Cloning of influenza cDNA ino M13: the sequence of the RNA segment encoding the A/PR/8/34 matrix protein

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Influenza viruses possess single-stranded RNA genomes, posing challenges for direct sequencing.
• Understanding viral gene sequences is crucial for developing antiviral strategies and vaccines.

Purpose of the Study:

• To establish a robust strategy for sequencing the RNA genes of influenza virus.
• To determine the complete nucleotide sequence of influenza virus segment 7.
• To identify the primary structure of the matrix protein and explore alternative reading frames.

Main Methods:

• Developed a sequencing strategy utilizing double-stranded complementary DNA (cDNA) copies of viral RNA.
• Cloned restriction fragments of cDNA into bacteriophage M13mp2 for dideoxy sequencing.
• Extended and overlapped sequences using virion RNA as a template and small restriction fragments as primers.

Main Results:

• Successfully sequenced the complete nucleotide sequence of influenza virus segment 7 (1027 nucleotides).
• Elucidated the primary structure of the matrix protein, with a calculated molecular weight of 27,861 daltons.
• Identified a second, partially overlapping long reading frame within segment 7.

Conclusions:

• The developed strategy is effective for sequencing influenza virus RNA genes.
• The complete sequence of segment 7 provides critical information about the matrix protein.
• The discovery of an overlapping reading frame suggests potential for novel viral protein functions.