Complete Genomic Sequence of the Amsacta Moorei Entomopoxvirus: Analysis and Comparison with Other Poxviruses

Virology 274, 120–139 (2000) doi:10.1006/viro.2000.0449, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Complete Genomic Sequence of the Amsacta moorei Entomopoxvirus: Analysis and Comparison with Other Poxviruses Alison L. Bawden,* Kathryn J. Glassberg,* James Diggans,† Regina Shaw,† William Farmerie,† and Richard W. Moyer*,1 *Department of Molecular Genetics and Microbiology, and †Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610 Received April 20, 2000; returned to author for revision May 5, 2000; accepted June 2, 2000 The genome of the genus B entomopoxvirus from Amsacta moorei (AmEPV) was sequenced and found to contain 232,392 bases with 279 unique open reading frames (ORFs) of greater than 60 amino acids. The central core of the viral chromosome is flanked by 9.4-kb inverted terminal repeats (ITRs), each of which contains 13 ORFs, raising the total number of ORFs within the viral chromosome to 292. ORFs with no known homology to other poxvirus genes were shown to constitute 33.6% of the viral genome. Approximately 28.6% of the AmEPV genome encodes homologs of the mammalian poxvirus colinear core genes, which are found dispersed throughout the AmEPV chromosome. There is also no significant gene order conservation between AmEPV and the orthopteran genus B poxvirus of Melanoplus sanguinipes (MsEPV). Novel AmEPV genes include those encoding a putative ABC transporter and a Kunitz-motif protease inhibitor. The most unusual feature of the AmEPV genome relates to the viral encoded poly(A) polymerase. In all other poxviruses this heterodimeric enzyme consists of a single large and a single small subunit. However, AmEPV appears to encode one large and two distinct small poly(A) polymerase subunits. AmEPV is one of the few entomopoxviruses which can be grown and manipulated in cell culture. The complete genomic sequence of AmEPV paves the way for an understanding and comparison of the molecular properties and pathogenesis between the entomopoxviruses of insects and the more intensively studied vertebrate poxviruses. © 2000 Academic Press INTRODUCTION The Entomopoxviridae historically comprise three genera based on host insect and virion morphology (Table Poxviruses are widespread and infect a variety of 1). Virus genera are designated as A (coleopteran), B vertebrate and invertebrate hosts. Phylogenetically, Pox- viridae comprise two subfamilies, the Chordopoxviridae (lepidopteran and orthopteran), and C (dipteran) (Arif and and Entomopoxviridae, which infect vertebrates and in- Kurstak, 1991; Goodwin et al., 1991; Moyer, 1999). The vertebrates (insects), respectively. Complete Chordopox- EPV from Melanoplus sanguinipes (MsEPV), a grasshop- viridae genomic sequences have been derived from four per (orthopteran)-derived genus B virus, was the first of the eight different Chordopoxvirus (ChPV)2 genera: the sequenced EPV (Afonso et al., 1999). The complete orthopoxviruses vaccinia (Goebel et al., 1990; Antoine et genomic sequence of MsEPV was informative and illu- al., 1998) and variola (Massung et al., 1994; Shchelkunov minated both similarities and differences between the et al., 1995; Shchelkunov et al., 2000); the leporipoxvi- insect and vertebrate poxviruses (Afonso et al., 1999). ruses myxoma (Cameron et al., 1999) and Shope fibroma The 236-kb MsEPV genome contains a subset of genes virus (Willer et al., 1999); the avipoxvirus fowlpox (Afonso shared between all sequenced poxviruses and allowed et al., 2000); and the molluscipoxvirus molluscum conta- the concept of a common, universally shared genetic giosum (Senkevich et al., 1997). core of poxvirus genes to be defined. Poxvirus core genes included many of those associated with RNA transcription, posttranscriptional modification, DNA rep- 1 To whom correspondence and reprint requests should be ad- lication, and core structural proteins. As informative as dressed at University of Florida, College of Medicine, Department of the MsEPV sequence has been, full exploitation of the Molecular Genetics and Microbiology, P.O. Box 100266, Gainesville, FL information has been difficult because of the inability to 32610-0266. Fax: (352) 392-3133. E-mail: [email protected]. 2 Abbreviations used: AmEPV, Amsacta moorei entomopoxvirus; grow and readily manipulate MsEPV in vitro. However, CbEPV, Choristoneura biennis entomopoxvirus; CfEPV, Choristoneura Amsacta moorei entomopoxvirus (AmEPV) is easily fumiferana entompoxvirus; ChPV, chordopoxvirus; CPV, cowpoxvirus; grown in cell culture and has already been genetically EPV, entomopoxvirus; FPV, fowlpoxvirus; HaEPV, Heliothis armigera manipulated (Winter et al., 1995; Palmer et al., 1995; entomopoxvirus; MCV, molluscum contagiosum virus; MmEPV, Bawden et al., 2000). Melolontha melolontha entomopoxvirus; MsEPV, Melanoplus sanguinipes entomopoxvirus; MYX, myxoma virus; SFV, Shope fibroma virus; The natural host of AmEPV is the red hairy caterpillar VV, vaccinia virus. Amsacta moorei, and it has been reported to infect ag- 0042-6822/00 $35.00 Copyright © 2000 by Academic Press 120 All rights of reproduction in any form reserved. AmEPV SEQUENCE 121 TABLE 1 The Entomopoxvirus Genera a After Moyer, 1999. b Estimates for genera B and C from Esposito et al., 1995. riculturally important pests such as Estigmene acrea vary from genus to genus within the vertebrate poxvi- (Hall and Hink, 1990) and Lymantria dispar (A. Bawden, ruses, is almost universally absent in MsEPV, providing a B. Arif, and R.W. Moyer, unpublished results). In the insect clear indication that a different repertoire of genes is host, infection follows ingestion of the occluded virus required to neutralize the defense responses of insects. and digestion of the occlusion body in the alkaline mid- We report here the complete genomic sequence of a gut of the insect. Infection of larvae by AmEPV is rela- genus B entomopoxvirus, from Amsacta moorei, AmEPV. tively instar specific, but generalized in the sense that While MsEPV and AmEPV are both currently classified many cells and tissues become involved, including the as genus B entomopoxviruses, MsEPV is a virus of fat body, hemocytes, nerve cells, midgut cells, hypoder- grasshoppers and AmEPV infects only lepidopteran mis, tracheoblasts, and muscle cells (Bawden et al., hosts. Analysis of the AmEPV genomic sequence corrob- 2000). The infected larvae become uncoordinated and orates the concept of a conserved core of poxvirus lethargic, with loss of mobility during the later phases of genes. Like MsEPV, there is no evidence of a colinear infection, which precedes death. The presence of occlu- organization of the AmEPV core genes similar to that sion bodies containing viral particles within a matrix observed among the vertebrate poxviruses. Surprisingly, consisting primarily of spheroidin is typical (Arif and there is also a lack of significant colinearity between Kurstak, 1991). Dissemination of the occluded virus into MsEPV and AmEPV, despite their grouping as genus B the environment occurs through regurgitation, defeca- viruses, and a relatively low overall degree of identity tion, and ultimately disintegration of dead larvae (Good- between comparable genes. However, as will become win et al., 1991). Hence exploring both the molecular evident from the data presented in this paper, the taxon- properties in tissue culture and the pathogenesis of omy, at least for the genus B entomopoxviruses, is inad- poxviruses in insects is readily approachable with the equate and needs to be reassessed. use of AmEPV. The genes encoded by AmEPV can be broadly divided Differences in sequence between MsEPV and verte- into three groups: genes belonging to the core of con- brate poxviruses were equally informative. One of the served poxvirus genes shared by all poxviruses; a sec- most distinctive features noted in the MsEPV gene se- ond group of genes shared by AmEPV and other insect quence was confirmation of the prediction that ento- viruses (e.g., MsEPV); and a third group composed of mopoxviruses lack a common colinear arrangement of genes totally unique to AmEPV. We propose that the common genes identified and found in the vertebrate collections of genes shared by the two entomopoxvi- poxviruses (Hall and Moyer, 1991; Sriskantha et al., 1997). ruses, but which are absent from the vertebrate poxvi- It became clear from the MsEPV sequence that, whereas ruses, comprise those genes necessary for growth the core genes themselves were conserved among the within a generalized insect environment. The third group various viruses, the colinear organization was not main- of genes, those completely unique to AmEPV, and the tained. A second major difference relates to those re- corresponding unique genes of MsEPV are likely to be maining genes not considered members of the con- required for growth in the specific insect host. served poxvirus core. In the vertebrate poxviruses, many of these genes encode various viroceptors and virokines, RESULTS which function to deflect the vertebrate host immune Organization of the AmEPV genome defenses (Smith et al., 1997, 1998, 1999; Alcami et al., 1998; Nash et al., 1999; Everett and McFadden, 1999; The AmEPV genome consists of 232,392 bases, which Barry and McFadden, 1997, 1998; McFadden and Barry, is approximately equal to published or estimated ge- 1998; McFadden et al., 1998; McFadden and Kelvin, nome sizes for other insect poxviruses [MsEPV,

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