Abstract
We recently demonstrated that the Autographa californica multinucleocapsid nuclear polyhedrosis virus homologous region (hr1) enhances transcription from the viral polyhedrin promoter and also functions as a putative origin of replication (ori). Hr1, carrying five 28-base pair core palindrome units, has also been mapped with respect to its enhancer and ori functions (Habib, S., Pandey, S., Chatterji, U., Burma, S., Ahmad, R., Jain, A., and Hasnain, S. E. (1996) DNA Cell Biol. 15, 737-747). A 38-kDa host factor termed hr1-binding protein (hr1-BP) binds with high specificity and affinity (Kd approximately 6.5 x 10(-11) M) to functionally important motifs within hr1. The core palindrome as well as sequences immediately flanking it are required for this interaction. Divalent cations are not essential, and ionic interactions play only a minor role in complex formation. hr1-BP binds through the minor groove of the double helix to multiple sites within hr1, and binding occurs as a function of the number of modules within hr1. Phosphorylation of hr1-BP is important for host factor-hr1 interaction. Hr1-BP differs in several respects from the other host factor, polyhedrin promoter-binding protein, described previously (Burma, S., Mukherjee, B., Jain, A., Habib, S., and Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750-2757). When hr1-BP was sequestered out, in vivo, by a plasmid carrying hr1 alone, the hr1-mediated enhancement of reporter expression was abolished, demonstrating that the binding of hr1-BP may be crucial for the enhancer activity of the dual function hr1 element.
Highlights
The genome of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (MNPV)1 is a double-stranded, covalently closed, circular DNA molecule consisting largely of unique sequences
Hr1, located ϳ3.7 kilobase pairs upstream of the A. californica MNPV very late polyhedrin gene promoter, lies within an 880-bp ClaI fragment and contains five imperfect 28-bp palindromes (Fig. 1). hr1 has been demonstrated to be a putative origin of viral replication [5,6,7,8], and preliminary data have suggested its role as an enhancer of the immediate-early ie-n gene and the delayed-early 39K gene (9 –11)
We recently demonstrated that it functions as a position- and orientationindependent enhancer of A. californica MNPV very late polyhedrin gene transcription in transient expression assays and that this enhancement effect is independent of the ori function of hr1 [1]
Summary
The genome of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (MNPV) is a double-stranded, covalently closed, circular DNA molecule consisting largely of unique sequences. Eight homologous region sequences or hrs (hr, hr1a, hr, hr3, hr4a, hr4b, hr4c, and hr5) are distributed throughout the genome These hrs vary in length from 150 to 800 bp and contain two to eight imperfect, 30-bp palindromes with an EcoRI site at the center of each palindromic core. The. hrs have been demonstrated to be putative origins (oris) of viral DNA replication as well as enhancers of delayed-early gene transcription [3, 4]. We recently demonstrated that it functions as a position- and orientationindependent enhancer of A. californica MNPV very late polyhedrin gene transcription in transient expression assays and that this enhancement effect is independent of the ori function of hr1 [1]. The abolition of hr1-mediated enhancement of reporter gene expression in an in vivo transient expression assay by the presence of a competing hr sequence in trans points to the importance of this factor in hr function
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