Determination of the Dna Sequence of a Small Plaque Isolate of Polyoma Virus.

Abstract

Crawford Small Plaque (CSP) is a strain of polyoma virus able to induce high levels of Tumor Specific Transplantation Antigen (TSTA). Several well characterized strains of polyoma whose sequences are known do not induce TSTA. To determine the basis for this difference, and to examine relationships between CSP and other strains of polyoma virus, the nucleotide sequence of CSP was determined. The DNA sequence of the noncoding, early coding and 87% of the late coding regions of CSP was determined by cloning the DNA in M13 single str and vectors and using the Sanger or "dideoxy" method of DNA sequence determination. The DNA sequence determined was compared to that of polyoma strain 3 and was found to have 58 base substitutions and 3 insertions. The most striking feature of the CSP genome is a duplication of 44bp to the late side of the viral origin of DNA replication starting 32 nt before the PvuII cleavage site at 67.4 mu. This duplication occurs within sequences known to be important in both DNA replication and early protein expression. The 44 bp are included in sequences duplicated in polyoma mutants able to grow in the embryonal carcinoma cell line, PCC4-aza-1. CSP was also found to grow in PCC4-aza-1 cells. This ability may be related to the sequence duplication. Base substitutions in the early coding region result in 5 predicted amino acid changes each for large and middle T-antigens. Secondary structure predictions for the CSP proteins indicate these amino acid changes alter predicted protein structure only slightly. Eight of the ten amino acid substitutions occur in sequences shared by middle and large T-antigens between 88 and 96 mu. Any or all of the early region changes could be responsible for the ability of CSP to induce TSTA. Base substitutions in the late region result in 5 amino acid changes in the major capsid protein VP1 Those which result in significant charge or structure changes are in regions found to vary among other polyoma virus strains. The sequence of CSP indicates that a moderate amount of sequence variation can occur in some regions of the polyoma genome while still maintaining the wild phenotype.