Genetic analyses of temperature-sensitive mutations in baculovirus late expression factors.

Two temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were characterized with respect to late and very late gene expression. Cells infected with ts317 or ts538 at the nonpermissive temperature revealed slightly reduced intracellular levels of late structural gene products including gp64 envelope, vp39 capsid, and p6.9 DNA binding proteins. However, with the exception of gp64, there was a dramatic reduction in extracellular levels of the structural proteins, suggesting that assembly or transport of extracellular virions was impaired. Electron microscopy confirmed the dramatic effect the mutations exhibited on viral morphogenesis. The ts538 mutation was physically mapped to the 60-65 mu region and DNA sequence analysis identified a single nucleotide alteration converting Leu to Phe in amino acid 105 of the late expression factor called lef-4. The mutation was positively correlated with the temperature-sensitive phenotype by analysis of DNA from ts+ revertants generated by marker rescue. Transient expression assays confirmed that lef-4 was required for activation of the late vp39 gene in virus infected cells. The defect in activation of the ts317 and ts538 polyhedrin genes could be overcome by coinfection with helper virus indicating that the mutations affected functions involved in activating the very late class of baculovirus genes as well. These results demonstrated a clear functional differentiation between genes regulating viral DNA replication which was normal in the mutant infected cells and those regulating late gene expression and extracellular virus production which were defective in these same infected cells.