Viral interference of HSV-1: Properties of the intracellular viral progeny DNA

Abstract

Intracellular progeny DNA was isolated and characterized from cells infected with standard herpes simplex virus or from cells coinfected with standard virus and with a virus stock obtained by serial passages at high multiplicity of infection (HP virus). The latter was shown to contain an excess of variant virus particles interfering with the production of infectious progeny virus. The ratio of plaque-forming to interfering virus in the HP virus stock used in this study was determined to be 1 10 . In both infections similar amounts of unit length viral DNA were synthesized. Restriction endonuclease digestion of intracellular viral progeny DNA yielded fragment patterns showing that the majority of the DNA molecules present in standard and HP HSV DNA yield the same restriction fragments. That the typical end fragments could be demonstrated suggests a correct processing of concatemeric precursor DNA into HSV unit length DNA. Despite the obvious similarity of the two DNA species the infectivity in transfection assays of progeny DNA formed in HP virus-infected cells was by three log lower than that of standard DNA. As shown by controls involving cotransfections with standard viral DNA and heterologous DNA, this difference can be attributed to the presence in HP DNA of HSV DNA molecules that interfere with the plaque formation by standard DNA. An alteration in various steps of DNA processing such as DNA methylation and incorporation of ribonucleotides into the DNA was demonstrated not to correlate with interference. Both in HP and standard virus DNA preparations methylation was below the level of detection of 10 5-methylcytosine residues per unit length of viral DNA. Low values were also obtained for the uridine content of the DNA. Almost 100% of the radioactivity incorporated could be recovered as deoxyribonucleosides and less than 0.07% as uridine.