David R. O'Reilly

Use of Whole Genome Sequence Data To Infer Baculovirus Phylogeny

ABSTRACT Several phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus (NPV) andGranulovirus (GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

Ancient Coevolution of Baculoviruses and Their Insect Hosts

ABSTRACT If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order of their hosts. This division highlighted the need to reconsider the classification of the baculoviruses to include one or possibly two new genera. Furthermore, the specialization of distinct virus lineages to particular insect orders suggests ancient coevolutionary interactions between baculoviruses and their hosts.

Baculovirus-encoded ecdysteroid UDP-glucosyltransferases

Abstract This review summarizes recent data on the regulation of host development by insect baculoviruses through the action of the viral egt gene. This gene was first identified in the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) and encodes the enzyme ecdysteroid UDP-glucosyl-transferase (EGT), which catalyses the conjugation of ecdysteroids with glucose or galactose. Egt genes have now been identified in 6 baculoviruses other than AcMNPV; the encoded proteins all display significant homology throughout their length. The baculovirus EGTs are also homologous to a variety of UDP-glycosyltransferases from animal, plant and bacterial species and five conserved domains have been identified that are shared by many of these proteins. Seven amino acids are absolutely conserved in all of these proteins. They all occur within domains III and IV; this region of the protein is thought to be involved in UDP-sugar binding. Comparison of insects infected by an egt-mutant of AcMNPV with those infected by wild-type virus demonstrated that EGT-catalysed conjugation of ecdysteroids causes a suppression of host moulting and pupation. Wild-type AcMNPV-infected insects do not experience the feeding arrest, normally associated with ecdysis and therefore become larger, and yield more virus, than those infected with egt-viruses. Thus, one function of egt may be to increase the yield of progeny virus by prolonging insect feeding. Other possible functions discussed in this review include the alteration of host behaviour or the suppression of adverse effects of ecdysteroids on viral replication at the cellular level. Because egt expression prolongs insect feeding, recombinant baculoviruses lacking egt appear to be more efficient biological insecticides. In addition to the reduced feeding, egt-AcMNPV-infected insects display accelerated mortality. Thus, deletion of egt represents a simple approach to the improvement of baculovirus pesticides by genetic engineering.

Whole genome analysis of the Epiphyas postvittana nucleopolyhedrovirus.

The nucleotide sequence of the Epiphyas postvittana nucleopolyhedrovirus (EppoMNPV) genome has been determined and analysed. The circular dsDNA genome contains 118584 bp, making it the smallest group I NPV sequenced to date. The genome has a G+C content of 40.7% and encodes 136 predicted open reading frames (ORFs), five homologous repeat regions and one unique repeat region. Of the genome, 92.9% encodes predicted ORFs and 2.2% is in repeat regions; the remaining 4.9% of the genome comprises nonrepeat intergenic regions. EppoMNPV encodes homologues of 126 Orgyia pseudotsugata MNPV (OpMNPV) ORFs and 120 Autographa californica MNPV ORFs, with average identities of 64.7 and 53.5%, respectively. Between the four sequenced group I NPVs, 117 ORFs are conserved, whereas 86 ORFs are conserved between all fully sequenced NPVs. A total of 62 ORFs is present in all baculoviruses sequenced to date, with EppoMNPV lacking a homologue of the superoxide dismutase (sod) gene, which has been found in all other fully sequenced baculoviruses. Whole genome phylogenetic analyses of the ten fully sequenced baculoviruses using the sequences of the 62 shared genes, gene content and gene order data sets confirmed that EppoMNPV clusters tightly with OpMNPV in the group I NPVs. The main variation between EppoMNPV and OpMNPV occurs where extra clusters of genes are present in OpMNPV, with sod occurring in one such cluster. EppoMNPV encodes one truncated baculovirus repeated ORF (bro) gene. The only repeated ORFs are the four iap genes. Eight, randomly distributed, unique ORFs were identified on EppoMNPV, none of which show any significant homology to genes in GenBank.

A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors

A novel baculovirus genome has been generated that can be maintained in Escherichia coli and which facilitates the rapid and efficient generation of recombinant baculovirus expression vectors. This genome, designated bAcGOZA, lacks a portion of the essential ORF1629 gene but includes a miniF replicon and selectable kanamycin-resistance marker. The bAcGOZA can replicate only in E. coli and can be rescued by recombination with a transfer vector containing an intact ORF1629. There is no background of non-recombinant virus.

Identification and characterization of the Cydia pomonella granulovirus cathepsin and chitinase genes.

A 3.2 kb BamHI-EcoRI fragment of the Cydia pomonella granulovirus (CpGV) genome was subcloned and characterized. Sequence analysis revealed two complete and one partial open reading frames (ORFs). ORF7L is predicted to encode a 66.7 kDa protein (594 amino acid residues) that is 57% identical (amino acid sequence) to the chiA gene (ORF126) of Autographa californica nucleopolyhedrovirus (AcMNPV), encoding a chitinase. ORF8R is 333 amino acids in length and shows high similarity (between 64% and 67%) with baculovirus cathepsins. The partial ORF, ORF5L, is related to AcMNPV ORF145 of unknown function. Phylogenetic trees were constructed for both chitinase and cathepsin sequences from baculoviruses and other species. In both cases, the baculovirus sequences were monophyletic but with a deep division between the GVs and NPVs, suggesting both genes were present in an ancestral virus prior to the separation of the two genera. However, these studies did not provide definitive evidence for the origin of either protein in baculoviruses. To investigate CpGV cathepsin function, a rescue experiment was performed using a Bombyx mori NPV (BmNPV) mutant (BmCysPD) which lacks a functional cathepsin (cath) gene. Larvae infected with BmCysPD-Cp.cat, a BmCysPD derivative carrying CpGV cath, showed similar symptoms to wild-type BmNPV infected insects, confirming that CpGV cath encodes a functional cathepsin. Primer extension analysis of mRNA from BmCysPD-Cp.cat infected cells showed that CpGV cath transcription was initiated from a consensus late transcription motif (ATAAG) within the CpGV sequences, indicating that a CpGV late promoter motif was recognized in this NPV system.

Characterization of Two Autographa californica Nucleopolyhedrovirus Proteins, Ac145 and Ac150, Which Affect Oral Infectivity in a Host-Dependent Manner

ABSTRACT The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.

Role of calcium in the stimulation of ecdysteroidogenesis by recombinant prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

Abstract The aim of this study was to clarify whether Ca 2+ plays a role in the mechanism by which recombinant prothoracicotropic hormone (PTTH) stimulates ecdysteroidogenesis by the prothoracic glands of the silkworm, Bombyx mori . Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect basal ecdysteroid secretion, but greatly inhibited the stimulation of ecdysteroidogenesis by PTTH. The addition of a Ca 2+ ionophore (ionomycin or A23187) stimulated ecdysteroid secretion in a dose-dependent manner. Furthermore, thapsigargin, a potent Ca 2+ -ATPase inhibitor, enhanced ecdysteroid secretion. In contrast, thimerosal, a thiol-oxidizing reagent, inhibited prothoracic gland activity. The mechanism of Ca 2+ action was examined further in relation to the cAMP effector system. Ca 2+ deprivation greatly suppressed the accumulation of cAMP in prothoracic glands treated with PTTH but had no effect on basal levels. Omission of Ca 2+ from the incubation medium, or inclusion of a Ca 2+ channel blocker (verapamil or lanthanum) did not affect the stimulation of ecdysteroid secretion in response to dibutyryl cAMP or 1-methyl-3-isobutylxanthine. The present study suggests that specific regulatory actions of Ca 2+ in prothoracic gland cells are exerted prior to cAMP generation.

Characterization of the ecdysteroid UDP-glucosyltransferase gene from Mamestra brassicae nucleopolyhedrovirus.

The ecdysteroid UDP-glucosyltransferase (egt) gene of Mamestra brassicae multinucleocapsid nucleopolyhedrovirus (MbMNPV) has been cloned and characterized. MbMNPV egt potentially encodes a protein of 528 amino acids. Analysis of the substrate specificity of the MbMNPV EGT protein showed that it mirrors that of Autographa californica MNPV (AcMNPV) EGT. MbMNPV EGT also appears to be secreted from infected cells. Confirmation that the cloned gene encodes an active EGT was obtained by transient expression assays. Phylogenetic trees of NPVs were generated based on the alignment of baculovirus EGT sequences. These phylogenies support the classification of MbMNPV as a group II NPV that is most closely related to Spodoptera exigua MNPV. Comparison of the EGT-based phylogenies with polyhedrin/granulin-based phylogenies shows that the position of AcMNPV is different in the two trees, possibly indicating that AcMNPV acquired its polyhedrin gene by recombination with another virus.

Tomato aspermy virus has an evolutionary relationship with other tripartite RNA plant viruses

The entire RNA 3 (2214 nucleotides) of a chrysanthemum isolate of tomato aspermy virus (C-TAV) has been cloned and its sequence determined. C-TAV possesses two open reading frames which encode a 3a protein (277 amino acids) and a coat protein (229 amino acids). Computer-assisted comparisons were made between C-TAV RNA 3 and its predicted protein sequences and those of two other tripartite RNA viruses, cucumber mosaic virus (CMV) and brome mosaic virus (BMV). Results from this study suggest that a close evolutionary relationship exists between C-TAV, Q-CMV and BMV. Divergence of nucleotide and amino acid sequences between these viruses is not reflected at the level of the predicted secondary structure of the encoded proteins, where conservation is strong.