Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis.

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.

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SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

Journal of Virology ◽

10.1128/jvi.78.23.13153-13162.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13153-13162 ◽

Cited By ~ 37

Author(s):

Keum S. Choi ◽

Akihiro Mizutani ◽

Michael M. C. Lai

Keyword(s):

Mammalian Cells ◽

Rna Synthesis ◽

Rna Binding ◽

Hepatitis Virus ◽

Affinity Purification ◽

Mouse Hepatitis Virus ◽

Viral Rna ◽

Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

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Polypyrimidine Tract-Binding Protein Binds to the Complementary Strand of the Mouse Hepatitis Virus 3′ Untranslated Region, Thereby Altering RNA Conformation

Journal of Virology ◽

10.1128/jvi.73.11.9110-9116.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9110-9116 ◽

Cited By ~ 45

Author(s):

Peiyong Huang ◽

Michael M. C. Lai

Keyword(s):

Conformational Change ◽

Viral Genome ◽

Untranslated Region ◽

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Complementary Strand ◽

Polypyrimidine Tract ◽

Mrna Transcription ◽

Polypyrimidine Tract Binding Protein

ABSTRACT Mouse hepatitis virus (MHV) RNA transcription is regulated mainly by the leader and intergenic (IG) sequences. However, a previous study has shown that the 3′ untranslated region (3′-UTR) of the viral genome is also required for subgenomic mRNA transcription; deletion of nucleotides (nt) 270 to 305 from the 3′-UTR completely abolished subgenomic mRNA transcription without affecting minus-strand RNA synthesis (Y.-J. Lin, X. Zhang, R.-C. Wu, and M. M. C. Lai, J. Virol. 70:7236–7240, 1996), suggesting that the 3′-UTR affects positive-strand RNA synthesis. In this study, by UV-cross-linking experiments, we found that several cellular proteins bind specifically to the minus-strand 350 nucleotides complementary to the 3′-UTR of the viral genome. The major protein species, p55, was identified as the polypyrimidine tract-binding protein (PTB, also known as heterogeneous nuclear RNP I) by immunoprecipitation of the UV-cross-linked protein and binding of the recombinant PTB. A strong PTB-binding site was mapped to nt 53 to 149, and another weak binding site was mapped to nt 270 to 307 on the complementary strand of the 3′-UTR (c3′-UTR). Partial substitutions of the PTB-binding nucleotides reduced PTB binding in vitro. Furthermore, defective interfering (DI) RNAs harboring these mutations showed a substantially reduced ability to synthesize subgenomic mRNA. By enzymatic and chemical probing, we found that PTB binding to nt 53 to 149 caused a conformational change in the neighboring RNA region. Partial deletions within the PTB-binding sequence completely abolished the PTB-induced conformational change in the mutant RNA even when the RNA retained partial PTB-binding activity. Correspondingly, the MHV DI RNAs containing these deletions completely lost their ability to transcribe mRNAs. Thus, the conformational change in the c3′-UTR caused by PTB binding may play a role in mRNA transcription.

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Mouse Hepatitis Virus Stem-Loop 4 Functions as a Spacer Element Required To Drive Subgenomic RNA Synthesis

Journal of Virology ◽

10.1128/jvi.05092-11 ◽

2011 ◽

Vol 85 (17) ◽

pp. 9199-9209 ◽

Cited By ~ 18

Author(s):

D. Yang ◽

P. Liu ◽

D. P. Giedroc ◽

J. Leibowitz

Keyword(s):

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Stem Loop ◽

Subgenomic Rna

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

Journal of Virology ◽

10.1128/jvi.74.12.5647-5654.2000 ◽

2000 ◽

Vol 74 (12) ◽

pp. 5647-5654 ◽

Cited By ~ 57

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.

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

Journal of Virology ◽

10.1128/jvi.74.7.3379-3387.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3379-3387 ◽

Cited By ~ 91

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.

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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

Journal of Virology ◽

10.1128/jvi.73.8.6862-6871.1999 ◽

1999 ◽

Vol 73 (8) ◽

pp. 6862-6871 ◽

Cited By ~ 74

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.

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