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Molecular cloning and sequencing of influenza virus A/Victoria/3/75 polymerase genes: sequence evolution and
S de la Luna1, C Martínez, J Ortín
1Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Abstract:
The influenza virus A/Victoria/3/75 (H3N2) polymerase genes encoding PB1, PB2 and PA have been cloned by cDNA synthesis and insertion into bacterial vectors. The complete sequence for each polymerase gene has been obtained from random M13 subclones and compared to other influenza virus polymerase genes. A total of 45, 74 and 78 nucleotide changes were fixed in the period 1968-1975, corresponding to 10, 12 and 9 amino acid changes, for PB1, PB2 and PA genes, respectively. The amino acid sequence of PB1 polypeptide contains motifs found in a series of positive- and negative-RNA virus polymerase genes and that of PA polypeptide share invariant residues common to DNA and presumptive RNA helicases.
Insights
Researchers cloned and sequenced influenza A virus (H3N2) polymerase genes (PB1, PB2, PA). Analysis revealed nucleotide and amino acid changes between 1968-1975, with conserved motifs in PB1 and PA sequences.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Influenza A virus polymerase complex is essential for viral replication.
- Understanding polymerase gene evolution provides insights into viral adaptation and pathogenicity.
- The H3N2 subtype has undergone significant evolution.
Purpose of the Study:
- To clone and sequence the PB1, PB2, and PA polymerase genes of A/Victoria/3/75 (H3N2) influenza virus.
- To compare these sequences with other influenza virus polymerase genes.
- To identify evolutionary changes and conserved functional motifs within the polymerase proteins.
Main Methods:
- Complementary DNA (cDNA) synthesis from viral RNA.
- Cloning of polymerase genes into bacterial vectors.
- Random M13 subcloning for complete gene sequencing.
- Bioinformatic analysis and comparison of nucleotide and amino acid sequences.
Main Results:
- Complete nucleotide sequences for PB1, PB2, and PA genes were obtained.
- Significant nucleotide (45, 74, 78) and amino acid (10, 12, 9) changes were identified between 1968 and 1975 for PB1, PB2, and PA, respectively.
- The PB1 polypeptide sequence exhibited motifs common to other RNA virus polymerases.
- The PA polypeptide sequence shared invariant residues with DNA and RNA helicases.
Conclusions:
- The study provides detailed sequence information for key influenza A virus polymerase genes.
- Evolutionary analysis reveals accumulated genetic changes in H3N2 polymerase during a specific period.
- Conserved motifs suggest functional importance and potential roles in viral replication and helicase activity.