Regulation of Mouse Hepatitis Virus RNA synthesis by Heterogeneous Nuclear Ribonucleoprotein Al

ABSTRACT The 3′-untranslated region (3′-UTR) of mouse hepatitis virus (MHV) RNA regulates the replication of and transcription from the viral RNA. Several host cell proteins have previously been shown to interact with this regulatory region. By immunoprecipitation of UV-cross-linked cellular proteins and in vitro binding of the recombinant protein, we have identified the major RNA-binding protein species as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). A strong hnRNP A1-binding site was located 90 to 170 nucleotides from the 3′ end of MHV RNA, and a weak binding site was mapped at nucleotides 260 to 350 from the 3′ end. These binding sites are complementary to the sites on the negative-strand RNA that bind another cellular protein, polypyrimidine tract-binding protein (PTB). Mutations that affect PTB binding to the negative strand of the 3′-UTR also inhibited hnRNP A1 binding on the positive strand, indicating a possible relationship between these two proteins. Defective-interfering RNAs containing a mutated hnRNP A1-binding site have reduced RNA transcription and replication activities. Furthermore, hnRNP A1 and PTB, both of which also bind to the complementary strands at the 5′ end of MHV RNA, together mediate the formation of an RNP complex involving the 5′- and 3′-end fragments of MHV RNA in vitro. These studies suggest that hnRNP A1-PTB interactions provide a molecular mechanism for potential 5′-3′ cross talks in MHV RNA, which may be important for RNA replication and transcription.

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Negative Strand RNA Synthesis by Temperature-Sensitive Mutants of Mouse Hepatitis Virus

Advances in Experimental Medicine and Biology - Coronaviruses and Arteriviruses ◽

10.1007/978-1-4615-5331-1_27 ◽

1998 ◽

pp. 221-227 ◽

Cited By ~ 5

Author(s):

D. R. Younker ◽

S. G. Sawicki

Keyword(s):

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Temperature Sensitive ◽

Negative Strand ◽

Temperature Sensitive Mutants

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Genetics of mouse hepatitis virus transcription: Identification of cistrons which may function in positive and negative strand RNA synthesis

Virology ◽

10.1016/0042-6822(90)90529-z ◽

1990 ◽

Vol 177 (2) ◽

pp. 634-645 ◽

Cited By ~ 27

Author(s):

Mary C. Schaad ◽

Stephen A. Stohlman ◽

James Egbert ◽

Karen Lum ◽

Kaisong Fu ◽

...

Keyword(s):

Rna Synthesis ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Negative Strand ◽

Virus Transcription

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

Journal of Virology ◽

10.1128/jvi.78.11.5957-5965.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5957-5965 ◽

Cited By ~ 40

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.

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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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