【病毒外文文献】2000 Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C-like Proteinase and Determination of a N

Further Characterization of the Coronavirus Infectious Bronchitis Virus 3C like Proteinase and Determination of a New Cleavage Site Lisa F P Ng and D X Liu 1 Institute of Molecular Agrobiology The National University of Singapore 1 Research Link Singapore 117604 Received December 10 1999 returned to author for revision March 14 2000 accepted March 29 2000 Coronavirus infectious bronchitis virus IBV encodes a trypsin like proteinase 3C like proteinase by ORF 1a which has been demonstrated to play a pivotal role in proteolytic processing of gene 1 encoded polyproteins In our previous studies the proteinase was identified as a 33 kDa protein in IBV infected cells and its catalytic center was shown to consist of H 2820 and C 2922 residues It is released from the 1a and 1a 1b polyproteins by autoprocessing at two Q S dipeptide bonds Q 2779 S 2780 and Q 3086 S 3087 In this report further characterization of the two cleavage sites demonstrates that the N terminal Q 2779 S 2780 site is tolerant to mutations at the P1 position Deletion of the C terminal region of the proteinase shows that a significant amount of the enzymatic activity is maintained upon deletion of up to 67 amino acids suggesting that the extreme C terminal region may be dispensable for the proteolytic activity of the proteinase Analysis of the autoprocessing kinetics in vitro reveals that proteolysis at the Q 2779 S 2780 site is the first cleavage event mediated by this proteinase This is followed by cleavage at the Q 3086 S 3087 site The occurrence of both cleavage events in intact cells is potentially rapid and efficient as no intermediate cleavage products covering the proteinase were detected in either IBV infected or transfected cells Immunofluorescence microscopy and subcellular fractionation studies further show differential subcellular localization of the proteinase in IBV infected cells and in cells expressing the 3C like proteinase alone indicating that additional roles in viral replication might be played by this protein Finally a Q A Q 3379 A 3380 dipeptide bond encoded by nucleotides 10 663 to 10 668 was demonstrated to be a cleavage site of the proteinase 2000 Academic Press INTRODUCTION The avian coronavirus infectious bronchitis virus IBV is now categorized under a new order Nidovirales Ca vanagh 1997 de Vries et al 1997 Its 27 6 kb genome length mRNA mRNA1 is composed of two large over lapping ORFs 1a and 1b at the 59 unique region and encodes a fusion polyprotein of 7 41 kDa by a unique frameshifting mechanism Boursnell et al 1987 Brierly et al 1987 1989 Proteolytic processing of the 441 kDa 1a and 7 41 kDa 1a 1b fusion polyproteins to smaller mature products is mediated by viral proteinases Three proteinase domains namely two overlapping papain like proteinase domains and a picornavirus 3C like protein ase domain 3C like proteinase Fig 1a have been predicted to be encoded by ORF 1a Boursnell et al 1987 Gorbalenya et al 1989 Lee et al 1991 In recent years progress has been made in the iden tification and characterization of the proteinase activities as well as their cleavage products The first papain like proteinase domain is involved in proteolytic cleavage of the N terminal region of the 1a and 1a 1ab polyproteins to release an 87 kDa protein and a 195 kDa protein and the N terminus of a 41 kDa product in IBV infected cells Liu et al 1995 Lim and Liu 1998 Lim et al 2000 Similar enzymatic activities were also reported for the first papain like proteinase domain of human and murine coronaviruses In mouse hepatitis virus MHV two N terminal cleavage products p28 and p65 have been reported Hughes et al 1995 Bonilla et al 1997 Baker et al 1989 1993 Dong and Baker 1994 Gao et al 1996 Schiller et al 1998 The same domain of human coro navirus HCV processes the two polyproteins to release p9 Herold et al 1998 Important roles in processing of the 1a and 1a 1b polyproteins are also played by the 3C like proteinase domain Characterization of the proteinase activities has shown that it cleaves the bulk of the polyproteins at conserved Q S G and N dipeptide bonds resulting in the release of more than 10 mature products Liu et al 1994 1995 1997 1998 Ng and Liu 1998 Tibbles et al 1999 Ziebuhr and Siddell 1999 Grotzinger et al 1996 Heusipp et al 1997a b Lu et al 1995 1998 Schiller et al 1998 The proteinase was identified as a 27 kDa protein for MHV Lu et al 1996 and a 34 kDa protein for HCV Ziebuhr et al 1995 The catalytic center of the proteinase consists of a His residue and a Cys residue Liu and Brown 1995 Lu et al 1995 Seybert et al 1997 Ziebuhr et al 1995 1997 The 3C like proteinase domain is flanked by hydropho bic regions in the coronaviral genomes In vitro studies in 1 To whom correspondence and reprint requests should be ad dressed E mail liudx ima org sg Virology 272 27 39 2000 doi 10 1006 viro 2000 0330 available online at on 0042 6822 00 35 00 Copyright 2000 by Academic Press All rights of reproduction in any form reserved 27MHV have shown that its enzymatic activity can be greatly enhanced by the addition of canine pancreatic microsomal membranes to the translation reaction mix ture Pinon et al 1997 Schiller et al 1998 The proteo lytic activity of the proteinase could be inhibited by a variety of cysteine and serine inhibitors and was elimi nated by the cysteine proteinase inhibitor E64d Lu et al 1996 Pinon et al 1997 Seybert et al 1997 More re cently immunofluorescence studies using region spe cific antisera against several 1a and 1a 1b cleavage products including the 3C like proteinase have sug gested that the proteinase may be associated with the viral RNA replication transcription machinery Schiller et al 1998 Ziebuhr and Siddell 1999 Shi et al 1999 Denison et al 1999 van der Meer et al 1999 In a previous study we identified the IBV 3C like pro teinase as a 33 kDa protein in IBV infected Vero cells Lim et al 2000 The proteinase was shown to be self cleaved from the polyproteins at the two previously predicted Q S dipeptide bonds Q 2779 S 2780 and Q 3086 S 3087 i na nin vitro processing assay Tibbles et al 1996 The in vitro proteolytic activity was shown to be strictly dependent on the addition of canine microsomal mem branes Tibbles et al 1996 Here we show by deletion and mutational analyses that the N terminal cleavage site of the 3C like proteinase is tolerant to mutations and the extreme C terminal region may be dispensable for the enzymatic activity The proteinase could be self cleaved to the 33 kDa mature form in vitro in rabbit reticulocyte lysate without the addition of canine micro somal membranes Immunofluorescence staining and cellular fractionation studies demonstrated that the pro teinase is associated mainly with the membrane fraction in virus infected cells However a free distribution pat tern was observed when the proteinase was overex pressed in intact cells Finally instead of the previously predicted Q 3365 G 3366 dipeptide bond the Q 3379 A 3380 dipeptide bond was identified as a cleavage site of the proteinase RESULTS Tolerance of the N terminal cleavage site Q 2779 S 2780 of the 3C like proteinase to mutations The previously predicted Q 2779 S 2780 dipeptide bond was shown to be the N terminal cleavage site of the 3C like proteinase in an in vitro processing assay Tibbles et al 1996 Substitution mutagenesis was car ried out to investigate the effects of mutations on the self cleavage efficiency of the proteinase at this position The Q 2779 S 2780 dipeptide bond was first mutated to E 2779 S 2780 giving rise to pIBV9D1Q 2779 E Fig 1a Expression of pIBV9D1Q 2779 E led to the detection of three protein species Fig 1b lane 2 In addition to the 33 kDa pro tein two products of approximately 43 and 48 kDa re spectively were also detected Fig 1b lane 2 The 43 kDa protein may represent a fusion product contain ing the 33 kDa protein and the product encoded by nucleotides 8693 to 8865 and the 48 kDa protein may represent the full length product encoded by this con struct Fig 1a As a significant amount of the 33 kDa protein was detected from the expression of this con struct Fig 1b lane 2 this result indicates that an E S dipeptide could be used efficiently by the 3C like pro teinase at this position Substitution of the Q 2779 residue with a K was then carried out giving rise to pIBV9D1Q 2779 K Fig 1a Expression of this construct resulted in more efficient detection of the 43 and 48 kDa products Fig 1b lane 3 However a trace amount of the 33 kDa protein was still detected Fig 1b lane 3 These results suggest that cleavage at the Q 2779 S 2780 dipeptide bond may be tolerant to mutations and the Q to E sub stitution is better tolerated than the Q K substitution Mutation and deletion analyses of the C terminal region of the 3C like proteinase Mutation of the C terminal cleavage site Q 3086 S 3087 dipeptide bond of the 3C like proteinase to E 3086 S 3087 was subsequently made giving rise to pIBV9Q 3086 E Fig 2a Expression of pIBV9Q 3086 E led to the detection of a novel product with an apparent molecular mass of ap proximately 35 kDa Fig 2b lane 2 No 33 kDa protein was observed Fig 2b lane 2 This was unexpected as no further cleavage site was predicted to be located 30 60 bp downstream of the Q 3086 S 3087 dipeptide bond To investigate the possibility that the C terminus of the 35 kDa protein may be derived from a novel cleavage site located 10 to 20 amino acid residues downstream of the Q 3086 residue two deletion plasmids pIBV9Q 3086 ED1 and pIBV9Q 3086 ED2 were constructed As can be seen in Fig 2a the IBV sequence present in pIBV9Q 3086 ED1 FIG 1 a Diagram of ORF1a illustrating the two overlapping papain like proteinase domains PLPDs and the 3C like proteinase domain 3CLP The Q 2779 S 2780 Q 3086 S 3087 Q 3379 A 3380 and Q 3462 S 3463 scissile bonds and viral products generated from cleavage by the 3C like proteinase are indicated Also shown are the IBV sequence recognized by anti 3C the stretches of hydrophobic residues encoded by ORF1a and the IBV sequences present in pIBV9 pIBV9D1Q 2779 E and pIBV9D1Q 2779 K The viral products generated from cleavage by the 3C like proteinase and the full length products encoded by some plasmids are illustrated The molecular masses of these products were estimated according to their migration on SDS PAGE The Q 2779 S 2780 Q 3086 S 3087 Q 3379 A 3380 and Q 3462 S 3463 residues are encoded by nucleotides 8863 8868 9784 9789 10 663 10 668 and 10 912 10 917 respectively b Mutational analysis of the Q 2779 S 2780 dipeptide bond Plasmids pIBV9 pIBV9D1Q 2779 E and pIBV9D1Q 2779 K respec tively were transiently expressed in Cos 7 cells using the vaccinia T7 expression system Cells were labeled with 35 S methionine cysteine lysates were prepared and polypeptides were immunoprecipitated with anti 3C Gel electrophoresis of the polypeptides was performed on an SDS 12 5 polyacrylamide gel and polypeptides were detected by fluorography 28 NG AND LIU29 3C LIKE PROTEINASE OF AVIAN CORONAVIRUS IBVwas terminated at nucleotide position 9911 Expression of this construct once again led to the detection of the 35 kDa protein Fig 2b lane 3 Meanwhile a novel product migrating more slowly than the 35 kDa protein was also detected Fig 2b lane 3 Its apparent molec ular mass of 39 kDa suggests that it is a fusion product containing the 35 kDa protein and the product encoded by nucleotides up to 9911 pIBV9Q 3086 ED2 was con structed by introducing a UGA termination codon imme diately downstream of the Q 3086 residue Fig 2a Inter estingly expression of this plasmid led to the detection of a single protein species comigrating with the 35 kDa protein processed from pIBV9Q 3086 ED1 Fig 2b lane 4 suggesting that the E 3086 residue is actually the C termi nus of the 35 kDa protein expressed from pIBV9Q 3086 E and pIBV9Q 3086 ED1 These results demonstrate that mu tation of the Q 3086 residue to an E only marginally reduces the cleavage efficiency occurring at the Q 3086 S 3087 dipep tide bond but alters the mobility of the 33 kDa protein on SDS PAGE To investigate the possibility that the C terminal region of the 3C like proteinase may contain sequences nones sential for the catalytic activity of the proteinase five deletion constructs pIBV9D19 pIBV9D20 pIBV9D21 pIBV9D22 and pIBV9D23 were made and expressed in intact cells These constructs encode proteins with de letions of 3 7 33 67 and 135 amino acid residues respectively from the C terminus of the 3C like protein ase Expression of pIBV9D19 and pIBV9D20 showed that almost full enzymatic activity was maintained upon the deletions As can be seen a 35 kDa product represent ing the C terminally truncated form of the 3C like protein ase was detected in both cases Fig 2b lanes 6 and 7 No full length product was observed from the expression of pIBV9D19 Fig 2b lane 6 but a trace amount of the full length product was detected from the expression of pIBV9D20 Fig 2b lane 7 Progressively less enzymatic activity was observed when pIBV9D21 and pIBV9D22 were expressed In each case two products represent ing the full length proteins and the C terminally truncated 3C like proteinase were detected Fig 2b lanes 8 and 9 Expression of pIBV9D23 showed the detection of the full length product only Fig 2b lane 10 indicating that the catalytic activity was abolished by this deletion These results suggest that the extreme C terminal region is not essential for the catalytic activity of the 3C like proteinase Characterization of the in vitro enzymatic activity of the 3C like proteinase Studies on the 3C like proteinase of human and mu rine coronaviruses have demonstrated that microsomal membranes are not strictly required for the in vitro cleav age activity but addition of the membranes can greatly enhance the autoprocessing activity of the proteinase FIG 2 a Diagram showing the IBV sequences present in pIBV9Q 3086 E pIBV9Q 3086 ED1 pIBV9Q 3086 ED2 pIBV9D19 pIBV9D20 pIBV9D21 pIBV9D22 and pIBV9D23 The viral products generated from cleavage by the 3C like proteinase and the full length products encoded by some plasmids are illustrated The molecular masses of these products were estimated according to their migration on SDS PAGE The Q 3086 residue is encoded by nucleotides 9784 9786 b Mutational and deletion analyses of the Q 3086 S 3087 dipeptide bond and the C terminal region of the 3C like proteinase Plasmids pIBV9 pIBV9Q 3086 E pIBV9Q 3086 ED1 pIBV9Q 3086 ED2 pIBV9D19 pIBV9D20 pIBV9D21 pIBV9D22 and pIBV9D23 respectively were transiently expressed in Cos 7 cells using the vaccinia T7 system The cells were labeled with 35 S methionine cysteine lysates were prepared and the labeled polypeptides were immunoprecipitated with anti 3C Gel electrophoresis of the polypeptides was performed on an SDS 12 5 polyacrylamide gel and polypeptides were de tected by fluorography 30 NG AND LIU Lu et al 1996 Pinon et al 1997 Schiller et al 1998 Heusipp et al 1997a However the microsomal mem branes were shown to be indispensable for the in vitro activity of the IBV 3C like proteinase Tibbles et al 1996 To clarify this discrepancy plasmid pIBV1a7D1 which covers nucleotides 7999 to 9786 and contains the up stream hydrophobic region M1 was expressed in vitro in a rabbit reticulocyte lysate system and pulse chased for up to 8 h Aliquots were removed at the indicated time and the reactions were stopped by the addition of RNase A The 33 kDa protein was detected after incubation for 1 h and its expression steadily increased toward the end of the time course Fig 3 The expression of the full length 54 kDa protein meanwhile decreased rapidly over time Fig 3 The addition of microsomal membranes did not significantly increase the autoprocessing rate of the 33 kDa protein data not shown These results demon strate that autoprocessing of the IBV 3C like proteinase could occur in vitro in reticulocyte lysate in the absence of the membranes In vitro autoprocessing kinetics of the 3C like proteinase The in vitro processing system was then used to study the autoprocessing kinetics of the IBV 3C like protein ase Plasmids pIBV9Q 3086 ED1 and its parental construct pIBV1a9D17 Fig 4a were expressed in reticulocyte ly sate and pulse chased for up to 2 h The full length 48 kDa product was observed from the expression of both constructs after chase for 10 min and its expression gradually decreased toward the end of the time course Fig 4b The appearance of a 39 kDa product repre senting the fusion protein containing the 3C like protein ase and the product encoded by nucleotides 9787 to 9910 was observed after chase for 20 min Fig 4b It peaks after chase for 60 min and then starts to decline Fig 4b The 33 kDa protein was first seen from the expression of pIBV1a9D17 after chase for 30 min and gradually increased toward the end of the time course Fig 4b The 35 kDa mutant 3C like proteinase ex pressed from pIBV9Q 3086 ED1 appeared slightly later than the wild type proteinase It was first seen after chase for 40 min Fig 4b lane 14 Differential subcellular localization of the 3C like proteinase in virus infected and transfected cells Determination of the subcellular localization of ORF 1a encoded viral products from the Nidovirales family namely MHV A59 MHV JHM HCV 229E and equine ar teritis virus EAV was the subject of several recent reports Shi et al 1999 Schiller et al 1998 Ziebuhr and Siddell 1999 van der Meer et al 1998 1999 It was interesting to note that immunofluorescence staining of viral infected cells using anti 3C antiserum showed dis tinct punctuate perinuclear staining patterns Schiller et al 1998 Shi et al 1999 Denison et al 1999 van der Meer et al 1999 suggesting that the proteinase may be associated with intracellular membranes To study the subcellular localization patterns of the IBV 3C like pro teinase the proteinase was tagged to an 11 amino acid T7 tag MASMTGGQQMG derived from the major capsid protein of bacteriophage T7 and a highly specific anti T7 monoclonal antibody Novagen was used to stain cells overexpressing the fusion protein This procedure is used to avoid the high background of the anti 3C poly clonal antiserum when it was used in indirect immuno fluorescence assays As can be seen in Figs 5A and 5B staining of Vero cells expressing the T7 tagged 3C like proteinase with anti T7 antibody showed a diffuse cyto plasmic staining profile Indirect immunofluorescence staining of IBV infected Vero cells with anti 3C antiserum showed a distinct perinuclear punctate pattern at 13 h postinfection Fig 5D although high background was also observed for the mock infected cells Fig 5C This distribution pattern is similar to those reported for other coronaviruses and arteriviruses Schiller et al 1998 Shi et al 1999 van der Meer et al 1998 These results suggest that the IBV 3C like proteinase may exhibit dif ferential subcellular distribution patterns in virus in fected cells and in transfected cells When it was ex pressed individually a diffuse distribution profile was observed However the proteinase was shown to be associated with intracellular membranous structures in virus infected cells Subcellular fractionation of pIBV3C transfected and IBV infected cells was then carried out to investigate this possibility further Two fractions namely the membrane and cytosol fractions were collected and subjected to immunoprecipitation with anti 3C antiserum Figure 6a shows that approximately equal amounts of the 33 kDa proteinase were detected from both fractions Fig 6a lanes 1 and 3 Quantification by densitometry confirmed that the membrane and cytosol fractions contain 50 19 FIG 3 Time course analysis of the autoprocessing of the 3C like proteinase expressed from pIBV1a7D1 in rabbit reticulocyte lysates A 10 fold excess of unlabeled methionine was added to the in vitro translation reaction after incubation at 30 C for 10 min Aliquots were subsequently taken from the reaction after incubation for 0 1 2 3 4 5 6 7 and 8 h The 35 S methionine labeled