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The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing
1Rutgers University, Department of Molecular Biology and Biochemistry, Piscataway, New Jersey 08855-1179.
Abstract:
We have shown previously that the influenza virus NS1 protein inhibits the nuclear export of mRNAs. Here we demonstrate that the NS1 protein also regulates another post-transcriptional step: It inhibits pre-mRNA splicing both in vivo and in vitro. The mode by which the NS1 protein inhibits pre-mRNA splicing is novel. The pre-mRNA forms spliceosomes, but subsequent catalytic steps in splicing are inhibited. Affinity selection experiments establish that the NS1 protein is associated with the spliceosomes that are formed. The RNA-binding domain of the NS1 protein is required for the inhibition of splicing and for the interaction of the protein with spliceosomes. Because the NS1 protein is associated with U6 snRNA in influenza virus-infected cells as well as in splicing extracts from uninfected cells, it is likely that the NS1 protein also inhibits pre-mRNA splicing in infected cells. Surprisingly, the splicing of the viral ns1 mRNA, the very mRNA that encodes the NS1 protein, was resistant to inhibition by the NS1 protein. This resistance is conferred by sequences in ns1 mRNA.
Insights
The influenza virus NS1 protein inhibits pre-messenger RNA (pre-mRNA) splicing by binding to spliceosomes. This novel mechanism affects viral gene expression, though the NS1 mRNA itself is resistant to inhibition.
Area of Science:
- Molecular Biology
- Virology
- Gene Expression Regulation
Background:
- The influenza virus NS1 protein is known to inhibit messenger RNA (mRNA) nuclear export.
- Post-transcriptional regulation plays a critical role in viral replication and host-pathogen interactions.
Purpose of the Study:
- To investigate the role of the influenza virus NS1 protein in pre-mRNA splicing.
- To elucidate the mechanism by which NS1 protein affects splicing and its implications for viral gene expression.
Main Methods:
- In vivo and in vitro splicing assays.
- Affinity selection experiments to identify protein-spliceosome interactions.
- Analysis of RNA-binding domain function.
- Investigation of NS1 protein association with U6 small nuclear RNA (snRNA).
Main Results:
- The NS1 protein inhibits pre-mRNA splicing both in vivo and in vitro.
- NS1 protein binds to spliceosomes, preventing catalytic splicing steps.
- The RNA-binding domain of NS1 is essential for splicing inhibition and spliceosome interaction.
- NS1 protein associates with U6 snRNA in infected cells and splicing extracts.
- Viral ns1 mRNA demonstrates resistance to NS1-mediated splicing inhibition due to specific mRNA sequences.
Conclusions:
- The influenza virus NS1 protein possesses a novel mechanism of inhibiting pre-mRNA splicing by binding to spliceosomes.
- This inhibition likely contributes to the regulation of viral gene expression in infected cells.
- The ns1 mRNA's resistance to inhibition suggests a self-regulatory mechanism within the virus.