Synthesis of Full-length, Virus Genomic DNA by Nuclei of Vaccinia-infected HeLa Cells

Abstract

It is well established that vaccinia virus infection induces the synthesis of virus-specific DNA in cytoplasmic factories, which are sites of virus-specific transcription. The present study demonstrates that vaccinia virus-specific DNA is synthesized also in the nuclei of infected human cervical carcinoma HeLa cells with a similar time course. Direct observation and radiolabeling confirm the integrity of isolated nuclei. Reconstitution experiments and inhibitor studies demonstrate that virus-induced DNA is synthesized de novo within nuclei and does not result from cytoplasmic contamination. Cell-specific DNA synthesis is inhibited completely after infection and nuclei of infected cells then synthesize DNA which co-sediments with virus genomic DNA in denaturing gradients. Restriction endonuclease cleavage and hybridization with a virus-specific probe indicate that this is full-length, virus genomic DNA. The biological implications of this are discussed.