Functional and structural characterisation of AgMNPV ie1

BILEN MARCOS; PILLOFF MARCELA; BELAICH MARIANO; DA ROS VANINA GABRIELA; RODRIGUES JULIO; RIBEIRO BERGMAN MORAIS; ROMANOWSKY VICTOR; LOZANO MARIO ENRIQUE; GHIRINGHELLI DANIEL

VIRUS GENES

SPRINGER

Lugar: Berlin; Año: 2007 vol. 2007 p. 549 - 562

0920-8569

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computational analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana Defective NPV and Orgyia pseudotsugata MNPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, amplified fragments of different length obtained from the ie1 upstream activating region (UAR) were cloned upstream of the E. coli lacZ ORF. The different plasmids were constructed substituting the ie1 ORF for the E. coli lacZ ORF. These constructs were used for transfection of UFL-AG-286 insect cells, to assess the time course and levels of expression of the lacZ reporter gene using a B-galactosidase activity assay, and to evaluate the regulatory functions of IE1