当前位置： SCI文献检索 > JOURNAL OF VIROLOGY期刊下所有文献 > Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63.

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63.

Abstract：

:Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.

journal_name

J Virol

journal_title

Journal of virology

authors

Chen Z,Wang Y,Ratia K,Mesecar AD,Wilkinson KD,Baker SC

doi

10.1128/JVI.02747-06

subject

Has Abstract

pub_date

2007-06-01 00:00:00

pages

6007-18

issue

eissn

0022-538X

issn

1098-5514

pii

JVI.02747-06

journal_volume

pub_type

杂志文章

在线工具

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63.