Herpes simplex virus defective genomes: structure of HSV-1 ANG defective DNA of class II and encoded polypeptides

Sequence organization and origin of HSV-1 strain Angelotti (ANG) class II defective DNA (HSV-1 ANG dDNA1) were examined in detail by establishing physical maps and by molecular cloning. dDNA1 consists of concatemers of tandem repeat units in which sequences from the UL region spanning map coordinates 0.37 to 0.415 of standard HSV ANG DNA are covalently linked to TRS/IRS sequences. The size of the repeat unit was determined to be about 8.9 kilobase pairs (kb), comprising sequences of 7.3 kb from UL and 1.6 kb from TRS/IRS regions. UL sequences were delineated by restriction enzyme sites KpnI N-P and EcoRI F-M, and were colinear with the corresponding sequences of the standard (wild-type) virus genome. Expression of dDNA1 was studied in African green monkey kidney cells and in Xenopus laevis oocytes. A major polypeptide of approx. mol. wt. 135 000 (135K) was overproduced, suggesting that this protein was encoded by dDNA1. By several parameters, e.g. size, immune cross-reactivity, and affinity for native and denatured DNA, the 135K polypeptide was identified as the major HSV DNA-binding protein. It was further shown that the repeat unit contains part of the DNA polymerase gene as demonstrated by its ability to rescue some mutations in this gene.