A Temperature-Sensitive IE1 Protein of Autographa californica Nuclear Polyhedrosis Virus Has Altered Transactivation and DNA Binding Activities

Abstract

The temperature-sensitive mutant tsB821 of Autographa californica nuclear polyhedrosis virus has two nucleotide transitions in the transregulatory ie 1 gene (Riblero et al. (1994) J. Virol. 68, 1075-1084). These mutations result in two conservative amino acid substitutions in the encoded protein. To determine whether the mutations affect the ability of IE1 to interact with viral enhancer elements, electrophoretic mobility shift assays were performed using extracts prepared from infected cells and a fragment of the viral enhancer element hr5. When binding reactions. Were assembled on ice, the extracts prepared from tsB821-infected cells showed wild-type levels of DNA binding activity, whether the infected cells were grown at the permissive or nonpermissive temperature. However, when binding reactions were incubated at 33°, enhancer binding activity was significantly reduced in the ts extract. This suggests that the ts phenotype results from reduced interactions of tsIE1 with the viral enhancer elements. Site-directed mutagenesis was used to construct plasmids encoding IE1 with both of the amino acid substitutions (tsIE1) and with each of the single substitutions. tsIE1 was expressed in transient assays and exhibited thermolabile enhancer binding activity. However, proteins with single substitutions did not accumulate to detectable levels and did not exhibit enhancer binding activity. Temperature shift-up experiments with tsB821 indicated that late genes were expressed at wt levels when cells were shifted to the nonpermissive temperature during the early phase. This suggests that IE1 is not directly and continuously required for expression of late genes. However, polyhedrin expression was decreased in the mutant-infected cells when the temperature was shifted during the early or late phases of infection. Together, these results suggest that IE1 may differentially affect expression of these two classes of genes.