSTRACT

Influenza virus and coronaviruses continue to cause pandemics across the globe. We now have a greater understanding of their functions. Unfortunately, the number of drugs in our armory to defend us against them is inadequate. This may require us to think about what mechanisms to address. Here, we review the biological properties of these viruses, their genetic evolution and antiviral therapies that can be used or have been attempted. We will describe several classes of drugs such as serine protease inhibitors, heparin, heparan sulfate receptor inhibitors, chelating agents, immunomodulators and many others. We also briefly describe some of the drug repurposing efforts that have taken place in an effort to rapidly identify molecules to treat patients with COVID-19. While we put a heavy emphasis on the past and present efforts, we also provide some thoughts about what we need to do to prepare for respiratory viral threats in the future.


Subject(s)
Antiviral Agents/therapeutic use , Coronavirus Infections/epidemiology , Coronavirus/drug effects , Drug Repositioning , Influenza, Human/epidemiology , Orthomyxoviridae/drug effects , Pandemics , Anticoagulants/therapeutic use , Antimalarials/therapeutic use , Antioxidants/therapeutic use , Chelating Agents/therapeutic use , Coronavirus/genetics , Coronavirus/growth & development , Coronavirus/pathogenicity , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Glycoconjugates/therapeutic use , Humans , Immunologic Factors/therapeutic use , Influenza, Human/drug therapy , Influenza, Human/virology , Orthomyxoviridae/genetics , Orthomyxoviridae/growth & development , Orthomyxoviridae/pathogenicity , Serine Proteinase Inhibitors/therapeutic use
10.
Cell ; 181(4): 865-876.e12, 2020 05 14.
Article in English | MEDLINE | ID: covidwho-684968

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , CRISPR-Cas Systems , Influenza A Virus, H1N1 Subtype/drug effects , RNA, Viral/antagonists & inhibitors , A549 Cells , Antibiotic Prophylaxis/methods , Base Sequence , Betacoronavirus/genetics , Betacoronavirus/growth & development , COVID-19 , Clustered Regularly Interspaced Short Palindromic Repeats , Computer Simulation , Conserved Sequence , Coronavirus/drug effects , Coronavirus/genetics , Coronavirus/growth & development , Coronavirus Infections/drug therapy , Coronavirus Nucleocapsid Proteins , Coronavirus RNA-Dependent RNA Polymerase , Epithelial Cells/virology , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/growth & development , Lung/pathology , Lung/virology , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Phylogeny , Pneumonia, Viral/drug therapy , RNA-Dependent RNA Polymerase/genetics , SARS-CoV-2 , Viral Nonstructural Proteins/genetics
11.
Virology ; 540: 45-56, 2020 01 15.
Article in English | MEDLINE | ID: covidwho-17451

ABSTRACT

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.


Subject(s)
Microbial Interactions , Nucleocapsid Proteins/metabolism , Porcine epidemic diarrhea virus/growth & development , Animals , Chlorocebus aethiops , Coinfection/virology , Coronavirus/growth & development , Coronavirus Nucleocapsid Proteins , Epithelial Cells/virology , HEK293 Cells , Humans , Transmissible gastroenteritis virus/growth & development , Vero Cells
12.
Drug Discov Today ; 25(4): 668-688, 2020 04.
Article in English | MEDLINE | ID: covidwho-2569

ABSTRACT

Human coronaviruses (CoVs) are enveloped viruses with a positive-sense single-stranded RNA genome. Currently, six human CoVs have been reported including human coronavirus 229E (HCoV-229E), OC43 (HCoV-OC43), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and MiddleEast respiratory syndrome (MERS) coronavirus (MERS-CoV). They cause moderate to severe respiratory and intestinal infections in humans. In this review, we focus on recent advances in the research and development of small-molecule anti-human coronavirus therapies targeting different stages of the CoV life cycle.


Subject(s)
Antiviral Agents/therapeutic use , Coronaviridae Infections/drug therapy , Coronavirus/drug effects , Drug Design , Drug Development , Molecular Targeted Therapy , Animals , Antiviral Agents/adverse effects , Antiviral Agents/chemistry , Coronaviridae Infections/diagnosis , Coronaviridae Infections/virology , Coronavirus/growth & development , Coronavirus/pathogenicity , Humans , Molecular Structure , Structure-Activity Relationship
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