The hr5 transcriptional enhancer stimulates early expression from the Autographa californica nuclear polyhedrosis virus genome but is not required for virus replication

Abstract

Autographa californica nuclear polyhedrosis virus (AcMNPV) contains five homologous regions (hrl through hr5) interspersed throughout its genome. Analysis of plasmid transfections indicates that the hrs function as transcriptional enhancers and possible origins of viral DNA replication. The role of these repetitive elements in regulating expression from the AcMNPV genome was examined by constructing a series of recombinant viruses that tested the effect of hr5 on expression of the adjacent p35 gene (p35). When embedded within the viral genome, hr5 stimulated transcription from the early p35 promoter in a position-and orientationindependent manner. Moreover, hr5 and the upstream activating region ofp35 were functionally interchangeable. A 28-bp imperfect palindrome, repeated six times within hr5, was the minimal sequence required forp35 promoter activation. hr5 also stimulated another early AcMNPV promoter but not a late promoter or a host-derived promoter, suggesting that enhancement is promoter specific during infection. To investigate its role during AcMNPV replication, hr5 was deleted from its normal position within the viral genome. The resulting hr5 mutants exhibited no apparent defects in replication, as judged by production of budded virus and levels of very late gene expression, even though steady-state levels of p35 RNA were reduced. These results indicated for the first time that hr5 functions as a transcriptional enhancer within the viral genome. However, the element is not required for AcMNPV replication in cultured cells. Thus, loss of one of five possible origins of DNA replication is not deleterious to viral growth. Since p26 was removed from the hr5 deletion mutants, this gene is also nonessential for viral replication.

Expression of PE38 and IE2, viral members of the C3HC4 finger family, during baculovirus infection: PE38 and IE2 localize to distinct nuclear regions

“…5). Previous studies have shown that the HRs stimulate expression of some early genes, even in uninfected S. frugiperda cells (26,28,34).…”

Precise excision of TTAA‐specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera

“…Insertion of the polhilac2 cartridge at the unique fstl site and subsequent homologous recombination at the FP locus generated the 5dl4(polh/lacZ) starting virus. the non-essential p26 gene (Rodems & Friesen, 1993;Fraser eta/., 1995). The insertion of the tagged element duplicated a TTAA target site present only once in the uninterrupted copy of the gene (Fraser eta/., 1995).…”

DNA-dependent transregulation by IE1 of Autographa californica nuclear polyhedrosis virus: IE1 domains required for transactivation and DNA binding

“…Current evidence indicates that immediate-early gene 1 (ie-1) is the principal transregulator of early gene expression by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The 67-kDa ie-1 gene product (IE1) is a potent transactivator of early viral genes in plasmid transfection assays (3,16,18,24,31,35,44). Moreover, ie-1 is required in plasmid replication assays and thus may contribute directly or indirectly to viral DNA replication (21,32).…”

“…As assayed by plasmid transfections, IE1-mediated transcriptional activation is significantly amplified when the affected promoter is cis linked to homologous region (hr) enhancer sequences from AcMNPV (3,15,17,31,35,39,44). Ranging in size from 30 to 800 bp, eight hr enhancer elements are dispersed throughout the circular AcMNPV genome (134 kbp) (1,8) and may function as origins of viral DNA replica-tion (22,28,36).…”

“…Ranging in size from 30 to 800 bp, eight hr enhancer elements are dispersed throughout the circular AcMNPV genome (134 kbp) (1,8) and may function as origins of viral DNA replica-tion (22,28,36). The 28-bp palindromic repeats (28-mer) comprising the hrs are the minimal sequence required for orientation-and position-independent enhancer activity (44,45) and hr-mediated plasmid DNA replication (28,36). IE1 interaction with the hrs is mediated in part by the palindromic 28-mers (6,13,14,26,45).…”