https://www.ncbi.nlm.nih.gov/pubmed/?term=CoV+Nsp5
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Items: 10
1.
Tomar S, Johnston ML, St John SE, Osswald HL, Nyalapatla PR, Paul LN, Ghosh AK, Denison MR, Mesecar AD.
J Biol Chem. 2015 Aug 7;290(32):19403-22. doi: 10.1074/jbc.M115.651463. Epub 2015 Jun 8.All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5
protease (also known as 3C-like protease, 3CL(pro)) during virus
replication, making it a high value target for the development of
anti-coronavirus therapeutics. Kinetic studies indicate that in contrast
to 3CL(pro) from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL(pro) is less efficient at processing a peptide substrate due to MERS-CoV 3CL(pro) being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL(pro) enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL(pro) is a weakly associated dimer (Kd ∼52 μm) with a slow off-rate.. .
Despite this structural similarity, substantial differences in the
dimerization ability suggest that long range interactions by the
nonconserved amino acids distant from the dimer interface may control
MERS-CoV 3CL(pro) dimerization. Activation of MERS-CoV
3CL(pro) through ligand-induced dimerization appears to be unique
within the genogroup 2c and may potentially increase the complexity in
the development of MERS-CoV 3CL(pro) inhibitors as antiviral agents.
2.
Wu A, Wang Y, Zeng C, Huang X, Xu S, Su C, Wang M, Chen Y, Guo D.
Virus Res. 2015 Oct 2;208:56-65. doi: 10.1016/j.virusres.2015.05.018. Epub 2015 May 31.
Coronavirus 3C-like protease (3CLpro) is responsible for the cleavage of
coronaviral polyprotein 1a/1ab (pp1a/1ab) to produce the mature
non-structural proteins (nsps) of nsp4-16. The nsp5 of the newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV)
was identified as 3CLpro and its canonical cleavage sites (between
nsps) were predicted based on sequence alignment, but the cleavability
of these cleavage sites remains to be experimentally confirmed and
putative non-canonical cleavage sites (inside one nsp) within the
pp1a/1ab awaits further analysis. Here, we proposed a method for
predicting coronaviral 3CLpro cleavage sites which balances the
prediction accuracy and false positive outcomes. By applying this method
to MERS-CoV,
the 11 canonical cleavage sites were readily identified and verified by
the biochemical assays. The Michaelis constant of the canonical cleavage
sites of MERS-CoV showed that the substrate specificity of MERS-CoV
3CLpro is relatively conserved. Interestingly, nine putative
non-canonical cleavage sites were predicted and three of them could be
cleaved by MERS-CoV nsp5. These results pave the way for identification and functional characterization of new nsp products of coronaviruses.
3. Konstruoitu virus pieneläimen (hiiren) saamiseksi koe-eläimeksi. BtCoV-HKU5- SE. Virus on erään lepakkolajin koronavirus, ja siihen on asetettu SRAS- viruksen s-piikistä ektodomeeni (SE) . Tämä julakistiin 2014, muta tänään päätin hakea nsp erikseen ja vuorossa random haku nsp5. Virus tuhoaa keuhkojen alveolitkin.
Agnihothram
S, Yount BL Jr, Donaldson EF, Huynh J, Menachery VD, Gralinski LE,
Graham RL, Becker MM, Tomar S, Scobey TD, Osswald HL, Whitmore A, Gopal
R, Ghosh AK, Mesecar A, Zambon M, Heise M, Denison MR, Baric RS.
mBio. 2014 Mar 25;5(2):e00047-14. doi: 10.1128/mBio.00047-14.
Cross-species transmission of
zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks.
Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5)
and the nucleocapsid (N) protein, which are significant drug and
vaccine targets. BtCoV HKU5 has been described in silico but has not
been shown to replicate in culture, thus hampering drug and vaccine
studies against subgroup 2c β-CoVs. We report the synthetic
reconstruction and testing of BtCoV HKU5 containing the severe acute
respiratory syndrome (SARS)-CoV
spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus
replicates efficiently in cell culture and in young and aged mice, where
the virus targets airway and alveolar epithelial cells. Unlike some
subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV
N-expressing Venezuelan equine encephalitis virus replicon particle
(VRP) vaccines do not cause extensive eosinophilia following BtCoV
HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in
enhanced virulence, causing 20% weight loss, diffuse alveolar damage,
and hyaline membrane formation in aged mice. Passaged virus was
characterized by mutations in the nsp13, nsp14, open reading frame 5
(ORF5) and M genes. Finally, we identified an inhibitor active against
the nsp5
proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of
reconstituting emerging zoonotic viral pathogens or their phylogenetic
relatives provide new strategies for identifying broad-based
therapeutics, evaluating vaccine outcomes, and studying viral
pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV
is related to BtCoV HKU5 in target regions that are essential for drug
and vaccine testing. Because no small animal model exists to evaluate
MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV
spike (S) glycoprotein, thereby allowing the recombinant virus to grow
in cell culture and in mice. We show that this recombinant virus targets
airway epithelial cells and causes disease in aged mice. We use this
platform to (i) identify a broad-spectrum antiviral that can potentially
inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV
N protein-based vaccines, and (iii) mouse adapt this virus to identify
viral genetic determinants of cross-species transmission and virulence.
This study holds significance as a strategy to control newly emerging
viruses.
4.
Stobart CC, Sexton NR, Munjal H, Lu X, Molland KL, Tomar S, Mesecar AD, Denison MR.
J Virol. 2013 Dec;87(23):12611-8. doi: 10.1128/JVI.02050-13. Epub 2013 Sep 11.Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV
replication is the translation and processing of replicase polyproteins
containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5
activity and their conservation across divergent CoVs are unknown, in
part due to challenges in cultivating many human and zoonotic CoVs. To
test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5
proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup,
genogroup 2a, with MHV, allowed for immediate viral recovery with
efficient replication albeit with impaired fitness in direct competition
with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.
5.
Stobart CC, Lee AS, Lu X, Denison MR.
J Virol. 2012 May;86(9):4801-10. doi: 10.1128/JVI.06754-11. Epub 2012 Feb 15.1
6.
Fang S, Shen H, Wang J, Tay FP, Liu DX.
J Virol. 2010 Jul;84(14):7325-36. doi: 10.1128/JVI.02490-09. Epub 2010 May 5.Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5),
processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11
specific sites to produce 12 mature nonstructural proteins (nsp5
to nsp16). Structural and biochemical studies suggest that a conserved
Gln residue at the P1 position is absolutely required for efficient
cleavage.
Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086,
nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu
substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the
nsp15-16/Q6327 site were tolerated and allowed recovery of infectious
mutant viruses, albeit with variable degrees of growth defects. In
contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868,
nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro
substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated
cleavage at the corresponding position and blocked the recovery of
infectious viruses. Analysis of the effects of these lethal mutations on
RNA synthesis suggested that processing intermediates, such as the
nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may
function in negative-stranded genomic RNA replication, whereas mature
proteins may be required for subgenomic RNA (sgRNA) transcription. More
interestingly, a mutant 3CLpro with either a P166S or P166L mutation was
selected when an IBV infectious cDNA clone carrying the Q6327N mutation
at the nsp15-16 site was introduced into cells. Either of the two
mutations was proved to enhance significantly the 3CLpro-mediated
cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and
compensate for the detrimental effects on recovery of infectious virus.
7.
von Brunn A, Teepe C, Simpson JC, Pepperkok R, Friedel CC, Zimmer R, Roberts R, Baric R, Haas J.PLoS One. 2007 May 23;2(5):e459.
8.
Lu JH, Zhang DM, Wang GL, Guo ZM, Li J, Tan BY, Ou-Yang LP, Ling WH, Yu XB, Zhong NS.
Acta Biochim Biophys Sin (Shanghai). 2005 Jul;37(7):473-9.
9.
Lu JH, Zhang DM, Wang GL, Guo ZM, Zhang CH, Tan BY, Ouyang LP, Lin L, Liu YM, Chen WQ, Ling WH, Yu XB, Zhong NS.
Chin Med J (Engl). 2005 May 5;118(9):707-13.The results suggest that polymerase nsp2 is relatively stable during the
phase of epidemic. The amino acid and secondary structure change may be
important for viral infection. The fact that majority of single
nucleotide variations (SNVs) are predicted to cause synonymous, as well
as the result of low mutation rate of nsp2 gene in the epidemic
variations, indicates that the nsp2 is conservative and could be a
target for anti-SARS drugs. The three-dimensional structure result
indicates that the nsp2 protein of (Guandong) GD strain is high homologous with
3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible
gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain,
which further suggests that nsp2 protein of GD strain possesses the
activity of 3CL(pro).
10.
Prentice E, McAuliffe J, Lu X, Subbarao K, Denison MR.
J Virol. 2004 Sep;78(18):9977-86... These results confirm the predicted protein processing pattern for
mature SARS-CoV replicase proteins, demonstrate localization of
replicase proteins to cytoplasmic complexes containing markers for
autophagosome membranes, and suggest conservation of protein epitopes in
the replicase and nucleocapsid of SARS-CoV and the group II
coronavirus, MHV. Further, the results demonstrate the ability of
replicase antibodies to detect SARS-CoV-infected cells as early as 6 h
postinfection and thus represent important tools for studies of SARS-CoV
replication, inhibition, and diagnosis.
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