Primary structure and translation of a defective interfering rna of murine coronavirus (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...
Virology, 2001
Expression of negative-strand murine coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNA transcripts in MHV-infected cells results in the accumulation of positive-strand DI RNAs (M. Joo et al., 1996, J. Virol. 70, 5769-5776). However, the expressed negative-strand DI RNA transcripts are poor templates for positive-strand DI RNA synthesis. The present study demonstrated that DI RNA accumulation from the expressed negative-strand DI RNA transcripts in MHVinfected cells was enhanced by the coexpression of complementary RNA transcripts that correspond to the 5Ј region of positive-strand DI RNA. The positive-strand RNA transcripts corresponding to the 5Ј end-most 0.7-2.0 kb DI RNA had a similar enhancement effect. The coexpressed positive-strand RNA transcripts lacking the leader sequence or those containing only the leader sequence failed to demonstrate this enhancement effect, demonstrating that the presence of the leader sequence in the coexpressed positive-strand RNA transcripts was necessary, but not sufficient, for the enhancement of DI RNA accumulation from the coexpressed negative-strand DI RNA transcripts. Negative-strand DI RNA transcripts that were coexpressed with the partial-length positive-strand RNA transcripts were no more stable than those expressed alone, suggesting that a higher stability of the expressed negative-strand RNA transcripts was an unlikely reason for the higher DI RNA accumulation in cells coexpressing two complementary DI RNA transcripts. Sequence analyses unexpectedly demonstrated that the leader sequence of the majority of accumulated DI RNAs switched to helper virus derived leader sequence, suggesting that enhancement of DI RNA accumulation was mediated by the efficient utilization of helper virus derived leader sequence for DI RNA synthesis. Furthermore, our data suggested that this leader switching, a type of homologous RNA-RNA recombination, occurred during positive-strand DI RNA synthesis and that MHV positive-strand RNA synthesis mechanism may have a preference toward recognizing double-stranded RNA structures over single-stranded negative-strand RNA to produce positive-strand DI RNAs.
A cis-acting function for the coronavirus leader in defective interfering RNA replication
Journal of Virology, 1994
To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of termi...
Coronavirus Transcription Mediated by Sequences Flanking the Transcription Consensus Sequence
Virology, 1996
In our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. Using our subgenomic DI system, we have studied the effects of sequences flanking the MHV transcription consensus sequence on subgenomic RNA transcription. We obtained the following results. (i) Insertion of a 12-nucleotide-long sequence including the UCUAAAC transcription consensus sequence at different locations of the DI RNA resulted in different efficiencies of subgenomic DI RNA synthesis. (ii) Differences in the amount of subgenomic DI RNA were defined by the sequences that flanked the 12-nucleotide-long sequence and were not affected by the location of the 12-nucleotide-long sequence on the DI RNA. (iii) Naturally occurring flanking sequences of intergenic sequences at gene 6-7, but not at genes 1-2 and 2-3, contained a transcription suppressive element(s). (iv) Each of three naturally occurring flanking sequences of an MHV genomic cryptic transcription consensus sequence from MHV gene 1 also contained a transcription suppressive element(s). These data showed that sequences flanking the transcription consensus sequence affected MHV transcription. ᭧ quence with a single-nucleotide mutation in the middle ences, Kyung-Hee University, Seoul, Korea. of the 0.3-kb-long intergenic region between genes 6 and 2 To whom reprint requests should be addressed. Fax: (512) 471-7088.
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.
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...
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...
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.