The Autographa californica Nuclear Polyhedrosis Virus Homologous Region 1a: Identical Sequences Are Essential for DNA Replication Activity and Transcriptional Enhancer Function

Abstract

Hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring EcoRI site at its core. Plasmid subclones of the hr1a-containing AcMNPV HindIII-N fragment were examined for their ability to replicate in virus-infected (Spodoptera frugiperda) Sf9 cells, and to stimulate transcription when linked in cis with a 39K gene promoter-beta-glucuronidase fusion and cotransfected into cells along with a plasmid (ple-1) containing the gene encoding the trans-acting factor IE-1. Only those plasmids containing hr1a underwent infection-dependent replication and were able to stimulate transcription. Sequences mapping to the left of hr1a were required for maximal levels of replication. A single palindrome from hr1a was sufficient for supporting plasmid replication and for stimulating transcription, although both activities were more efficient when both palindromes were present. Plasmids containing only one-half of a palindrome or disruptions of the central EcoRI core either did not replicate, or replicated very poorly, and did not exhibit enhanced transcriptional activity from the 39K gene promoter. Gel retardation experiments showed that labeled hr1a-containing DNA fragments had retarded migration 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. Fragments 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.

Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143).

“…The most well-characterized baculovirus, Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV), has a circular dsDNA genome of 134 kb, which encodes approximately 150 genes (Ayres et al, 1994). It has eight homologous regions (hrs) which have been shown to act as early gene transcriptional enhancers (Guarino & Summers, 1986 ;Leisy et al, 1995 ;Rodems & Friesen, 1993 and may function as origins of DNA replication (Leisy et al, 1995 ;Leisy & Rohrmann, 1993 ;Pearson et al, 1992).…”

“…Six of these genes appear to be required for DNA replication and encode the following proteins : a DNA polymerase homologue (Tomalski et al, 1988) ; P143, a helicase homologue (Lu & Carstens, 1991) that is capable of binding dsDNA (Laufs et al, 1997) ; LEF-3, a homotrimeric ssDNA-binding (SSB) protein Hang et al, 1995) that appears to be involved in transporting P143 into the nucleus (Wu & Carstens, 1998) ; IE-1, a transcriptional activator (Guarino & Summers, 1987) which also binds to hrs (Choi & Guarino, 1995 ;Guarino & Dong, 1991 ;Kovacs et al, 1992 ;Leisy et al, 1995 ;Rodems & Friesen, 1995) ; and two proteins, LEF-1 (a putative primase) and LEF-2, which have been shown to interact with each other . The three gene products, which are stimulatory for DNA replication, include two additional transcriptional activators, IE-2 (Carson et al, 1991) and PE-38 (Krappa & Knebel-Mo$ rsdorf, 1991), and P35, which blocks apoptosis (Clem et al, 1991 ;Herschberger et al, 1992) and may therefore influence DNA replication in an indirect manner.…”

Specificity of multiple homologous genomic regions in Spodoptera exigua nucleopolyhedrovirus DNA replication.

“…AcMNPV IE1 binds as a dimer to the palindromic sequences of an hr (Rodems & Friesen, 1995). Interaction of IE1 with these sequences is essential for hrs to function as transcriptional enhancers (Leisy et al, 1995). The 28 bp core of the palindrome acts as ori, whereas additional flanking sequences are required for enhancer activity (Leisy et al, 1995 ;Habib et al, 1996).…”

Recent Advances in Our Knowledge of Baculovirus Molecular Biology and Its Relevance for the Registration of Baculovirus-Based Products for Insect Pest Population Control

“…Six AcMNPV genes were found to be essential for DNA replication in transient DNA replication assays (Kool et al, 1994;Lu & Miller, 1995a). The genes directly involved in DNA replication (reviewed in Rohrman, 2011) encode for a homologous-region (hr) binding protein and transcriptional activator (ie-1) (Leisy et al, 1995;Rodems & Friesen., 1995), a single-stranded DNA binding protein (lef-3) (Hang et al, 1995), DNA binding helicase (p143) (McDougal & Guarino, 2000), a putative primase (lef-1), a primase-associated protein (lef-2) (Mikhailov & Rohrmann, 2002) and a DNA polymerase (dnapol) (Hang & Guarino, 1999;McDougal & Guarino, 1999). Four of these genes, dnapol, p143, lef-1, and lef-2 are core genes found in all baculoviruses (Okano et al, 2006).…”