THE AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS HOMOLOGOUS REGION 1A - IDENTICAL SEQUENCES ARE ESSENTIAL FOR DNA-REPLICATION ACTIVITYAND TRANSCRIPTIONAL ENHANCER FUNCTION

Hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring EcoRI site a t its core. Plasmid subclones of the hr1a-containing AcMNPV HindIII-N fragment were examined for their ability to replicate in virus-infecte d (Spodoptera frugiperda) Sf9 cells, and to stimulate transcription wh en linked in cis with a 39K gene promoter-beta-glucuronidase fusion an d cotransfected into cells along with a plasmid (ple-1) containing the gene encoding the trans-acting factor IE-1. Only those plasmids conta ining hr1a underwent infection-dependent replication and were able to stimulate transcription. Sequences mapping to the left of hr1a were re quired for maximal levels of replication. A single palindrome from hr1 a was sufficient for supporting plasmid replication and for stimulatin g transcription, although both activities were more efficient when bot h palindromes were present. Plasmids containing only one-half of a pal indrome or disruptions of the central EcoRI core either did not replic ate, or replicated very poorly, and did not exhibit enhanced transcrip tional activity from the 39K gene promoter. Gel retardation experiment s showed that labeled hr1a-containing DNA fragments had retarded migra tion after incubation with extracts from cells transfected with ple-1. Supershift experiments using polyclonal antibodies to IE-1 indicated that IE-1 is a component of the protein complex bound to hr1a. Fragmen ts containing disruptions of the EcoRI-core still bound IE-1, as shown by gel retardation assays, indicating that IE-1 binding alone is not sufficient to allow replication and transcriptional enhancement. (C) 1 995 Academic Press, Inc.