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Characterization of the mRNA of influenza virus
Abstract:
The kinetics of the appearance of influenza mRNA, the distribution of mRNA between free and membrane-associated polyribosomes, its poly(A) content, and the extent to which the genome was transcribed into mRNA early in infection were determined. Polyribosomes were prepared from influenza virus-infected cells labeled for 30-min periods at various times after infection with [3H]uridine. Most of the 3H-labeled RNA extracted from these polyribosomes sedimented as a heterogeneous 8S to 20S peak in sucrose gradients, and it was largely complementary to virion RNA. By the following criteria, the complementary RNA had properties normally ascribed to mRNA: (i) it labeled rapidly with [3H]uridine; (ii) after glutaraldelyde treatment, it banded with polyribosomes in CsCl density gradients; and (iii) it contained poly(A). In chick cells at 37 C, virus mRNA was first detectable at 45 min postinfection and reached its maximal rate of appearance at 2 to 2.5 h postinfection. The free and membrane-bound polyribosomes of infected cells were separated and were found to contain the same classes of mRNA. There was no absolute segregation of mRNA sequences into either polyribosome class although each probably contained distinct ratios of the different mRNA's. From 45 min postinfection onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80% of the genome RNA, whereas poly(A)-minus RNA contained sequences complementary to 90 to 100% of the genome. There was no evidence for the temporal control of transcription of influenza mRNA. At 31 C, when virus development was slowed relative to 37 C,complementary RNA first appeared at 1 h postinfection. At this time, total polysomal RNA contained sequences complementary to the whole genome.
Insights
Influenza virus mRNA synthesis begins 45 minutes post-infection, with both free and membrane-bound polyribosomes containing similar mRNA classes. Viral mRNA transcription shows no temporal control during early infection.
Area of Science:
- Virology
- Molecular Biology
- Gene Expression
Background:
- Influenza virus replication involves complex gene expression mechanisms.
- Understanding messenger RNA (mRNA) kinetics and localization is crucial for deciphering viral replication strategies.
Purpose of the Study:
- To investigate the kinetics of influenza mRNA appearance and its distribution within infected cells.
- To analyze the poly(A) content and transcriptional extent of viral mRNA early in infection.
Main Methods:
- Infected chick cells were labeled with [3H]uridine at various time points post-infection.
- Polyribosomes were isolated and separated into free and membrane-associated fractions.
- RNA was extracted and analyzed using sucrose gradient sedimentation, CsCl density gradients, and hybridization techniques.
Main Results:
- Influenza mRNA was first detected at 45 minutes post-infection at 37°C, with maximal appearance rate between 2-2.5 hours.
- Both free and membrane-bound polyribosomes contained similar mRNA classes, with no absolute segregation.
- Polyadenylated RNA contained sequences from at least 80% of the genome, while poly(A)-minus RNA represented 90-100%.
- No temporal control of influenza mRNA transcription was observed.
Conclusions:
- Influenza mRNA synthesis initiates relatively early in infection and is distributed across different polyribosome fractions.
- The findings suggest a comprehensive transcription of the viral genome into mRNA early in the infectious cycle.
- Viral mRNA production is not subject to strict temporal regulation during the initial stages of infection.