A TEMPERATURE-SENSITIVE IE1 PROTEIN OF AUTOGRAPHA-CALIFORNICA NUCLEARPOLYHEDROSIS-VIRUS HAS ALTERED TRANSACTIVATION AND DNA-BINDING ACTIVITIES

The temperature-sensitive mutant tsB821 of Autographa californica nucl ear polyhedrosis virus has two nucleotide transitions in the transregu latory ie 1 gene (Riblero at al. (1994) J. Virol. 68, 1075-1084). Thes e mutations result in two conservative amino acid substitutions in the encoded protein. To determine whether the mutations affect the abilit y of IE1 to interact with viral enhancer elements, electrophoretic mob ility shift assays were performed using extracts prepared from infecte d cells and a fragment of the viral enhancer element hr5. When binding reactions were assembled on ice, the extracts prepared from tsB821-in fected cells showed wild-type levels of DNA binding activity, whether the infected cells were grown at the permissive or nonpermissive tempe rature. However, when binding reactions were incubated at 33 degrees, 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 aci d substitutions (tsIE1) and with each of the single substitutions. tsI E1 was expressed in transient assays and exhibited thermolabile enhanc er binding activity. However, proteins with single substitutions did n ot accumulate to detectable levels and did not exhibit enhancer bindin g activity. Temperature shift-up experiments with tsB821 indicated tha t late genes were expressed al wt levels when cells were shifted to th e 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-infe cted cells when the temperature was shifted during the early or late p hases of infection. Together, these results suggest that IE1 may diffe rentially affect expression of these two classes of genes. (C) 1995 Ac ademic Press, Inc.