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Regulated M1 mRNA splicing in influenza virus-infected cells
J Valcárcel1, A Portela, J Ortín
1Centro Nacional de Biotechnologia (CSIC), Universidad Autónoma, Madrid, Spain.
Abstract:
Influenza virus RNA segment 7 generates three poly(A)+ RNAs, M1 mRNA, M2 mRNA and mRNA3, the last of which has almost no coding capacity; M2 mRNA and mRNA3 derive from M1 mRNA by removal of a single intron. The kinetics of M1 and M2 mRNA accumulation in the cytoplasm of productively infected cells were studied by means of a quantitative RNA protection assay; the ratio of M2 mRNA to M1 mRNA increased 2.7-fold during the course of infection. To analyse the basis for this change, the kinetics of M1 and M2 mRNA synthesis and nuclear accumulation, their stability and nucleocytoplasmic transport were studied. Under the experimental conditions used, the synthesis of segment 7-specific RNA showed a peak at 4 h post-infection and continued later at a slower rate. The half-lives of M1 and M2 mRNAs were indistinguishable (2.73 h for M1 mRNA and 2.70 h for M2 mRNA) and the kinetics of nucleocytoplasmic transport in vivo or in vitro showed no preference for either mRNA early or late in infection. Consequently, regulation at the level of mRNA splicing is proposed. Using the mRNA synthesis and stability data, a simulation was performed to predict the change in splicing efficiency required to account for the mRNA accumulation results. The best fit was obtained when splicing efficiency changed about 20 times during a period in which viral gene expression was maximal.
Insights
Influenza virus RNA segment 7 produces M1 and M2 mRNAs. Splicing efficiency significantly increases during infection, leading to a higher M2 mRNA to M1 mRNA ratio in the cytoplasm.
Area of Science:
- Molecular Virology
- Gene Expression Regulation
Background:
- Influenza virus RNA segment 7 encodes multiple related mRNAs.
- M2 mRNA and mRNA3 are derived from M1 mRNA via intron removal.
- Differential accumulation of M1 and M2 mRNAs occurs during infection.
Purpose of the Study:
- To investigate the regulatory mechanisms behind the changing ratio of M1 and M2 mRNAs in infected cells.
- To determine if changes in synthesis, stability, or transport explain M2 mRNA accumulation.
Main Methods:
- Quantitative RNA protection assay to measure mRNA levels.
- Analysis of mRNA synthesis, nuclear accumulation, stability, and nucleocytoplasmic transport.
- Computer simulation to model splicing efficiency changes.
Main Results:
- The ratio of M2 mRNA to M1 mRNA increased 2.7-fold during infection.
- M1 and M2 mRNAs exhibited similar half-lives and transport kinetics.
- Viral gene expression peaked at 4 hours post-infection.
Conclusions:
- Regulation occurs at the level of mRNA splicing.
- Splicing efficiency must increase approximately 20-fold to explain observed mRNA accumulation.
- This highlights post-transcriptional regulation in influenza virus replication.