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The segments of influenza viral mRNA
Abstract:
Influenza viral mRNA, i.e., complementary RNA (cRNA), isolated from infected cells , was resolved into six different species by electrophoresis in 2.1% acrylamide gels containing 6 M urea. The cRNA's were grouped into three size classes: L (large), M (medium-size), and S (small). Similarly, when gels were sliced for analysis, the virion RNA (vRNA) also distributed into six peaks because the three largest vRNA segments were closely spaced and were resolved only when the gels were autoradiographed or stained. Because of their attached polyadenylic acid [poly(A)]sequences, the cRNA segments migrated more slowly than did the corresponding vRNA segments during gel electrophoresis. After removal of the poly(A) by RNase H, the cRNA and vRNA segments comigrated, indicating that they were approximately the same size. One of the cRNA segments, S2, was shown by annealing to contain the genetic information in the vRNA segment with which it comigrated, strongly suggesting that each cRNA segment was transcribed from the vRNA segment of the same size. In contrast to the vRNA segments, which when isolated from virions were present in approximately 1:1 molar ratios, the segments of the isolated cRNA were present in unequal amounts, with the segments M2 and S2 predominating, suggesting that different amounts of the cRNA segments were synthesized in the infected cell. The predominant cRNA segments, M2 and S2, and also the S1 segment, were active as mRNA's in wheat germ extracts. The M2 cRNA was the mRNA for the nucleocapsid protein; S1 for the membrane protein; and S2 for the nonstructural protein NS1.
Insights
Influenza virus complementary RNA (cRNA) segments were isolated and characterized. Different amounts of cRNA segments were synthesized, with some segments functioning as messenger RNA (mRNA) to produce viral proteins.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Influenza viruses possess segmented RNA genomes, crucial for their replication and pathogenesis.
- Understanding the transcription and translation of viral RNA is fundamental to virology.
- Complementary RNA (cRNA) represents a key intermediate in viral RNA synthesis.
Purpose of the Study:
- To characterize the different species of influenza viral complementary RNA (cRNA) isolated from infected cells.
- To compare the size and abundance of cRNA segments with virion RNA (vRNA) segments.
- To investigate the translational activity of cRNA segments as messenger RNA (mRNA).
Main Methods:
- Electrophoresis of RNA in polyacrylamide gels containing urea to resolve different RNA species.
- RNase H treatment to remove polyadenylic acid [poly(A)] sequences from cRNA.
- RNA-RNA annealing assays to determine genetic relatedness between cRNA and vRNA segments.
- Wheat germ extract translation assays to assess mRNA activity of cRNA segments.
Main Results:
- Influenza cRNA resolved into six species grouped into L, M, and S size classes.
- cRNA segments migrated slower than vRNA due to poly(A) tails, but comigrated after poly(A) removal.
- Unequal molar ratios of cRNA segments were observed, with M2 and S2 predominating.
- Specific cRNA segments (M2, S2, S1) demonstrated mRNA activity, encoding nucleocapsid, nonstructural NS1, and membrane proteins, respectively.
Conclusions:
- Each cRNA segment is transcribed from a vRNA segment of the same size.
- Differential synthesis of cRNA segments occurs within infected cells.
- Certain cRNA segments function directly as mRNA, directing the synthesis of viral proteins.