Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome

Abstract

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.

FIG. 1.

Linear map of the NeleNPV genome. Arrows indicate the position and direction of transcription for potential ORFs, with polyhedrin (ph) shown as ORF 1. NeleNPV ORF numbers and potential gene names are shown below the arrows, and AcMNPV ORF numbers (7) are shown above the arrows.

FIG. 2.

Alignment of NeleNPV direct repeat sequences. Direct repeat regions are numbered according to their order in the genome. The repeat sequences are aligned to obtain maximum similarity, and their locations within the genome are indicated. Sequences within repeat 4 that are found within nl2 and nl36 are bolded and underlined. Arrows indicate palindromes.

FIG. 3.

Gene parity plots. Comparison of NeleNPV with AcMNPV (A), HaSNPV (B), CuniNPV (C), and PxGV (D). AcMNPV ORFs are renumbered, with polyhedrin as ORF 1. The CuniNPV gene order is as listed in GenBank and not by ORF number for lef-5, 38k, ac96, and helicase. The graphic representations display the colinearity of gene arrangement between two genomes. ORFs unique to each virus are shown on the x axis and y axis, respectively. Most conserved clusters are circled: cluster 1, p6.9 (ac100), p40 (ac101), and p48 (ac103); cluster 2, ac76, vlf-1 (ac77), ac78, and gp41 (ac80); cluster 3, lef-5 (ac99), 38k (ac98), ac96, and helicase (ac95); cluster 4, p49 (ac142), odv-ec27 (ac143), odv-ec27 (ac144), and ac145.

FIG. 4.

Baculovirus phylogeny based on complete genome data. Most-parsimonious tree based on analysis of the combined sequences of 29 conserved genes found in 24 fully sequenced baculovirus genomes. Bootstrap values for 1,000 replicates are given. Sequences include those for AcMNPV (7), Orgyia pseudotsugata MNPV (OpMNPV) (3), BmNPV (25), LdMNPV (40), SeMNPV (37), XcGV (30), PxGV (28), HaSNPV G4 (13), Helicoverpa armigera NPV (HaNPV) (GenBank accession number NC_003094), SpltMNPV (50), CpGV (46), CuniNPV (2), E. postvittana MNPV (EppoMNPV) (36), H. zea SNPV (HzSNPV) (14), MacoNPV A (44), MacoNPV B (43), PhopGV (GenBank accession number AF499596), RoMNPV (27), AdhoNPV (GenBank accession number AP006270), C. fumiferana defective NPV (CfDEFNPV) (GenBank accession number AY327402), Choristoneura fumiferana MNPV (CfMNPV) (GenBank accession number NC_004778), CrleGV (42), AdorGV (69), and NeleNPV (GenBank accession number AY349019).