MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA

Terada, Yutaka; Kawachi, Kengo; Matsuura, Yoshiharu; Kamitani, Wataru

doi:10.1016/j.virol.2017.08.026

Cited by 58 publications

(66 citation statements)

References 62 publications

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“…Northern blot analysis. RNAs from C3663-infected or rC3663-infected Fcwf-4 cells were used for Northern blot analysis as described elsewhere (32). RNA samples were diluted to 2 g in 5 l by the use of UltraPure distilled water (DW) (Invitrogen) and then mixed with 5 l 2ϫ loading dye (New England Biolabs, Ipswich, MA).…”

Section: Discussionmentioning

confidence: 99%

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663

Terada

Kuroda

Morikawa

et al. 2019

J Virol

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Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific-pathogenfree cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed growth kinetics similar to those shown by the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h postinfection. Furthermore, we found that A72 cells derived from canine fibroblasts permitted FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.IMPORTANCE Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is a lethal disease in cats. Tissue culture-adapted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that type I FCoV strain C3663 efficiently induces FIP in specificpathogen-free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.

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Section: Discussionmentioning

confidence: 99%

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663

Terada

Kuroda

Morikawa

et al. 2019

J Virol

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“…Generation of recombinant MERS-CoV from BAC plasmids. A bacterial artificial chromosome (BAC) clone carrying the full-length infectious genome of the MERS-CoV EMC2012 strain (termed pBAC-MERS-wt) was used to generate recombinant MERS-CoV (22). Amino acid substitutions at the P1 site within the fusion cleavage sites in the S protein were generated by modification of the pBAC-MERS-wt (the template) using a Red/ET recombination system counterselection BAC modification kit (Gene Bridges, Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…4B). MERS-CoV mutants in which the S protein lacks furin cleavage sites at R748 (the S1/S2 site) and/or R884 (the S2= site) were generated using a recently developed reverse genetics system (22). Western blot analysis of S proteins harboring mutations at R748 (R748S or R748S/R884S) detected no 80-kDa cleavage product on virions (Fig.…”

Section: Calu-3mentioning

confidence: 99%

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

Matsuyama

Shirato

Kawase

et al. 2018

J Virol

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Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin. Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

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“…Nsp3, 4, and 6 of SARS-CoV contain transmembrane domains and are all responsible for the formation of DMVs [45]. In addition, the cisacting element at the 5 0 end of the nsp1 coding region facilitates the specific recognition of viral RNA, which is necessary for efficient virus replication [46]. MERS-CoV helicase (nsp13) is one of the most important viral replication enzymes and can profoundly affect tropism and virulence [47].…”

Section: Viral Replicationmentioning

confidence: 99%

“…The mRNA degradation activity of MERS-CoV nsp1 is separate from its translational inhibitory function. Furthermore, certain residues of MERS-CoV nsp1 are crucial for evading translational shutdown [46]. The escape of MERS-CoV mRNAs from inhibition by MERS-CoV nsp1 is promoted by their cytoplasmic origin [60].…”

Section: Inhibition Of the Expression And Translation Of Host Mrnamentioning

confidence: 99%

Molecular Characteristics, Functions, and Related Pathogenicity of MERS-CoV Proteins

et al. 2019

Engineering

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a b s t r a c tMiddle East respiratory syndrome (MERS) is a viral respiratory disease caused by a de novo coronavirus-MERS-CoV-that is associated with high mortality. However, the mechanism by which MERS-CoV infects humans remains unclear. To date, there is no effective vaccine or antibody for human immunity and treatment, other than the safety and tolerability of the fully human polyclonal Immunoglobulin G (IgG) antibody (SAB-301) as a putative therapeutic agent specific for MERS. Although rapid diagnostic and public health measures are currently being implemented, new cases of MERS-CoV infection are still being reported. Therefore, various effective measures should be taken to prevent the serious impact of similar epidemics in the future. Further investigation of the epidemiology and pathogenesis of the virus, as well as the development of effective therapeutic and prophylactic anti-MERS-CoV infections, is necessary. For this purpose, detailed information on MERS-CoV proteins is needed. In this review, we describe the major structural and nonstructural proteins of MERS-CoV and summarize different potential strategies for limiting the outbreak of MERS-CoV. The combination of computational biology and virology can accelerate the advanced design and development of effective peptide therapeutics against MERS-CoV. In summary, this review provides important information about the progress of the elimination of MERS, from prevention to treatment.

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MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA

Cited by 58 publications

References 62 publications

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663

Establishment of a Virulent Full-Length cDNA Clone for Type I Feline Coronavirus Strain C3663

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells

Molecular Characteristics, Functions, and Related Pathogenicity of MERS-CoV Proteins

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