Abstract
A previous report (P. Mavromara-Nazos and B. Roizman, Proc. Natl. Acad. Sci. USA 86:4071-4075, 1989) demonstrated that substitution of sequences of the thymidine kinase (tk) gene, a beta gene, extending from -16 to +51 with sequences extending from -12 to +104 of the gamma 2 UL 49.5 gene in viral recombinant R3820 conferred upon the chimeric gene gamma 2 attributes in the context of the viral genome in a productive infection. The UL49.5 gene sequences extending from -179 to +104 contain four DNA binding sites for the major regulatory protein ICP4. Of these sites, two map between nucleotides +20 and +80 within the sequence which confers gamma 2 regulation upon the chimeric gene. To determine the role of these ICP4 binding sites in conferring the gamma 2 gene attributes, sequences comprising the two ICP4 binding sites were mutagenized and used to reconstruct the R3820 recombinant virus. In addition, a new recombinant virus (R8023) was constructed in which tk sequences extending from -240 to +51 were replaced with wild-type or mutated sequences contained between nucleotides -179 to +104 of the UL 49.5 gene. Vero cells infected with the recombinant viruses in the presence or absence of phosphonoacetate, a specific inhibitor of viral DNA synthesis, were then tested for accumulation of tk RNA by using an RNase protection assay. The results indicate that in the recombinant R3820, a mutation which destroyed one of the two UL49.5 ICP4 DNA binding sites significantly reduced the accumulation of tk RNA at both early and late times after infection. The effect of this mutation was less pronounced in cells infected with the R8023 virus, whose chimeric tk gene contains the two upstream UL49.5 ICP4 binding sites. None of the mutations affected the sensitivity of the chimeric genes to phosphonoacetate. The mutated site appears to be involved in the accumulation of RNA.