scholarly journals Four Proteins Processed from the Replicase Gene Polyprotein of Mouse Hepatitis Virus Colocalize in the Cell Periphery and Adjacent to Sites of Virion Assembly

2000 ◽  
Vol 74 (7) ◽  
pp. 3379-3387 ◽  
Author(s):  
Anne Gibson Bost ◽  
Robert H. Carnahan ◽  
Xiao Tao Lu ◽  
Mark R. Denison
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Protein Localization ◽  
Mouse Hepatitis Virus ◽  
Cell Periphery ◽  
Replicase Gene ◽  
Reading Frame ◽  
Virion Assembly ◽  
Amino Terminal ◽  
Infected Cells

ABSTRACT The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.

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 Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication

2004 ◽  
Vol 78 (11) ◽  
pp. 5957-5965 ◽  
Author(s):  
Mark R. Denison ◽  
Boyd Yount ◽  
Sarah M. Brockway ◽  
Rachel L. Graham ◽  
Amy C. Sims ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Wild Type ◽  
Virus Growth ◽  
Amino Terminal ◽  
Normal Expression ◽  
Infected Cells ◽  
Critical Residues

ABSTRACT The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1), 247Gly↓Val248, in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.

scholarly journals Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

1998 ◽  
Vol 72 (3) ◽  
pp. 2265-2271 ◽  
Author(s):  
Xiao Tao Lu ◽  
Amy C. Sims ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Reading Frame ◽  
Amino Terminal ◽  
Infected Cells ◽  
Carboxy Terminal ◽  
A Site

ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

scholarly journals Mouse Hepatitis Virus Replicase Proteins Associate with Two Distinct Populations of Intracellular Membranes

2000 ◽  
Vol 74 (12) ◽  
pp. 5647-5654 ◽  
Author(s):  
Amy C. Sims ◽  
Joachim Ostermann ◽  
Mark R. Denison
Keyword(s):  
High Speed ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Virus Assembly ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Replicase Gene ◽  
Electron Microscopic ◽  
Amino Terminal ◽  
Late Endosomes

ABSTRACT The coronavirus replicase gene (gene 1) is translated into two co-amino-terminal polyproteins that are proteolytically processed to yield more than 15 mature proteins. Several gene 1 proteins have been shown to localize at sites of viral RNA synthesis in the infected cell cytoplasm, notably on late endosomes at early times of infection. However, both immunofluorescence and electron microscopic studies have also detected gene 1 proteins at sites distinct from the putative sites of viral RNA synthesis or virus assembly. In this study, mouse hepatitis virus (MHV)-infected cells were fractionated and analyzed to determine if gene 1 proteins segregated to more than one membrane population. Following differential centrifugation of lysates of MHV-infected DBT cells, gene 1 proteins as well as the structural N and M proteins were detected almost exclusively in a high-speed small membrane pellet. Following fractionation of the small membrane pellet on an iodixanol density gradient, the gene 1 proteins p28 and helicase cofractionated with dense membranes (1.12 to 1.13 g/ml) that also contained peak concentrations of N. In contrast, p65 and p1a-22 were detected in a distinct population of less dense membranes (1.05 to 1.09 g/ml). Viral RNA was detected in membrane fractions containing helicase, p28, and N but not in the fractions containing p65 and p1a-22. LAMP-1, a marker for late endosomes and lysosomes, was detected in both membrane populations. These results demonstrate that multiple gene 1 proteins segregate into two biochemically distinct but tightly associated membrane populations and that only one of these populations appears to be a site for viral RNA synthesis. The results further suggest that p28 is a component of the viral replication complex whereas the gene 1 proteins p1a-22 and p65 may serve roles during infection that are distinct from viral RNA transcription or replication.

scholarly journals Coronavirus Transcription Early in Infection

1998 ◽  
Vol 72 (11) ◽  
pp. 8517-8524 ◽  
Author(s):  
Sungwhan An ◽  
Akihiko Maeda ◽  
Shinji Makino
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Replication ◽  
Intergenic Sequence ◽  
Constant Ratio ◽  
Negative Strand ◽  
Infected Cells ◽  
Accumulation Kinetics ◽  
Kinetics Of

ABSTRACT We studied the accumulation kinetics of murine coronavirus mouse hepatitis virus (MHV) RNAs early in infection by using cloned MHV defective interfering (DI) RNA that contained an intergenic sequence from which subgenomic DI RNA is synthesized in MHV-infected cells. Genomic DI RNA and subgenomic DI RNA accumulated at a constant ratio from 3 to 11 h postinfection (p.i.) in the cells infected with MHV-containing DI particles. Earlier, at 1 h p.i., this ratio was not constant; only genomic DI RNA accumulated, indicating that MHV RNA replication, but not MHV RNA transcription, was active during the first hour of MHV infection. Negative-strand genomic DI RNA and negative-strand subgenomic DI RNA were first detectable at 1 and 3 h p.i., respectively, and the amounts of both RNAs increased gradually until 6 h p.i. These data showed that at 2 h p.i., subgenomic DI RNA was undergoing synthesis in the cells in which negative-strand subgenomic DI RNA was undetectable. These data, therefore, signify that negative-strand genomic DI RNA, but not negative-strand subgenomic DI RNA, was an active template for subgenomic DI RNA synthesis early in infection.

scholarly journals RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

2002 ◽  
Vol 76 (8) ◽  
pp. 3697-3708 ◽  
Author(s):  
Rainer Gosert ◽  
Amornrat Kanjanahaluethai ◽  
Denise Egger ◽  
Kurt Bienz ◽  
Susan C. Baker
Keyword(s):  
Electron Microscopy ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Viral Rna ◽  
Double Membrane ◽  
Ultrastructural Studies ◽  
Infected Cells

ABSTRACT The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

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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