scholarly journals Identification of Severe Acute Respiratory Syndrome Coronavirus Replicase Products and Characterization of Papain-Like Protease Activity

2004 ◽  
Vol 78 (24) ◽  
pp. 13600-13612 ◽  
Author(s):  
Brian H. Harcourt ◽  
Dalia Jukneliene ◽  
Amornrat Kanjanahaluethai ◽  
John Bechill ◽  
Kari M. Severson ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Protease Activity ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Sars Coronavirus ◽  
Hydrophobic Domain ◽  
Amino Terminal ◽  
Viral Proteases ◽  
Infected Cells ◽  
Lumenal Domain

ABSTRACT Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a ≈300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.

scholarly journals Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches

2009 ◽  
Vol 83 (13) ◽  
pp. 6957-6962 ◽  
Author(s):  
Surendranath Baliji ◽  
Stephen A. Cammer ◽  
Bruno Sobral ◽  
Susan C. Baker
Keyword(s):  
Viral Replication ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Nonstructural Proteins ◽  
Hydrophobic Domain ◽  
Transmembrane Topology ◽  
Molecular Approaches ◽  
Viral Proteases ◽  
Infected Cells ◽  
Membrane Spanning

ABSTRACT Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.

scholarly journals ORF1a-Encoded Replicase Subunits Are Involved in the Membrane Association of the Arterivirus Replication Complex

1998 ◽  
Vol 72 (8) ◽  
pp. 6689-6698 ◽  
Author(s):  
Yvonne van der Meer ◽  
Hans van Tol ◽  
Jacomine Krijnse Locker ◽  
Eric J. Snijder
Keyword(s):  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Membrane Association ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Replication Complex ◽  
Viral Proteases ◽  
Double Label ◽  
Membrane Bound ◽  
Triton X

ABSTRACT Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Proteins 3, 4, and 6 Induce Double-Membrane Vesicles

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Megan M. Angelini ◽  
Marzieh Akhlaghpour ◽  
Benjamin W. Neuman ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Host Cell ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Full Length ◽  
Nonstructural Proteins ◽  
Human Coronavirus ◽  
Double Membrane ◽  
Infected Cells ◽  
Proliferation Ability

ABSTRACTCoronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6.IMPORTANCEAlthough the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.

scholarly journals Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS

Virology ◽  
2007 ◽  
Vol 363 (2) ◽  
pp. 288-302 ◽  
Author(s):  
Benoît Callendret ◽  
Valérie Lorin ◽  
Pierre Charneau ◽  
Philippe Marianneau ◽  
Hugues Contamin ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Dna Vaccines ◽  
Protective Efficacy ◽  
Viral Rna ◽  
Spike Protein ◽  
Sars Coronavirus ◽  
Rna Export

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Coronavirus Infections

2006 ◽  
Vol 81 (3) ◽  
pp. 1220-1229 ◽  
Author(s):  
Chandra Tangudu ◽  
Heidi Olivares ◽  
Jason Netland ◽  
Stanley Perlman ◽  
Thomas Gallagher
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Open Reading Frames ◽  
Viral Rna ◽  
Protein Accumulation ◽  
Sars Coronavirus ◽  
Viral Rnas ◽  
Growth Properties ◽  
Murine Coronavirus

ABSTRACT One or more of the unique 3′-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

scholarly journals Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1106 ◽  
Author(s):  
Mira Laajala ◽  
Minna M. Hankaniemi ◽  
Juha A. E. Määttä ◽  
Vesa P. Hytönen ◽  
Olli H. Laitinen ◽  
...  
Keyword(s):  
Mass Spectrometry Analysis ◽  
Structural Proteins ◽  
Calpain Inhibitor ◽  
Nonstructural Proteins ◽  
Vitro Assay ◽  
Spectrometry Analysis ◽  
Viral Proteases ◽  
Infected Cells ◽  
Calpain 2

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

scholarly journals A Novel System to Study Dengue Virus Replication Organelle Formation Independent from Viral RNA Replication

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 139
Author(s):  
Berati Cerikan ◽  
Sarah Goellner ◽  
Christopher John Neufeldt ◽  
Uta Haselmann ◽  
Mirko Cortese ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Replication ◽  
Host Factors ◽  
Viral Rna ◽  
Replication Machinery ◽  
Nonstructural Proteins ◽  
Independent System ◽  
Specific Host ◽  
Infected Cells ◽  
Positive Strand Rna

Positive-strand RNA viruses, such as dengue virus (DENV), induce the extensive rearrangement of intracellular membranes that serve as a scaffold for the assembly of the viral replication machinery. In the case of DENV, the main endomembrane ultrastructure produced in infected cells consists of invaginations of the endoplasmic reticulum, designated vesicle packets (VPs), which are the assumed sites of viral RNA replication. VPs are observed as arrays of vesicles surrounded by an outer membrane, the formation of which is induced by the viral nonstructural proteins, presumably in conjunction with specific host factors. However, little is known about the mechanisms governing VP formation, which is mainly due to the lack of a replication-independent system supporting the biogenesis of these membranous structures. Here we describe an expression-based, viral RNA replication-independent, DENV polyprotein system, designated as pIRO (plasmid-induced replication organelle), which is sufficient to induce VP formation. We show that VPs induced by pIRO expression are morphologically indistinguishable from those found in infected cells, suggesting that DENV replication organelle formation does not require RNA replication. We conclude that the pIRO system is a novel and valuable tool that can be used to dissect the mechanisms underlying DENV replication organelle formation.

scholarly journals The Putative Helicase of the Coronavirus Mouse Hepatitis Virus Is Processed from the Replicase Gene Polyprotein and Localizes in Complexes That Are Active in Viral RNA Synthesis

1999 ◽  
Vol 73 (8) ◽  
pp. 6862-6871 ◽  
Author(s):  
Mark R. Denison ◽  
Willy J. M. Spaan ◽  
Yvonne van der Meer ◽  
C. Anne Gibson ◽  
Amy C. Sims ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Helicase ◽  
Viral Rna ◽  
Replicase Gene ◽  
Reading Frame ◽  
N Protein ◽  
Amino Terminal ◽  
Infected Cells

ABSTRACT The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.

scholarly journals Localization of Mouse Hepatitis Virus Nonstructural Proteins and RNA Synthesis Indicates a Role for Late Endosomes in Viral Replication

1999 ◽  
Vol 73 (9) ◽  
pp. 7641-7657 ◽  
Author(s):  
Yvonne van der Meer ◽  
Eric J. Snijder ◽  
Jessika C. Dobbe ◽  
Sibylle Schleich ◽  
Mark R. Denison ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Multilayered Structures ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Infected Cells

ABSTRACT The aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (MHV). Antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3C-like protease (3CLpro), the putative polymerase (POL), helicase, and a recently described protein (p22) derived from the C terminus of the open reading frame 1a protein (CT1a), were used to probe MHV-infected cells by indirect immunofluorescence (IF) and electron microscopy (EM). At early times of infection, all of these proteins showed a distinct punctate labeling by IF. Antibodies to the nucleocapsid protein also displayed a punctate labeling that largely colocalized with the replicase proteins. When infected cells were metabolically labeled with 5-bromouridine 5′-triphosphate (BrUTP), the site of viral RNA synthesis was shown by IF to colocalize with CT1a and the 3CLpro. As shown by EM, CT1a localized to LAMP-1 positive late endosomes/lysosomes while POL accumulated predominantly in multilayered structures with the appearance of endocytic carrier vesicles. These latter structures were also labeled to some extent with both anti-CT1a and LAMP-1 antibodies and could be filled with fluid phase endocytic tracers. When EM was used to determine sites of BrUTP incorporation into viral RNA at early times of infection, the viral RNA localized to late endosomal membranes as well. These results demonstrate that MHV replication occurs on late endosomal membranes and that several nonstructural proteins derived from the gene 1 polyprotein may participate in the formation and function of the viral replication complexes.

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