Sequence analysis of the genome of the Neodiprion sertifer nucleopolyhedrovirus

Abstract

The genome of the Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), which infects the European pine sawfly, N. sertifer (Hymenoptera: Diprionidae), was sequenced and analyzed. The genome was 86,462 bp in size. The C+G content of 34% was lower than that of the majority of baculoviruses. A total of 90 methionine-initiated open reading frames (ORFs) with more than 50 amino acids and minimal overlapping were found. From those, 43 ORFs were homologous to other baculovirus ORFs, and 29 of these were from the 30 conserved core genes among all baculoviruses. A NeseNPV homolog to the ld130 gene, which is present in all other baculovirus genomes sequenced to date, could not be identified. Six NeseNPV ORFs were similar to non-baculovirus-related genes, one of which was a trypsin-like gene. Only one iap gene, containing a single BIR motif and a RING finger, was found in NeseNPV. Two NeseNPV ORFs (nese18 and nese19) were duplicates transcribed in opposite orientations from each other. NeseNPV did not have an AcMNPV ORF 2 homolog characterized as the baculovirus repeat ORF (bro). Six homologous regions (hrs) were located within the NeseNPV genome, each containing small palindromes embedded within direct repeats. A phylogenetic analysis was done to root the tree based upon the sequences of DNA polymerase genes of NeseNPV, 23 other baculoviruses, and other phyla. Baculovirus phylogeny was then constructed with 29 conserved genes from 24 baculovirus genomes. Culex nigripalpus nucleopolyhedrovirus (CuniNPV) was the most distantly related baculovirus, branching to the hymenopteran NeseNPV and the lepidopteran nucleopolyhedroviruses and granuloviruses.

FIG. 1.

Linear map of the HindIII sites of the NeseNPV genome. The number and transcriptional direction of each ORF are labeled as a black arrow. Known baculovirus homologs are labeled in bold by the NeseNPV ORF number. Nonbaculovirus homologs are indicated in parentheses. The homologous regions (hr) are shown as triangles, and the direct repeats (dr) are shown as circles (•).

FIG. 2.

Alignment of NeseNPV ORF 7 (nese7) and trypsins from other organisms. Bos taurus, cow; Hypoderma lineatum, early cattle grub; Sarcophaga bullata, grey fleshfly; Drosophila erecta, a fruit fly; Aedes aegypti, yellow fever mosquito; Bombyx mori, silkworm. Regions highlighted as conserved amino acids by the conserved domain database (CDD) are boxed with a consensus sequence indicated on the top line. # represents the trypsin catalytic triad, histidine, aspartic acid, and serine. * indicates the conserved cysteines.

FIG. 3.

(A) hrs of NeseNPV. The nucleotide sequence of three NeseNPV hrs are aligned to exemplify the direct repeats found in units of 65 bp (hr2), 45 bp (hr5), or alternating 65 and 45 bp (hr1). The consensus sequence of the hrs is shown in bold, with uppercase letters representing 75% or more conserved nucleotides (60). Gaps (−) were inserted to optimize the alignment of the repeat units. The numbers on the left hand side of each hr indicate the nucleotide position in the genome. For each hr, the nucleotides forming a perfect palindrome (10 to 12 nucleotides) are marked with a solid arrow. Imperfect palindromes, formed by 70% or more complementary bases, are indicated by dots that extend 31 to 40 bases. The late promoter motif ATAAG and the potential GATA factor-binding site are double underlined and boxed, respectively. (B) Schematic representation of the repeats units of either 65 bp (solid arrows) or 45 bp (double-line arrows) found in each hr. (C) Comparison of the consensus sequences of the six NeseNPV hrs with the perfect palindrome region underlined and the extended imperfect palindrome dotted. An asterisk in hr6 indicates that the repeat sequence is different from the other hrs.

FIG. 4.

GeneParity plots comparing the NeseNPV gene order to six other baculoviruses: AcMNPV (A), LdMNPV (B), MacoMNPV-A (C), HzSNPV (D), CpGV (E), and CuniNPV (F). Eight clusters are underlined and/or boxed in the graphs, which include the following genes or AcMNPV homologs: 1, ac92, ac93; 2, p45, p40, p6.9; 3, vlf-1, ac78, gp41, ac81; 4, lef-5, 38K, ac96, helicase and lef-4; 5, ac142, odv-e18, odv-ec27*, ac148; 6, lef-9, ac68; 7, p47, p74; and 8, lef-1, ac115. Asterisks represent genes that are not part of a specific cluster in certain genomes. Straight lines mark clusters of two or more genes (underlined above) that are sequential in both NeseNPV and the genome being compared. Relaxed clusters that include genes that are not sequential in the compared genome are indicated with a box.

FIG. 5.

DNA polymerase phylogeny (A) indicates distinct lineages when all sequenced baculoviruses and the Hz-1 DNA polymerases are included. The root position of the DNA polymerase gene suggested that the lepidopteran NPV and GV share a common ancestral lineage with the NeseNPV after its split from the one leading to the CuniNPV. (B) A global maximum-likelihood phylogeny for 29 conserved genes from 24 baculoviruses supports the DNA polymerase tree in its separation of NeseNPV and CuniNPV. The numbers near nodes indicate the percentage of time a partition was found by the quartet method and is considered the level of support for the tree. Only nodes appearing more than 50% of the time were resolved.

FIG. 5.

DNA polymerase phylogeny (A) indicates distinct lineages when all sequenced baculoviruses and the Hz-1 DNA polymerases are included. The root position of the DNA polymerase gene suggested that the lepidopteran NPV and GV share a common ancestral lineage with the NeseNPV after its split from the one leading to the CuniNPV. (B) A global maximum-likelihood phylogeny for 29 conserved genes from 24 baculoviruses supports the DNA polymerase tree in its separation of NeseNPV and CuniNPV. The numbers near nodes indicate the percentage of time a partition was found by the quartet method and is considered the level of support for the tree. Only nodes appearing more than 50% of the time were resolved.