Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver

Abstract

We found that livers from woodchucks chronically infected with woodchuck hepatitis virus (WHV) contained covalently closed circular DNA (cccDNA) molecules with deletions and insertions indicative of their formation from linear viral DNA by nonhomologous recombination, as we previously described for the duck hepatitis B virus (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). However, evidence for two different types of linear precursors was obtained by analysis of the recombination joints in WHV cccDNA. Type 1 linear precursors possessed the structural properties that correspond to those of in situ-primed linear DNA molecules, which constitute between 7 and 20% of all viral DNA replicative intermediates synthesized in the liver. Type 2 linear precursors are hypothetical species of linear DNAs with a terminal duplication of the cohesive-end region, between DR1 and DR2. This type of linear DNA has not been previously described and was not detected among the DNA species present in nucleocapsids. A fraction of cccDNAs formed from both type 1 and type 2 linear DNAs are predicted to be functional for further DNA synthesis, and some evidence for the formation of two or more generations of cccDNA from linear DNA was observed.