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A murine coronavirus MHV-S isolate from persistently infected cells has a leader and two consensus sequences between
Abstract:
A plaque-cloned mouse hepatitis virus mutant, MHV-S No. 8, was isolated from Ki-BALB cells persistently infected with MHV-S. The mRNAs 1 to 6 were larger in the mutant, whereas there was no difference between the two viruses in the size of the smallest mRNA, mRNA 7. Sequence analyses of the genomic RNA, mRNA 6, and mRNA 7 of the two viruses revealed that an additional 111 nt were inserted just upstream of the intergenic consensus sequence preceding the N gene in MHV-S No. 8. The inserted region consisted of two different parts; the 3'-most 30 nt corresponded to nucleotides 28 to 57 of the leader sequence and the 5'-most 81 nt corresponded to nucleotides 58 to 138 of mRNA 7. This structure of No. 8 was most likely generated by RNA-RNA recombination between genomic RNA and subgenomic RNA species. The nucleotide insertion in the intergenic sequence between genes M and N resulted in two consensus sequences separated by 111 nt. Primer extension analysis revealed that the amount of a slightly larger, subgenomic mRNA resulting from initiation of synthesis at the upstream consensus sequence was only 5% of the usual sized mRNA 7 initiated from the downstream consensus sequence.
Insights
A novel mouse hepatitis virus (MHV-S) mutant, MHV-S No. 8, acquired an 111-nucleotide insertion via RNA-RNA recombination. This genetic alteration impacts viral gene expression, affecting mRNA sizes and transcription efficiency.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Persistent infections with mouse hepatitis virus (MHV-S) can lead to the emergence of viral mutants.
- Understanding the genetic basis of viral adaptation is crucial for controlling infectious diseases.
Purpose of the Study:
- To characterize a plaque-cloned MHV-S mutant, MHV-S No. 8, isolated from persistently infected cells.
- To elucidate the molecular mechanisms underlying the observed changes in viral RNA and gene expression.
Main Methods:
- Isolation and characterization of MHV-S No. 8 from infected Ki-BALB cells.
- Comparative sequence analysis of genomic RNA and subgenomic mRNAs (mRNAs 1-7) between wild-type MHV-S and the mutant.
- RNA-RNA recombination analysis to determine the origin of the inserted sequence.
- Primer extension analysis to quantify mRNA transcription from alternative initiation sites.
Main Results:
- MHV-S No. 8 exhibited larger mRNAs 1-6 compared to wild-type MHV-S, while mRNA 7 size remained unchanged.
- A 111-nucleotide insertion was identified upstream of the N gene's intergenic consensus sequence in MHV-S No. 8.
- Sequence analysis revealed the insertion comprised leader sequence and mRNA 7-derived elements, suggesting RNA-RNA recombination.
- The insertion created two consensus sequences, leading to reduced transcription (5%) from the upstream site for mRNA 7.
Conclusions:
- The MHV-S No. 8 mutant arose from RNA-RNA recombination between genomic and subgenomic RNAs.
- The nucleotide insertion significantly altered intergenic sequences, impacting transcription initiation and efficiency of subgenomic mRNA synthesis.
- This study provides insights into viral evolution and the generation of genetic diversity in coronaviruses.