scholarly journals Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection

2000 ◽  
Vol 74 (9) ◽  
pp. 4039-4046 ◽  
Author(s):  
Ralph S. Baric ◽  
Boyd Yount
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Full Length ◽  
Host Cells ◽  
Positive Strand ◽  
Negative Strand ◽  
Molar Ratios ◽  
Infected Cells ◽  
And Function ◽  
Hepatitis Virus Infection

ABSTRACT Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenomic-length positive- and negative-strand RNAs. The origin and function of the subgenomic negative-strand RNAs is controversial. In this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these RNAs function as important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of positive-strand synthesis. Using cycloheximide, which preferentially inhibits negative-strand and to a lesser extent positive-strand synthesis, we demonstrate that cycloheximide treatment equally inhibits full-length and subgenomic-length negative-strand synthesis. Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.

scholarly journals Characterization of an Autonomous Subgenomic Pestivirus RNA Replicon

1998 ◽  
Vol 72 (3) ◽  
pp. 2364-2372 ◽  
Author(s):  
Sven-Erik Behrens ◽  
Claus W. Grassmann ◽  
Heinz-Jürgen Thiel ◽  
Gregor Meyers ◽  
Norbert Tautz
Keyword(s):  
Mammalian Cells ◽  
Rna Replication ◽  
Full Length ◽  
Host Cells ◽  
Rnase Protection ◽  
Positive Strand ◽  
Negative Strand ◽  
Diarrhea Virus ◽  
Positive Strand Rna

ABSTRACT As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of theFlaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5′ and 3′ untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3′ end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5′ third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5′ end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

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.

Mouse Hepatitis Virus Infection Suppresses Modulation of Mouse Spleen T- Cell Activation

1988 ◽  
Author(s):  
Joan M. Cook-Mills ◽  
Hidayatulla G. Munshi ◽  
Robert L. Perlman ◽  
Donald A. Chambers
Keyword(s):  
T Cell ◽  
Virus Infection ◽  
T Cell Activation ◽  
Cell Activation ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Mouse Spleen ◽  
Hepatitis Virus Infection

The Role of B Cells in Mouse Hepatitis Virus Infection and Pathology

2001 ◽  
pp. 363-368 ◽  
Author(s):  
Amy E. Matthews ◽  
Susan R. Weiss ◽  
Ehud Lavi ◽  
Mark Shlomchik ◽  
Yvonne Paterson
Keyword(s):  
B Cells ◽  
Virus Infection ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Hepatitis Virus Infection

scholarly journals Characterization of the Coronavirus Mouse Hepatitis Virus Strain A59 Small Membrane Protein E

2000 ◽  
Vol 74 (5) ◽  
pp. 2333-2342 ◽  
Author(s):  
Martin J. B. Raamsman ◽  
Jacomine Krijnse Locker ◽  
Alphons de Hooge ◽  
Antoine A. F. de Vries ◽  
Gareth Griffiths ◽  
...  
Keyword(s):  
Virus Strain ◽  
Hepatitis Virus ◽  
Expression System ◽  
Mouse Hepatitis Virus ◽  
Viral Envelope ◽  
Membrane Structures ◽  
Electron Microscopic ◽  
E Protein ◽  
Infected Cells ◽  
Virus Expression

ABSTRACT The small envelope (E) protein has recently been shown to play an essential role in the assembly of coronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E protein and the membrane (M) protein are required. Since little is known about this generally low-abundance virion component, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residue polypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, we found that the E protein was synthesized in infected cells with similar kinetics as the other viral structural proteins. The protein appeared to be quite stable both during infection and when expressed individually using a vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was found to become integrated in membranes without involvement of a cleaved signal peptide, nor were any other modifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E protein demonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of the protein could not be detected on the outside of virions but appeared to be inside, where it was protected from proteolytic degradation. The results lead to a topological model in which the polypeptide is buried within the membrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain. Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced the formation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently consisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies to E and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E protein accumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shown earlier to assemble by budding.

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