Synthesis of herpes simplex virus type 1 (HSV-1) DNA in isolated nuclei. II. Covalent linkage of RNA to nascent viral DNA.

Abstract

Nascent HSV-1 DNA synthesized in vitro by isolated nuclei of infected rat embryo fibroblasts was found to be covalently linked to stretches of ribonucleotides. The nature of ribo- and deoxyribonucleotides at the RNA-DNA joint was investigated by assaying for the transfer of [32P]-radioisotope from an [α-32P]-deoxyribonucleotide to a 2’(3’)-ribonucleotide after alkali hydrolysis of the nascent HSV-1 DNA. All four common ribo-and deoxyribonucleotides were present at the RNA-DNA junction. To provide additional proof that the RNA pieces were linked to the newly synthesized DNA by a phosphodiester bond, the viral DNA was synthesized in the presence of labeled substrates for both DNA (α-32P-dCTP) and RNA (14C-GTP and14C-CTP). Analysis of the native and heat-denatured viral DNA-RNA copolymers by equilibrium centrifugation in CS2SO4 density gradients indicated that the [14C]-RNA did not dissociate upon denaturation and banded with the [³²P]-DNA.