Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Polyomavirus early proteins (large, middle, and small T antigens) play crucial roles in viral replication and cellular transformation.
• Previous studies established the middle T protein gene, but constructs for individual large and small T proteins were needed.

Purpose of the Study:

• To construct and characterize modified polyomavirus genomes encoding individual large and small T proteins.
• To assess the efficacy of these constructs and the previously described middle T protein gene using SV40-polyomavirus recombinants.
• To determine the cellular localization and relative synthesis rates of these polyomavirus early proteins.

Main Methods:

• Construction of modified polyomavirus genomes by exchanging restriction endonuclease fragments between cDNA and genomic DNA.
• Generation of simian virus 40 (SV40)-polyomavirus recombinants replacing the SV40 late region with modified polyomavirus early genes.
• Analysis of protein synthesis rates and cellular localization using infected CV-1 cells and immunofluorescence studies.

Main Results:

• Recombinant viruses successfully induced synthesis of only the expected polyomavirus early protein.
• Synthesis rates of large, middle, and small T proteins were approximately 1.5, 4.0, and 9.0 times that of SV40 large T protein, respectively.
• Deletion of introns did not impair mRNA biogenesis; however, a cryptic splice site led to an aberrant middle T protein form in one construct.

Conclusions:

• Modified polyomavirus genomes can be effectively used to express individual large and small T proteins.
• The deletion of introns is compatible with efficient mRNA biogenesis for these viral proteins.
• Cellular localization studies confirmed the distinct intracellular distribution of polyomavirus early proteins, with no direct correlation between middle T protein overproduction and tyrosine phosphokinase activity.