Poxvirus DNA

Abstract

Abstract Vaccinia virions labeled with [3H]thymidine or [14C]thymidine were isolated and purified using two methods. In Method I, the virion preparations were sonicated during purification, banded repeatedly in sucrose gradients until their specific activity (cpm/μg viral DNA) was constant, and were frozen and thawed at least once before their DNA (DNA-I) was analyzed. In Method II virions were separated from other cellular constituents by a single banding in 20–40% linear sucrose gradients. The preparations were neither sonicated nor were they frozen before analysis of their DNA (DNA-II). DNA-I was released by solubilizing virions with detergents and 2-mercaptoethanol. In 5–20% neutral sucrose gradients DNA-I, released in this way, sedimented at about 72 S relative to Ad2 DNA. DNA-I denatured with alkali fragmented into three size classes of molecules, sedimenting at about 56–60, 45–51, and 27–32 S in alkaline sucrose gradients. Analysis of DNA-I purified by phenol extraction on hydroxyapatite or benzoylated napthoylated DEAE-cellulose (BND-cellulose) columns showed that while most of the sequences in DNA-I were base paired, DNA-I molecules also contained ssDNA or weakly hydrogen bonded regions which eluted from BND-cellulose with 2% caffeine and 0.1 N NaOH. DNA-II molecules released from virions by lysis with detergents and 2-mercaptoethanol sedimented at 68 S in neutral sucrose gradients relative to Ad2 DNA. After denaturation with alkali and analysis in alkaline sucrose gradients, DNA-II molecules sedimenting at 102–106 and 90–92 S, appropriate for closed ssDNA circular molecules and linear ssDNA molecules, respectively, with approximately twice the molecular weight expected for a single complimentary DNA strand from vaccinia genomes of MW 120–150 × 106 were detected. This result and other experimental data support the view that the DNA-II genome was cross-linked. Analysis of DNA-II preparations on hydroxyapatite and BND-cellulose columns demonstrated that 80% or more of the DNA-II genomes analyzed were completely double-stranded. Freezing and thawing or sonication of virions prepared by Method II resulted in alterations to the genomes so that they behaved upon sedimentation analysis like DNA-I genomes.