scholarly journals Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus

2005 ◽  
Vol 69 (4) ◽  
pp. 635-664 ◽  
Author(s):  
Susan R. Weiss ◽  
Sonia Navas-Martin
Keyword(s):  
Animal Models ◽  
Severe Acute Respiratory Syndrome ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Hepatitis Virus ◽  
Emerging Pathogen ◽  
Murine Hepatitis Virus ◽  
Veterinary Importance ◽  
Positive Strand Rna ◽  
Human Viruses

SUMMARY Coronaviruses are a family of enveloped, single-stranded, positive-strand RNA viruses classified within the Nidovirales order. This coronavirus family consists of pathogens of many animal species and of humans, including the recently isolated severe acute respiratory syndrome coronavirus (SARS-CoV). This review is divided into two main parts; the first concerns the animal coronaviruses and their pathogenesis, with an emphasis on the functions of individual viral genes, and the second discusses the newly described human emerging pathogen, SARS-CoV. The coronavirus part covers (i) a description of a group of coronaviruses and the diseases they cause, including the prototype coronavirus, murine hepatitis virus, which is one of the recognized animal models for multiple sclerosis, as well as viruses of veterinary importance that infect the pig, chicken, and cat and a summary of the human viruses; (ii) a short summary of the replication cycle of coronaviruses in cell culture; (iii) the development and application of reverse genetics systems; and (iv) the roles of individual coronavirus proteins in replication and pathogenesis. The SARS-CoV part covers the pathogenesis of SARS, the developing animal models for infection, and the progress in vaccine development and antiviral therapies. The data gathered on the animal coronaviruses continue to be helpful in understanding SARS-CoV.

scholarly journals Murine Hepatitis Virus Nonstructural Protein 4 Regulates Virus-Induced Membrane Modifications and Replication Complex Function

2009 ◽  
Vol 84 (1) ◽  
pp. 280-290 ◽  
Author(s):  
Mark J. Gadlage ◽  
Jennifer S. Sparks ◽  
Dia C. Beachboard ◽  
Reagan G. Cox ◽  
Joshua D. Doyle ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Complex Function ◽  
Microscopic Analysis ◽  
Murine Hepatitis Virus ◽  
Electron Microscopic ◽  
Virus Growth ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

scholarly journals COVID-19 Basics and Vaccine Development with a Canadian Perspective

2020 ◽  
Author(s):  
Marina Liu ◽  
Xiongbiao Chen
Keyword(s):  
Animal Models ◽  
Severe Acute Respiratory Syndrome ◽  
Clinical Evaluation ◽  
Vaccine Development ◽  
Specific Treatment ◽  
Vaccine Candidates ◽  
Preclinical Evaluation ◽  
Canadian Perspective ◽  
Basic Biology

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a rapidly evolving situation. New discoveries about COVID-19 and its causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continue to deepen the understanding of this novel disease. As there is currently no COVID-19 specific treatment, isolation is the most effective method to prevent transmission. Moreover, development of a safe and effective COVID-19 vaccine will be instrumental in reinstating pre-COVID-19 conditions. As of July 31, 2020, there are at least 139 vaccine candidates from around the globe are in preclinical evaluation, with another 26 undergoing clinical evaluation. This paper aims to review the basics of COVID-19, including epidemiology, basic biology of SARS-CoV-2, and transmission. We also review COVID-19 vaccine development, including animal models, platforms under development, and vaccine development in Canada.

scholarly journals Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mice Models

2020 ◽  
Author(s):  
Yingjun Li ◽  
Liu Cao ◽  
Ge Li ◽  
Feng Cong ◽  
Yunfeng Li ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Drug Administration ◽  
Half Life ◽  
Hepatitis Virus ◽  
Food And Drug Administration ◽  
Drug Candidate ◽  
Murine Hepatitis Virus ◽  
Global Pandemic ◽  
Plasma Distribution ◽  
Viral Titers

AbstractThe outbreak of coronavirus disease 2019 (COVID-19) rapidly spreads across worldwide and becomes a global pandemic. Remdesivir is the only COVID-19 treatment approved by U.S. Food and Drug Administration (FDA); however, its effectiveness is still under questioning as raised by the results of a large WHO Solidarity Trial. Herein, we report that the parent nucleotide of remdesivir, GS-441524, potently inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero E6 and other cells. It exhibits good plasma distribution and longer half-life (t1/2=4.8h) in rat PK study. GS-441524 is highly efficacious against SARS-CoV-2 in AAV-hACE2 transduced mice and murine hepatitis virus (MHV) in mice, reducing the viral titers in CoV-attacked organs, without noticeable toxicity. Given that GS-441524 was the predominant metabolite of remdesivir in the plasma, the anti-COVID-19 effect of remdesivir may partly come from the effect of GS-441524. Our results also supported that GS-441524 as a promising and inexpensive drug candidate in the treatment of COVID-19 and future emerging CoVs diseases.

scholarly journals Crystal Structure of Nonstructural Protein 10 from the Severe Acute Respiratory Syndrome Coronavirus Reveals a Novel Fold with Two Zinc-Binding Motifs

2006 ◽  
Vol 80 (16) ◽  
pp. 7894-7901 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Kumar Singh Saikatendu ◽  
Vanitha Subramanian ◽  
Benjamin W. Neuman ◽  
Alexei Brooun ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Severe Acute Respiratory Syndrome ◽  
Structural Integrity ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Critical Role ◽  
Sequence Specificity ◽  
Nucleic Acid Binding ◽  
Murine Hepatitis Virus

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 “nonstructural” proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular β-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the “gag-knuckle fold group” of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

scholarly journals The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

2005 ◽  
Vol 79 (21) ◽  
pp. 13399-13411 ◽  
Author(s):  
Rachel L. Graham ◽  
Amy C. Sims ◽  
Sarah M. Brockway ◽  
Ralph S. Baric ◽  
Mark R. Denison
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Replication ◽  
Cleavage Site ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Retroviral Transduction ◽  
Coding Sequence ◽  
And Function

ABSTRACT The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.

scholarly journals Switching Species Tropism: an Effective Way To Manipulate the Feline Coronavirus Genome

2003 ◽  
Vol 77 (8) ◽  
pp. 4528-4538 ◽  
Author(s):  
Bert Jan Haijema ◽  
Haukeliene Volders ◽  
Peter J. M. Rottier
Keyword(s):  
Recombinant Virus ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Rna Recombination ◽  
Recombination System ◽  
Lethal Infection ◽  
Positive Strand Rna ◽  
Targeted Recombination ◽  
Selection Of

ABSTRACT Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVΔ7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVΔ7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.

scholarly journals Replication of Murine Hepatitis Virus Is Regulated by Papain-Like Proteinase 1 Processing of Nonstructural Proteins 1, 2, and 3

2006 ◽  
Vol 80 (23) ◽  
pp. 11610-11620 ◽  
Author(s):  
Rachel L. Graham ◽  
Mark R. Denison
Keyword(s):  
Deletion Mutant ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Protein Processing ◽  
Mutant Virus ◽  
Viral Titer ◽  
Wild Type ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Positive Strand Rna

ABSTRACT Coronaviruses are positive-strand RNA viruses that translate their genome RNA into polyproteins that are co- and posttranslationally processed into intermediate and mature replicase nonstructural proteins (nsps). In murine hepatitis virus (MHV), nsps 1, 2, and 3 are processed by two papain-like proteinase activities within nsp3 (PLP1 and PLP2) to yield nsp1, an nsp2-3 intermediate, and mature nsp2 and nsp3. To determine the role in replication of processing between nsp2 and nsp3 at cleavage site 2 (CS2) and PLP1 proteinase activity, mutations were engineered into the MHV genome at CS2, at CS1 and CS2, and at the PLP1 catalytic site, alone and in combination. Mutant viruses with abolished cleavage at CS2 were delayed in growth and RNA synthesis but grew to wild-type titers of >107 PFU/ml. Mutant viruses with deletion of both CS1 and CS2 exhibited both a delay in growth and a decrease in peak viral titer to ∼104 PFU/ml. Inactivation of PLP1 catalytic residues resulted in a mutant virus that did not process at either CS1 or CS2 and was severely debilitated in growth, achieving only 102 PFU/ml. However, when both CS1 and CS2 were deleted in the presence of inactivated PLP1, the growth of the resulting mutant virus was partially compensated, comparable to that of the CS1 and CS2 deletion mutant. These results demonstrate that interactions of PLP1 with CS1 and CS2 are critical for protein processing and suggest that the interactions play specific roles in regulation of the functions of nsp1, 2, and 3 in viral RNA synthesis.

scholarly journals Processing of Open Reading Frame 1a Replicase Proteins nsp7 to nsp10 in Murine Hepatitis Virus Strain A59 Replication

2007 ◽  
Vol 81 (19) ◽  
pp. 10280-10291 ◽  
Author(s):  
Damon J. Deming ◽  
Rachel L. Graham ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Proteolytic Processing ◽  
Mutant Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Murine Hepatitis Virus ◽  
Reading Frame ◽  
Genes Encoding

ABSTRACT Coronaviruses express open reading frame 1a (ORF1a) and ORF1b polyproteins from which 16 nonstructural proteins (nsp) are derived. The highly conserved region at the carboxy terminus of ORF1a is processed by the nsp5 proteinase (Mpro) into mature products, including nsp7, nsp8, nsp9, and nsp10, proteins with predicted or identified activities involved in RNA synthesis. Although continuous translation and proteolytic processing of ORF1ab by Mpro is required for replication, it is unknown whether specific cleavage events within the polyprotein are dispensable. We determined the requirement for the nsp7 to nsp10 proteins and their processing during murine hepatitis virus (MHV) replication. Through use of an MHV reverse genetics system, in-frame deletions of the coding sequences for nsp7 to nsp10, or ablation of their flanking Mpro cleavage sites, were made and the effects upon replication were determined. Viable viruses were characterized by analysis of Mpro processing, RNA transcription, and growth fitness. Deletion of any of the regions encoding nsp7 to nsp10 was lethal. Disruption of the cleavage sites was lethal with the exception of that of the nsp9-nsp10 site, which resulted in a mutant virus with attenuated replication. Passage of the attenuated nsp9-nsp10 cleavage mutant increased fitness to near-wild-type kinetics without reversion to a virus capable of processing nsp9-nsp10. We also confirmed the presence of a second cleavage site between nsp7 and nsp8. In order to determine whether a distinct function could be attributed to preprocessed forms of the polyprotein, including nsp7 to nsp10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus was not viable, suggesting that the uncleaved protein may be essential for replication or proteolytic processing.

scholarly journals Crystal Structure of a Monomeric Form of Severe Acute Respiratory Syndrome Coronavirus Endonuclease nsp15 Suggests a Role for Hexamerization as an Allosteric Switch

2007 ◽  
Vol 81 (12) ◽  
pp. 6700-6708 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Kumar Singh Saikatendu ◽  
Vanitha Subramanian ◽  
Benjamin W. Neuman ◽  
Michael J. Buchmeier ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Active Site ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Full Length ◽  
Structural Basis ◽  
Monomeric Form ◽  
Murine Hepatitis Virus ◽  
Catalytic Loop ◽  
Intersubunit Interactions

ABSTRACT Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn2+-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 Å. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by ∼120° into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

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