A mutant baculovirus with a temperature-sensitive IE-1 transregulatory protein.

Abstract

We have mapped the mutation responsible for the temperature-sensitive (ts) phenotype of tsB821, a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus (H. H. Lee and L. K. Miller, J. Virol. 31:240-252, 1979), to a single nucleotide which changes alanine 432 of the multifunctional regulatory protein IE-1 to a valine. Mapping was done with a combination of marker rescue and transient expression assays, hybrid gene construction by overlap PCR gene splicing, and nucleotide sequence analysis. Cells infected with tsB821 at high multiplicities of infection showed a spectrum of responses from severe cytopathic effects, including apoptosis, to a lack of obvious signs of infection. Protein synthesis in tsB821-infected cells at the restrictive temperature appeared similar to uninfected cell protein synthesis, but viral DNA replication and budded virus production were observed, albeit in a delayed manner. The dependence of early and late promoter activity on the wild-type IE-1 gene, ie-1, was observed in transient expression assays. However, the dependence of early promoter activity on ie-1 was strongest in the absence of other viral genes. Thus, other viral genes appear to be able to compensate, at least in part, for the lack, or low levels, of ie-1 in transient expression assays using early promoters. The mutant should prove useful in further defining the function(s) of IE-1.