Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jun;81(11):6007-18.
doi: 10.1128/JVI.02747-06. Epub 2007 Mar 28.

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

Zhongbin Chen  1 , Yanhua Wang, Kiira Ratia, Andrew D Mesecar, Keith D Wilkinson, Susan C Baker
Affiliations

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

Zhongbin Chen et al. J Virol. 2007 Jun.

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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Detection of replicase products from HCoV-NL63-infected cells. (A) Schematic diagram illustrating HCoV-NL63 ORFs, the predicted processing of replicase polyprotein to nsp's, and regions used to generate anti-R3 (R3) and anti-R4 (R4) sera. The PLP domain (PLP1 and PLP2) and picornavirus 3C-like protease domain (3CLpro) are indicated. (B) Indirect immunofluorescence assay for HCoV-NL63 nsp3 and nsp4. LLC-MK2 cells were infected with HCoV-NL63, fixed, permeabilized at 24 hpi, and stained with anti-R3 or anti-R4 sera, anti-rabbit Ig Alexa-Fluor-488 secondary antibody, and Sytox orange to stain nuclei as described in Materials and Methods. (C) Detection of nsp3 by immunoprecipitation. HCoV-NL63 (NL)- or mock (M)-infected cells were radiolabeled with [35S]methionine for 24 h from 72 to 96 hpi. Cells were harvested, and the cell lysates were subjected to immunoprecipitation with anti-R3 sera or preimmune sera. Products were analyzed by 10% SDS-PAGE and subjected to autoradiography. (D) Detection of nsp3 and nsp4 by a Western blot assay with anti-R3 and anti-R4 antibodies. LLC-MK2 cells infected with HCoV-NL63 were lysed at 96 hpi, and whole-cell lysates were separated by SDS-PAGE. Immunoblotting was performed using anti-R3 and anti-R4 antibodies. Molecular mass markers (in kDa) are shown on the left of each gel.
FIG. 2.
FIG. 2.
Identification of proteases that process the amino-terminal region of the HCoV-NL63 replicase polyprotein. (A) Schematic representation of predicted processing at CS1 and CS2 by PLP1 and PLP2 (top panel) and constructs tested in the cis-cleavage assay. nsp3 conserved domains: Ac, acidic domain; ADRP, ADP-ribose-1′′-phosphatase (49); TM, transmembrane domain; Y, Y domain (70). The proposed catalytic residues (C1602 for PLP1 and C1678 for PLP2) are indicated. (B) cis-Cleavage assay for CS1 and CS2. Constructs were transfected into HeLa-MHVR cells infected with vTF7.3. Newly synthesized proteins were labeled with [35S]methionine from 4.5 to 9.5 hpi. Lysates were prepared and subjected to immunoprecipitation (IP) with anti-V5 (top) or anti-Flag M2 (bottom) antibody. Immunoprecipitated proteins were separated by 10% SDS-PAGE or 8 to 16% Criterion gel and subjected to autoradiography. The precursors and cleaved products are indicated. Molecular mass markers (in kDa) are shown on the left of the gel.
FIG. 3.
FIG. 3.
Processing of HCoV-NL63 replicase CS3 by PLP2. (A) Schematic diagram of the predicted processing at CS3 by PLP2 and the constructs used in the cis-cleavage assay. The PLP2 catalytic residues (C1678 and H1836) are indicated. (B) Detection of processing at CS3 by PLP2 and identification of PLP2 catalytic sites. cis-Cleavage assays were performed as described for Fig. 2. The processed product (nsp4) and the precursors are indicated. The precursors and processed products were detected with anti-V5 antibody from cells transfected with wild-type PLP2 (WT) (lane 1), the C1678A mutant (lane 2), the C1684A mutant (lane 3), or the H1836A mutant (lane 4). (C) Identification of HCoV-NL63 PLP2 core domain. The processing activities, as detected by immunoprecipitation (IP) of nsp4, from pNL-4 (lane 1), pNL-5 (lane 2), and pNL-6 (lane 3), are shown. Molecular mass markers (in kDa) are shown on the left of each gel.
FIG. 4.
FIG. 4.
Identification of CS2 and CS3 cleavage sites. (A) Purified wild-type HCoV-NL63 six-His-PLP2 (lane 2) and mutant PLP2-C1678A (lane 3) are shown resolved on a Coomassie-stained SDS-PAGE gel. Twelve micrograms of purified protein was loaded in each lane. The molecular masses of the marker proteins (lane 1) are shown to the left of the gel in kDa. The expected molecular mass of six-His-PLP2 is 38.6 kDa. HPLC separation of cleavage products from 12-mer peptides representing CS2 (FTKLAGGKISFS) (B) and CS3 (VAKQGAGFKRTY) (C) incubated in the presence (dashed lines) and absence (solid lines) of purified HCoV-NL63 PLP2. The identity of each cleavage product was confirmed by MS (data not shown), as indicated above the peaks.
FIG. 5.
FIG. 5.
Effect of mutagenesis of critical determinants in CS2 and CS3 on PLP2 recognition and processing. (A) Cleavage site sequences of CS2 and CS3. The arrow indicates the cleavage site identified in the peptide-based cleavage assay. The P1 and P2 residues are highlighted. (B) P1 and P2 residues are critical for processing at CS2. pNL-3 constructs encoding the wild-type (WT) sequence or amino acid substitutions were tested for processing activity as described for Fig. 2. The site of substitution is indicated above each lane. (C) Critical determinants for processing at CS3. pNL-6 constructs (lanes 1 to 4) and pNL-4 constructs (lanes 5 and 6) were analyzed for processing at CS3. Specific substitutions are indicated above each lane. IP, immunoprecipitation.
FIG. 6.
FIG. 6.
HCoV-NL63 PLP2 has DUB activity. (A) Processing of K48-Ub6 by PLP2. Lane 1, K48-Ub6 incubated in the absence of PLP2. Lane 2, a molecular-mass (MW) ladder with 250-, 150-, 100-, 75-, 50-, 37-, 25-, 20-, 15-, and 10-kDa markers. Lanes 3 to 5, K48-Ub6 incubated in the presence of wild-type (WT) PLP2 for the indicated time points. Lane 6, K48-Ub6 incubated in the presence of PLP2-C1678A for 60 min. The positions of bovine serum albumin and the various Ub multimers are indicated to the right of the gel. (B) Schematic diagram of the adduct formed between DUBs and HA-Ub-VS. (C) Western blot detection of HA-Ub-PLP2. Purified protein and substrate were incubated for 10 min at 37°C, and the products were separated by SDS-PAGE and analyzed by Western blot analysis using anti-HA antibody.
FIG. 7.
FIG. 7.
Model for processing the HCoV-NL63 replicase polyprotein by PLP1 and PLP2. PLP1 processing at CS1 and PLP2 processing at CS2 and CS3 are indicated by arrows. Critical determinants at the P1 and P2 positions of the cleavage sites are indicated in bold.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Almeida, J. D., and D. A. Tyrrell. 1967. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture. J. Gen. Virol. 1:175-178. - PubMed
    1. Arden, K. E., M. D. Nissen, T. P. Sloots, and I. M. Mackay. 2005. New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia. J. Med. Virol. 75:455-462. - PMC - PubMed
    1. Balakirev, M. Y., M. Jaquinod, A. L. Haas, and J. Chroboczek. 2002. Deubiquitinating function of adenovirus proteinase. J. Virol. 76:6323-6331. - PMC - PubMed
    1. Barretto, N., D. Jukneliene, K. Ratia, Z. Chen, A. D. Mesecar, and S. C. Baker. 2005. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J. Virol. 79:15189-15198. - PMC - PubMed
    1. Bastien, N., K. Anderson, L. Hart, P. Van Caeseele, K. Brandt, D. Milley, T. Hatchette, E. C. Weiss, and Y. Li. 2005. Human coronavirus NL63 infection in Canada. J. Infect. Dis. 191:503-506. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-