SARS-2-CoV nsp5 (3CL-PRO) proteiini-proteiini interaktioista

* Mitä tietoja Swiss modul antaa nyt nsp5  koronavirusproteiinista, joka on SARS-virusten pääentsyymi.  Nsp5 pystyy pilkkomaan  replikaasi-polyproteiinia 11 eri kohdasta, joten se tuottaa  useimmat   viruksen työkaluproteiineista, kun on päässyt itse  pilkkoutumaan  replikaasista esiin. https://covid-19.uniprot.org/uniprotkb/P0DTD1

 3C-like proteinase (EC:3.4.22.691 Publication)

Short name: 3CL-PRO

Short name: 3CLp

Alternative name(s):

nsp5 ( sequence)

>sp|P0DTD1|3264-3569
SGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIR
KSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNG
SPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGN
FYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYE
PLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQC
SGVTFQ

3C-like proteinase:Cleaves the C-terminus of replicase polyprotein at 11 sites. Recognizes substrates containing the core sequence [ILMVF]-Q-|-[SGACN] (PubMed:32198291, PubMed:32272481). Also able to bind an ADP-ribose-1''-phosphate (ADRP).

Inhibited by pyridone-containing alpha-ketoamides compounds 13a and 13b. In turn, alpha-ketoamide 13b (tert-butyl (1-((S)-1-(((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate) inhibits SARS-CoV-2 replication in human lung cells (PubMed:32198291). Inhibited ex vivo by michael acceptor inhibitor N3 (PubMed:32272481)"

* https://www.biorxiv.org/content/10.1101/2020.03.22.002386v1.full.pdf 

"The SARS-CoV-2 interactome reveals novel aspects of SARS-CoV-2 biologyOur study highlighted interactions between SARS-CoV-2 proteins and human proteins with a range of functions including DNA replication (Nsp1), epigenetic and gene expression regulators (Nsp5, Nsp8, Nsp13, E), vesicle trafficking (Nsp6, Nsp7, Nsp10, Nsp13, Nsp15, Orf3a, E, Orf8), lipid modification (Spike), RNA processing and regulation (Nsp8, N), ubiquitin ligases (Orf10), signaling (Nsp8, Nsp13, N, Orf9b), nuclear transport machinery (Nsp9, Nsp15, Orf6), cytoskeleton (Nsp1, Nsp13), mitochondria (Nsp4, Nsp8, Orf9c), and extracellular matrix (Nsp9) (Fig.3)"

 SARS-2-CoV  nsp5  proteiiniinteraktio  ihmisen  HDAC2-proteiinien kanssa.
( histonideasetylaasi 2)  
 ja  
SARS-2-CoV nsp5) proteiininteraktio  ihmisen TRMT1-proteiinin kanssa  (tRNA-metyylitransferaasi 1)

"We identified protein-protein interactions with the main protease Nsp5, using both wild-type and catalytic dead(C145A) constructs. 
For wild-type Nsp5, we identified one high-confidence interaction, the epigenetic regulator histone deacetylase 2 (HDAC2), and predicted a cleavage site between the HDAC domain and the nuclear localization sequence, suggesting that Nsp5 may inhibit HDAC2 transport into the nucleus (Extended Data Fig. 7), potentially impacting the published functions of HDAC2 in mediating inflammation and interferon response38,39.
 We also identified an interaction of Nsp5 (C145A) with tRNA methyltransferase 1 (TRMT1), which is responsible for synthesis of the dimethylguanosine (m2,2G) base modification on both nuclear and mitochondrial tRNAs40. We predict TRMT1 is also cleaved by Nsp5, removing its zinc finger and nuclear localization signal and likely resulting in an exclusively mitochondrial localization (Extended Data Fig. 7).

• HDAC2 (6q21)

(Histonideasetylaasi2.

Tämän entsyymin tehtävänä on poistaa  acetyl ryhmiä (Ac)  lysiinitähteistä (de-asetyloida histonien  N-terminaalisia lysiineitä(K).  Lisäksi entsyymissä on nitroso-cysteiinikohtia ( nitrosyloituvia). HDAC2:n    aktiivissa kohdassa on sinkkiä sitovaa kykyä, mikä on olennaista  entsyymiaktiivisuudelle.  Tämä HDAC2 -proteiini muodostaa transkriptiota vaimentavan kompleksin liittyessään  esim.  YY1 -sinkkisormiproteiiniin.  SARS- CoV nsp5  mutiloi näitä  stabiileja sinkkisormimuodostumia, mikä  epästabiloi proteiinin struktuuria ja samalla  hävittää entsyymiaktiivisuuden. Myös  tumaan lokalisoiva  signaali  häviää proteiinista ja se ei pääse DNA:n  histoneille, vaan jäänee mitokondriaan). 

 https://www.ncbi.nlm.nih.gov/gene/3066
 Histone deacetylase 2. Also known as HD2; RPD3; YAF1; KDAC2
Summary This gene product belongs to the histone deacetylase family. Histone deacetylases act via the formation of large multiprotein complexes, and are responsible for the deacetylation of lysine residues at the N-terminal regions of core histones (H2A, H2B, H3 and H4). This protein forms transcriptional repressor complexes by associating with many different proteins, including YY1, a mammalian zinc-finger ( Znf) transcription factor. Thus, it plays an important role in transcriptional regulation, cell cycle progression and developmental events. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2010] Expression. Ubiquitous expression in testis (RPKM 14.8), endometrium (RPKM 10.6) and 25 other tissues See more
Preferred Names:histone deacetylase 2.
Names: YY1-associated factor 1.
transcriptional regulator homolog RPD3. 
FEATURES:https://www.ncbi.nlm.nih.gov/protein/NP_001518.3
Active site: Comment:Active site consists of a long narrow tunnel (that apparently serves for substrate binding) and a cavity with Zn ion (that is important for catalysis). In this structure, the tunnel is filled by the aliphatic chain of the inhibitor.
  Histone deacetylase 2 (HDAC2) is a Zn-dependent class I enzyme that catalyzes hydrolysis of N(6)-acetyl-lysine residue of a histone to yield a deacetylated histone (EC 3.5.1.98). Histone acetylation/deacetylation process is important for mediation of transcriptional regulation of many genes. HDAC2 is involved in regulation through association with DNA binding proteins to target specific chromatin regions. It forms transcriptional repressor complexes by associating with several proteins, including the mammalian zinc-finger transcription factor YY1, thus playing an important role in transcriptional regulation, cell cycle progression and developmental events. Additionally, a few non-histone HDAC2 substrates have been found. HDAC2 plays a role in embryonic development and cytokine signaling important for immune response, and is over-expressed in several solid tumors including oral, prostate, ovarian, endometrial and gastric cancer. It participates in DNA-damage response, along with HDAC1; together, they can promote DNA non-homologous end-joining. HDAC2 is considered an important cancer prognostic marker. Inhibitors specifically targeting HDAC2 could be a therapeutic drug option.
https://www.ncbi.nlm.nih.gov/pubmed/31545224

Biomed Pharmacother. 2019 Oct;118:109380. doi: 10.1016/j.biopha.2019.109380. Epub 2019 Aug 30. Expression of GR-α and HDAC2 in steroid-Sensitive and steroid-Insensitive interstitial lung disease.Bin YF¹, Wu LJ¹ et al.

(Tämä proteiiniperhe  kiinnostaa muitakin viruksia:Esim: 

HIV-1 Vpr depletes HDAC1, 2, 3 and 8 to overcome HIV-1 proviral latency in macrophages).

• TRMT1 (19p13.13)

(tRNA- metyylitransferaasi 1.
 Muita nimiä TRM1 ja MRT68. Tämä geeni koodaa tRNA:ta modifioivaa entsyymiä ja toimii  dimetyylitransferaasina,  joka  modifioi tRNA:n guaniinitähteen 26- asemassa. Entsyymi voi siirtää   yhden  tai  kaksi metyyliryhmää (Me)  ja käyttää  aktivoitua metioniinia(SAM) metyylin (Me) antajana.  Geenimutaatio assosioituu leukomalasiaan ja mikrokefaliaan)


https://www.ncbi.nlm.nih.gov/gene/55621 
 tRNA methyltransferase 1. Also known as TRM1; MRT68.
Summary. This gene encodes a tRNA-modifying enzyme that acts as a dimethyltransferase, modifying a single guanine residue at position 26 of the tRNA. The encoded enzyme has both mono- and dimethylase activity when exogenously expressed, and uses S-adenosyl methionine (SAM) as a methyl donor. The C-terminal region of the encoded protein has both a zinc finger motif (Znf), and an arginine/proline-rich region. Mutations in this gene have been implicated in autosomal recessive intellectual disorder (ARID). Alternative splicing results in multiple transcript variants encoding different isoforms. There is a pseudogene of this gene on the X chromosome. [provided by RefSeq, May 2017] Expression Ubiquitous expression in spleen (RPKM 12.4), lymph node (RPKM 11.5) and 25 other tissues. Preferred Names tRNA (guanine(26)-N(2))-dimethyltransferase
Names:N(2),N(2)-dimethylguanosine tRNA methyltransferase,
TRM1 tRNA methyltransferase 1 homolog,
tRNA 2,2-dimethylguanosine-26 methyltransferase,
tRNA methyltransferase 1 homolog,
tRNA(guanine-26,N(2)-N(2)) methyltransferase,
tRNA(m(2,2)G26)dimethyltransferase.

FEATURES: NP_001129507.1  tRNA (guanine(26)-N(2))-dimethyltransferase isoform 1.

Conserved Domains (3) summary

smart00356
Location:600 → 626

ZnF_C3H1; zinc finger

pfam02005
Location:45 → 500

TRM; N2,N2-dimethylguanosine tRNA methyltransferase

cl25637
Location:571 → 624

DFRP_C; DRG Family Regulatory Proteins, Tma46.

Ref.   https://www.ncbi.nlm.nih.gov/pubmed/30289604/



 Lähdeartikkelin listasta  löytyy maininta  myös:

SARS-2- CoV nsp5 (C145A) 
 tekee interaktion
ihmisen  GPX1 -proteiinin kanssa ( joka on glutationiperoksidaasi 1) .

• GPX1 (3p21.31) 

(Glutationiperoksidaasi 1 on   selenoproteiini  solun redoxsysteemissä.  Sen rakenteessa on harvinainen aminohappo selenocysteiini (Sec) entsyymin atiivissa kohdassa . Se koodautuu UGA-koodilla, joka tavallisesti merkitsee translaation lopetusta. Tällä entsyymin  hienosääteisellä yksityiskohdalla  on kliinistä merkitystä.  

Seleeni on maaperästä  ravintoon tuleva aine ja esim aiemmin  Suomessa oli vajetta seleenistä maaperän seleeniköyhyyden takia.  Joissain paikoissa maailmassa on  taas hyvin  runsaasti seleeniä sisältävää maaperää, mikä ei sekään ole hyvä asia. Seleeni kuuluu ravintosuosituksissa mainittuihin hivenaineisiin. Tässä yhteydessä  ei selviä, miten  SARS-2- CoV nsp5 (C145A) . Selitys lienee lähdeartikkelin tekstissä. Kiinan Anhui provinssissa on korkea seleenipitoisuus. Se on tutkittu Dashan kylästä.  Anhuiprovinssista tuli ensimmäiset COVID-19 potilaat, ja he saivat antiviraalin hoidon eikä kukaan heistä kuollut. https://www.sciencedirect.com/science/article/pii/S1201971220302034. 

Dashan kylän  seleenipitoisuus maaperässä:  https://www.sciencedirect.com/science/article/pii/S1201971220302034

Rakenteellinen aminohappo selenocysteiini (asemassa 49) näyttää merkatun  U kirjaimella. Ensimmistä kertaa huomaan tällaisen merkinnän peptidisekvenssissä).    https://www.ncbi.nlm.nih.gov/pubmed/32179940The dietary Se intake of local residents was estimated to be 261.2 µg day^- 1 and hair Se content varied from 0.34 to 1.35 mg kg^- 1, suggesting that the potential health risk should be concerned. Weathering of carbonaceous rock was speculated to be the primary source of soil Se according to the contents of Se in rocks, the distribution of Se in soil profiles and the relationships between Se and other elements in soils and parent rocks.  
 (Ps. Kun seleeni-innostus tuli Suomeen, oli tarjolla 300 ug tableteita immunoseleenia. Sain jopa ilmaiseksi  apteekista tällaisia! Olin aloitamassa keliakidieettiä siihen aikaan).
Brasilian  selenium tilanteesta. https://www.ncbi.nlm.nih.gov/pubmed/20646739
 https://edition.cnn.com/2020/05/07/americas/amazon-manaus-coronavirus-intl/index.html
COVID 19 on aivan  akuutin katastrofin astetta Brasiliassa vielä päivästä päivään.  Manaus- nimisessä kaupungissa lienee epicentrum.   Tästä seleeniasiasta  johduin   nihin maaperäseikkoihin.

glutathione peroxidase 1

Also known as GPXD; GSHPX1

Summary: The protein encoded by this gene belongs to the glutathione peroxidase family, members of which catalyze the reduction of organic hydroperoxides  and hydrogen peroxide (H2O2) by glutathione, and thereby protect cells against oxidative damage. Other studies indicate that H2O2 is also essential for growth-factor mediated signal transduction, mitochondrial function, and maintenance of thiol redox-balance; therefore, by limiting H2O2 accumulation, glutathione peroxidases are also involved in modulating these processes. Several isozymes of this gene family exist in vertebrates, which vary in cellular location and substrate specificity. This isozyme is the most abundant, is ubiquitously expressed and localized in the cytoplasm, and whose preferred substrate is hydrogen peroxide. It is also a selenoprotein, containing the rare amino acid selenocysteine (Sec) at its active site. Sec is encoded by the UGA codon, which normally signals translation termination. The 3' UTRs of selenoprotein mRNAs contain a conserved stem-loop structure, designated the Sec insertion sequence (SECIS) element, that is necessary for the recognition of UGA as a Sec codon, rather than as a stop signal. This gene contains an in-frame GCG trinucleotide repeat in the coding region, and three alleles with 4, 5 or 6 repeats have been found in the human population. The allele with 4 GCG repeats has been significantly associated with breast cancer risk in premenopausal women. Alternatively spliced transcript variants have been found for this gene. Pseudogenes of this locus have been identified on chromosomes X and 21. [provided by RefSeq, Aug 2017] Expression Ubiquitous expression in fat (RPKM 212.4), spleen (RPKM 100.0) and 24 other tissues See more Orthologs.

FEATURES:https://www.ncbi.nlm.nih.gov/protein/NP_000572.2

ORIGIN      
        1 mcaarlaaaa aaaqsvyafs arplaggepv slgslrgkvl lienvaslug ttvrdytqmn
       61 elqrrlgprg lvvlgfpcnq fghqenakne eilnslkyvr pgggfepnfm lfekcevnga
      121 gahplfaflr ealpapsdda talmtdpkli twspvcrndv awnfekflvg pdgvplrrys
      181 rrfqtidiep dieallsqgp sca
//

MERS-CoV, 3CL proteaasi, (nsp5) pilkkoo esiin nsp4- nsp16 proteiinit MERS-koronaviruspolyproteiinista ..

Kun tekee haun "CoV nsp5", saa PubMed hakulaitteella 10 löytöä. Otan niistä sitaatin.
https://www.ncbi.nlm.nih.gov/pubmed/?term=CoV+Nsp5 

Search results

Items: 10

Select item 260557151.

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS.

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.

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Select item 260367872.

Prediction and biochemical analysis of putative cleavage sites of the 3C-like protease of Middle East respiratory syndrome coronavirus.

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.

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Select item 246677063. 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.

A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant.

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.

DOI:

10.1128/mBio.00047-14

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Select item 240273354.

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

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.

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Select item 223454515.

Temperature-sensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3CLpro) define residues involved in long-distance communication and regulation of protease activity.

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

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Select item 204448936.

Functional and genetic studies of the substrate specificity of coronavirus infectious bronchitis virus 3C-like proteinase.

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.

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Select item 175200187.

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

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.

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Select item 159992088.

Sequence analysis and structural prediction of the severe acute respiratory syndrome coronavirus nsp5.

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.

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Select item 158991309.

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model.

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).

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Select item 1533173110.

Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins.

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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