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Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein
K Watanabe1, H Handa, K Mizumoto
1Tokyo Institute of Technology, Yokohama, Japan.
Abstract:
Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.
Insights
Influenza virus M1 protein inhibits viral RNA synthesis by binding to RNA and interfering with early transcription steps. A specific M1 region (residues 91-111) is crucial for this RNA polymerase inhibition.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Influenza virus M1 protein is known to inhibit viral RNA transcription.
- The precise mechanism of this inhibition requires further elucidation.
Purpose of the Study:
- To investigate the mechanism by which influenza virus M1 protein inhibits viral RNA synthesis.
- To identify the specific regions of M1 protein responsible for RNA binding and transcriptional inhibition.
Main Methods:
- Recombinant M1 protein purification from Escherichia coli.
- RNA mobility shift assays to assess M1-RNA binding.
- In vitro RNA synthesis assays using exogenous RNA templates.
- Analysis of M1 mutant proteins with amino acid deletions.
Main Results:
- Recombinant M1 protein binds to RNA, forming complexes that promote cooperative M1 binding.
- M1 protein binds to preformed RNA-nucleocapsid protein complexes.
- M1 inhibits viral RNA synthesis primarily during early transcription steps.
- The M1 region between amino acid residues 91 and 111 is essential for RNA binding, oligomerization, and anti-RNA synthesis activity.
Conclusions:
- Influenza virus M1 protein inhibits viral RNA polymerase activity by binding to RNA and interfering with early transcription.
- The M1 region spanning residues 91-111 plays a critical role in M1's RNA binding, oligomerization, and inhibitory function.