《2012 Temperature-Sensitive Mutants and Revertants in the Coronavirus Nonstructural Protein 5 Protease (3CLpro) Define Re》由会员分享，可在线阅读，更多相关《2012 Temperature-Sensitive Mutants and Revertants in the Coronavirus Nonstructural Protein 5 Protease (3CLpro) Define Re（10页珍藏版）》请在金锄头文库上搜索。
1、Temperature-Sensitive Mutants and Revertants in the Coronavirus Nonstructural Protein 5 Protease (3CLpro) Defi ne Residues Involved in Long-Distance Communication and Regulation of Protease Activity Christopher C. Stobart,b,cAlice S. Lee,a,cXiaotao Lu,a,cand Mark R. Denisona,b,c Departments of Pedia
2、tricsaand Pathology, Microbiology and Immunology,band The Elizabeth B. Lamb Center for Pediatric Research,cVanderbilt University Medical Center, Nashville, Tennessee, USA Positive-strandRNAvirusgenomesaretranslatedintopolyproteinsthatareprocessedbyviralproteasestoyieldfunctionalin- termediateandmatu
3、reproteins.Coronaviruses(CoVs)carrygenesthatencodeannsp5protease(alsoknownas3CLproor Mpro)responsiblefor11maturationcleavages.Thensp5structurecontainstwochymotrypsin-likedomains(D1andD2)anda uniquedomain(D3),andformsfunctionaldimers.However,littleisknownofinteractionsorcommunicationacrossthestruc- t
4、ureoftheproteaseduringnsp5activity.UsingreversegeneticmutagenesisoftheCoVmurinehepatitisvirus(MHV)nsp5,we identifi edanewtemperature-sensitive(ts)mutationinD2ofnsp5(Ser133Ala)andconfi rmeda tsresidueinD3(Phe219Leu). BothD2-tsS133AandD3-tsF219Lwereimpairedforviralreplicationandnsp5-mediatedpolyprotei
5、nprocessingatthenonper- missivetemperature.Passageof tsS133AandtsF219Latthenonpermissivetemperatureresultedinemergenceofmultiplesec- ond-sitesuppressormutations,singlyandincombinations.Amongthesecond-sitemutations,aD2His134Tyrchangesup- pressedthetsphenotypeofD2-tsS133AandD3-tsF219L,aswellastheprevi
6、ouslyreportedD2-tsV148A.Analysisofmultiple CoVnsp5structures,andalignmentofnonredundantnsp5primarysequences,demonstratedthat tsandsuppressorresiduesare notconservedacrossCoVsandarephysicallydistant(10)fromeachother,fromcatalyticandsubstrate-bindingresidues, andfromthensp5dimerinterface.Thesefi nding
7、sdemonstratethatlong-distancecommunicationpathwaysbetweenmultiple residuesanddomainsofnsp5playasignifi cantroleinnsp5activityandviralreplication,suggestingpossiblenoveltargetsfor non-activesiteinhibitorsofnsp5. P ositive-strand RNA viruses are responsible for prevalent and epidemicdiseasesinawideran
8、geofvertebratehosts,aswellas new and emerging viruses, such as severe acute respiratory syn- dromecoronavirus(SARS-CoV),WestNilevirus,andChikungu- nya virus. The rapid evolution, host species movement, and dis- eases of positive-strand RNA viruses demonstrate the need to develop novel strategies to
9、prevent and treat present and new dis- easescausedbytheseviruses.Akeydeterminantofpositive-strand RNA viruses is the requirement for processing of translated poly- proteins by virus gene-encoded proteases. RNA virus proteases therefore have been high-profi le targets for development of anti- viral a
10、gents, with most protease inhibitors targeted to active sites or substrate-binding sites (21, 26, 32, 35). However, due to the potential for viral escape mutants, it is critical to identify addi- tional noncatalytic, non-substrate-binding determinants of pro- tease activity as potential targets for
11、inhibition that are less prone to development of resistance. To date, fi ve CoVs have been shown to be associated with hu- man respiratory diseases of different degrees of severity: human coronavirus HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV- HKU1, and SARS-CoV (10, 15, 30, 31, 44, 45). CoVs contain the
12、 largest known positive-strand RNA genomes, ranging from 26 to 32 kb in length. Murine hepatitis virus (MHV) strain A59 is an established model for study of CoV replication and pathogenesis. The 32-kb genome of MHV contains seven genes, with the repli- case gene (22 kb) encoding 16 nonstructural pro
13、teins (nsp1 to nsp16) (Fig. 1A) (20, 25). The replicase gene is translated into polyprotein 1a (pp1a; nsp1 to nsp11) or, via a ribosomal frame- shift, pp1ab (nsp1 to nsp16) (9, 25, 33). MHV encodes two papa- in-like proteases (PLP1 and PLP2) responsible for cleavages of nsp1 to nsp3, and an nsp5 pro
14、tease, also known as 3CLpro or Mpro,thatmediatesmaturationcleavagesofnsp4tonsp16andis required for virus replication (33, 50). TheCoVnsp5isacysteineproteasepresentinallknownCoVs and is structurally similar to the nsp4 protease of distantly related arteriviruses (6, 33, 49). The crystal structure of
15、nsp5 has been solved for divergent CoVs from every genus, including SARS- CoV, infectious bronchitis virus (IBV), human HCoV-HKU1, and human HCoV-229E. Comparison of solved nsp5 structures demonstratesconservationoftertiarystructuredespitenumerous differences in primary sequences (1, 2, 5, 46, 47, 4
16、9). The X-ray crystalstructureofMHVnsp5hasyettobedetermined;however, the structure of the closely related HCoV-HKU1 nsp5 (84% se- quence identity) has been resolved to 2.5 (Fig. 1B and C) (49). The nsp5 proteases of all CoVs exhibit a three-domain structure, with domains 1 and 2 forming a chymotrypsin-like fold contain- ing the His41-Cys145 catalytic dyad and substrate-binding sites (Fig.1B)(1,2,5,28,46,47).Incontrast,domain3isuniquetothe CoV nsp5 protease among chymotrypsin-like enzymes and al
