Expression of Murine Coronavirus Genes 1 and 7 is Sufficient for Viral RNA Synthesis (original) (raw)
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Two murine coronavirus genes suffice for viral RNA synthesis
Journal of virology, 1995
We identified two mouse hepatitis virus (MHV) genes that suffice for MHV RNA synthesis by using an MHV-JHM-derived defective interfering (DI) RNA, DIssA. DIssA is a naturally occurring self-replicating DI RNA with nearly intact genes 1 and 7. DIssA interferes with most MHV-JHM-specific RNA synthesis, except for synthesis of mRNA 7, which encodes N protein; mRNA 7 synthesis is not inhibited by DIssA. Coinfection of MHV-JHM containing DIssA DI particles and an MHV-A59 RNA- temperature-sensitive mutant followed by subsequent passage of virus at the permissive temperature resulted in elimination of most of the MHV-JHM helper virus. Analysis of intracellular RNAs at the nonpermissive temperature demonstrated efficient synthesis of DIssA and mRNA 7 but not of the helper virus mRNAs. Oligonucleotide fingerprinting analysis demonstrated that the structure of mRNA 7 was MHV-JHM specific and therefore must have been synthesized from the DIssA template RNA. Sequence analysis revealed that DIss...
Primary structure and translation of a defective interfering rna of murine coronavirus
Virology, 1988
An intracellular defective-interfering (DI) RNA, DlssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DlssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5' end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the S'end of the parental MHV genome. The DlssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DIinfected cells. Sequence comparison of DlssE and the corresponding parts of the parental virus genome revealed that DlssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5' end of DlssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.
Journal of virology, 1999
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 la...
Journal of Virology, 2011
The RNA replication and transcription complex of coronaviruses is associated with an elaborate reticulovesicular network (RVN) of modified endoplasmic reticulum. Using cycloheximide and puromycin, we have studied the effect of translation inhibition on the RNA synthesis of severe acute respiratory syndrome coronavirus and mouse hepatitis virus. Both inhibitors prevented the usual exponential increase in viral RNA synthesis, with immunofluorescence and electron microscopy indicating that RVN development came to a standstill. Nevertheless, limited RNA synthesis was supported, implying that continued translation is not an absolute requirement and suggesting a direct link between RVN formation and accumulation of coronavirus proteins.
Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins
PLOS Pathogens, 2005
The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (nonstructural proteins nsp1-nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12-nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II-VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicasetranscriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the nonpermissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive-and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved. Citation: Sawicki SG, Sawicki DL, Younker D, Meyer Y, Thiel V, et al. (2005) Functional and genetic analysis of coronavirus replicase-transcriptase proteins. PLoS Pathog 1(4): e39.
Virology, 1998
Seven to eight species of viral subgenomic mRNAs are produced in coronavirus-infected cells. These mRNAs are produced in different quantities, and their molar ratios remain constant during viral replication. We studied RNA elements that affect coronavirus transcription efficiency by characterizing a series of cloned coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNAs containing an inserted intergenic sequence, from which subgenomic DI RNA is transcribed in MHV-infected cells. Certain combinations of upstream and downstream flanking sequences of the intergenic sequence suppressed subgenomic DI RNA transcription, yet changing one of the flanking sequences to a different sequence eliminated transcription suppression. The suppressive effect of certain combinations of flanking sequences, but not all combinations, could be counteracted by altering the intergenic sequence. Thus, the combination of intergenic sequence and flanking sequence affected transcription efficiency. We also characterized another set of DI RNAs designed to clarify which transcription step determines the relative molar ratios of coronavirus mRNAs. Our study indicated that if subgenomic mRNAs were exclusively synthesized from negative-strand genomic RNA, then the relative molar ratios of coronavirus mRNAs were most likely determined after synthesis of the genomic-sized template RNA. If negative-strand subgenomic RNAs were templates for subgenomic mRNAs, then the relative molar ratios of coronavirus mRNAs probably were determined after synthesis of the genomic-sized template RNA used for subgenomic-sized RNA transcription but prior to the completion of the synthesis of subgenomic-sized RNAs containing the leader sequence. The relative molar ratios of coronavirus mRNAs, therefore, seem to have been established prior to a putative replicon-type amplification of subgenomic mRNAs.
Virus Research, 1998
The (−)-strand viral RNAs that result from after infection of cells with coronaviruses, which possess RNA genomes of message polarity, are genomic-sized and subgenomic-sized. Each of the (−)-strand subgenomic RNAs corresponds in size to each of the subgenomic mRNA species that are made in infected cells. We tested whether (−)-strand subgenomic RNAs might initially be synthesized from the input single-stranded (+)-strand genomic RNA prior to the production of subgenomic mRNAs. We used a mouse hepatitis virus (MHV) defective interfering (DI) RNA, from which subgenomic RNA was produced in DI RNA-replicating cells, because this DI RNA had a functional MHV intergenic region inserted in its interior. MHV samples containing the DI particles were irradiated with UV-light and then superinfected into cells that had been infected with MHV 4 h prior to superinfection. Northern blot analysis of intracellular RNAs that were extracted 3 h after superinfection showed that genomic DI RNA and subgenomic DI RNA had similar UV-target sizes, indicating that (−)-strand genomic DI RNA synthesis from input genomic DI RNA probably occurred prior to the subgenomic-size DI RNA synthesis. We discuss why, in the course of coronavirus transcription, (−)-strand genomic-length coronavirus RNA synthesis might occur before subgenomic-sized RNAs of either polarity are made.
Coronavirus Transcription Early in Infection
Journal of Virology, 1998
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, si...