Expression of murine coronavirus recombinant papain-like proteinase:: Efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides

被引:23
作者
Teng, H [1 ]
Piñón, JD [1 ]
Weiss, SR [1 ]
机构
[1] Univ Penn, Sch Med, Dept Microbiol, Philadelphia, PA 19104 USA
关键词
D O I
10.1128/JVI.73.4.2658-2666.1999
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication, In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for PLP-1, We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates, Subsequent characterization of the recombinant PLP-1 revealed that in vitro trans cleavage is more efficient at 22 degrees C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa, In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans, In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage in cis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.
引用
收藏
页码:2658 / 2666
页数:9
相关论文
共 30 条
[1]  
[Anonymous], 1993, Soc. Sci. Rev. D Physiochem. Biol
[2]  
Ausubel F. M., 1994, CURRENT PROTOCOLS MO
[3]   IDENTIFICATION OF A DOMAIN REQUIRED FOR AUTOPROTEOLYTIC CLEAVAGE OF MURINE CORONAVIRUS GENE-A POLYPROTEIN [J].
BAKER, SC ;
SHIEH, CK ;
SOE, LH ;
CHANG, MF ;
VANNIER, DM ;
LAI, MMC .
JOURNAL OF VIROLOGY, 1989, 63 (09) :3693-3699
[4]   IDENTIFICATION OF THE CATALYTIC SITES OF A PAPAIN-LIKE CYSTEINE PROTEINASE OF MURINE CORONAVIRUS [J].
BAKER, SC ;
YOKOMORI, K ;
DONG, S ;
CARLISLE, R ;
GORBALENYA, AE ;
KOONIN, EV ;
LAI, MMC .
JOURNAL OF VIROLOGY, 1993, 67 (10) :6056-6063
[5]   CHARACTERIZATION OF THE LEADER PAPAIN-LIKE PROTEINASE OF MHV-A59 - IDENTIFICATION OF A NEW IN-VITRO CLEAVAGE SITE [J].
BONILLA, PJ ;
HUGHES, SA ;
PINON, JD ;
WEISS, SR .
VIROLOGY, 1995, 209 (02) :489-497
[6]   MOUSE HEPATITIS-VIRUS STRAIN A59 RNA-POLYMERASE GENE ORF 1A - HETEROGENEITY AMONG MHV STRAINS [J].
BONILLA, PJ ;
GORBALENYA, AE ;
WEISS, SR .
VIROLOGY, 1994, 198 (02) :736-740
[7]   Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59 [J].
Bonilla, PJ ;
Hughes, SA ;
Weiss, SR .
JOURNAL OF VIROLOGY, 1997, 71 (02) :900-909
[8]   The genome organization of the nidovirales: Similarities and differences between arteri-, toro-, and coronaviruses [J].
de Vries, AAF ;
Horzinek, MC ;
Rottier, PJM ;
de Groot, RJ .
SEMINARS IN VIROLOGY, 1997, 8 (01) :33-47
[9]   PROCESSING AND EVOLUTION OF THE N-TERMINAL REGION OF THE ARTERIVIRUS REPLICASE ORF1A PROTEIN - IDENTIFICATION OF 2 PAPAINLIKE CYSTEINE PROTEASES [J].
DENBOON, JA ;
FAABERG, KS ;
MEULENBERG, JJM ;
WASSENAAR, ALM ;
PLAGEMANN, PGW ;
GORBALENYA, AE ;
SNIJDER, EJ .
JOURNAL OF VIROLOGY, 1995, 69 (07) :4500-4505
[10]   IDENTIFICATION AND CHARACTERIZATION OF A 65-KDA PROTEIN PROCESSED FROM THE GENE-1 POLYPROTEIN OF THE MURINE CORONAVIRUS MHV-A59 [J].
DENISON, MR ;
HUGHES, SA ;
WEISS, SR .
VIROLOGY, 1995, 207 (01) :316-320