Journal of General Virology (1992), 73, 1049-1056. Printed in GreatBritain 1049 The Anticarsia gemmatalis nuclear polyhedrosis virus polyhedrin gene region: sequence analysis, gene product and structural comparisons Paolo M. de A. Zanotto,lt Maria Jose A. Sampaio, 2 Dave W. Johnson, 1 Talles L. Rocha 2 and James E. Maruniak 1. 1Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32611, U.S.A. and 2EMBRAPA-EMPRASA Brasiliera de Pesquisa Agropecuaria, CENARGEN-Centro Nacional de Recursos Geneticos e Biotecnologia, Sain, Parque Rural C.P. 102372, CEP 70770 Brasilia, DF-Brazil The genomic region of the Anticarsia gemmatalis multiple nucleocapsid nuclear polyhedrosis virus (AgMNPV) strain 2D encoding the polyhedrin gene was cloned and mapped, and a 2085 bp SphI-Pstl fragment containing the gene was sequenced. The polyhedrin polypeptide of the parental isolate AgMNPV was manually sequenced, and the amino acid sequence obtained agreed with that deduced from the DNA coding region sequence. AgMNPV and Orgyia pseudotsugata MNPV (OpMNPV) are similar in terms of promoter structure and polyhedrin primary sequence, and the polyhedrin gene of both viruses is transcribed in the anti-clockwise direction in relation to their physical maps. The region upstream from the polyhedrin gene of AgMNPV, OpMNPV, Bombyx mori NPV and ,4utographa californica MNPV (AcMNPV) was compared and this showed that the open reading frame (ORF) common to all four viruses (ORF 5) has sequence homology with the AcMNPV 25K gene. The sequences between ORF 5 and the poly- hedrin gene were found to be variable among the polyhedrin gene loci compared. Additionally, con- served elements in the promoters of the very late genes encoding polyhedrin and granulin, and those encoding two pl0 proteins were found to share sequence homology and positional similarity with consensus regions in the conserved boxes A and C, responsible for binding transcription factors to eukaryotic 5S riboso- mal RNA genes, and to box C of tRNA genes. Introduction Baculoviruses are enveloped dsDNA viruses belonging to a large family of occluded viruses, the Baculoviridae, which are arthropod pathogens (Brown, 1986). The biology of baculoviruses has been reviewed elsewhere (Granados & Williams, 1986; Blissard & Rohrmann, 1990). The baculoviruses are being investigated owing to their potential for use as biological control agents and as vectors for expression of foreign genes in insect cells (Smith et al., 1983; Luckow & Summers, 1988), and because they constitute an important model for inverte- brate virology. The Anticarsia gemmatalis nuclear polyhedrosis virus (AgMNPV) is being used extensively for biological control of the velvetbean caterpillar A. gemmatalis (Hiibner) in Brazil (Moscardi & CorrSa Ferreira, 1985; Moscardi, 1989) and is under evaluation for use in the United States. The AgMNPV genome is estimated to t Present address: NERC Institute of Virologyand Environmental Microbiology,MansfieldRoad, Oxford OX1 3SR, U.K. 0001-0432© 1992SGM contain 133 kb, and several restriction enzyme maps have been reported (Johnson & Maruniak, 1989). Studies with genotypic variants plaque-purified from field isolates and host range variants, as well as studies of genomic organization are under way (Maruniak, 1989). AgMNPV replicates in Trichoplusia ni TN368 (Hink, 1970) and Spodoptera frugiperda IPLB-Sf21-AE cells (Vaughn et al., 1977), as well as in the A. gemmatalis UFL-AG-286 embryo-derived cell line (Sieburth & Maruniak, 1988). The relationship between AgMNPV and other baculoviruses has been investigated previously by means of DNA hybridization, which indicates that the AgMNPV genome has DNA sequence similarity to viruses related to Autographa californica MNPV (AcMNPV) (Smith & Summers, 1982; Johnson & Maruniak, 1989). We chose to study the AgMNPV polyhedrin gene and flanking regions in order to compare them to those of other baculoviruses for which molecular data on the polyhedrin gene region are available. The HindlII G fragment of 7196 bp, shown to hybridize with AcMNPV polyhedrin gene probes