The genome of Melanoplus sanguinipes entomopoxvirus

Abstract

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.

FIG. 1

Linear map of the MsEPV genome. ORFs are numbered from left to right based on initiation codon position. ORFs transcribed to the right are located above the horizontal lines; ORFs transcribed to the left are below. ChPV homologues are indicated with red italicized numbers. Genes with similar functions and members of gene families are colored according to the figure key. ITRs are represented as heavy black bars underneath the ORF map (numbers indicate sizes [in base pairs] of nucleotide repeats).

FIG. 2

Multiple amino acid sequence alignments of MsEPV ORFs with DNA repair and replication enzymes. Boldfaced letters represent active site residues, asterisks mark residues that match Prosite signatures, and shaded residues represent amino acids with identity to those of the corresponding MsEPV ORF. Amino acid positions are indicated on the right. (A) Alignment of MSV235 with class 2 CPD photolyases; regions I and II represent class 2 CPD photolyase Prosite signatures PS01083 and PS01084, respectively. Abbreviations: Monodelph, Monodelphis domestica, accession no. D31902; Oryzias, Oryzias latipes, accession no. S52048; Drosoph, Drosophila melanogaster, accession no. S52047; Arab, Arabidopsis thaliana, accession no. X99301; Methano, Methanobacterium thermoautotrophicum, accession no. D30752. (B) Alignment of MSV162 with NAD⁺-dependent DNA ligases; regions I and II represent Prosite signatures PS01055 and PS01056 for NAD⁺-dependent DNA ligases, respectively. Abbreviations: Thermus, Thermus aquaticus, accession no. P26996; Ecoli, E. coli, accession no. P15042; Mycoplas, Mycoplasma pneumoniae, accession no. AE000047.

FIG. 3

Multiple amino acid sequence alignments of MSV061 with RTs. The seven RT motifs are indicated with roman numbers I to VII (210). Boldfaced letters indicate invariant amino acids, shaded letters indicate amino acids that are identical to corresponding ones in MSV061, and consensus residues are indicated at the bottom as follows: h, hydrophobic; p, small polar; c, charged; and x, any amino acid. Uppercase letters indicate the one-letter amino acid code. Amino acid positions are indicated on the right. Abbreviations: LINE, LINE type of RT; Intron, group II intron; MsDNA, multicopy single-stranded DNA; Cele, Caenorhabditis elegans, accession no. U00063; Rat, Rattus norvegicus, accession no. X61294; Xenla, Xenopus laevis, accession no. P14381; Dictyo, Dictyostelium discoideum, accession no. X57031; Aedes, Aedes aegypti, accession no. M95171; Yeast, Saccharomyces cerevisiae, accession no. P21325; Ecoli, E. coli, accession no. V00694.

FIG. 4

Multiple amino acid sequence alignments of MsEPV ORFs with protein modification enzymes. (A) Alignment of MSV175, MSV176, and MSV179 with the catalytic or zinc-binding regions of zinc-dependent proteases. Boldfaced letters represent amino acids which are either histidine zinc ligands or glutamic acid catalytic residues, and shaded residues represent amino acids with identity to the corresponding MsEPV ORF. The consensus for the metzincin (Metzn) subfamily (81) is exhibited underneath. (where b is any bulky hydrophobic amino acid and x is any amino acid). Abbreviations: Xenop, Xenopus laevis, accession no. L49412; Human, Homo sapiens, accession no. P39900; Mus, Mus musculus, accession no. L36244; Gmax, Glycine max, accession no. U63725; Bfrag, Bacteroides fragilis, accession no. U90931. (B) Alignment of MSV081 and MSV135 with eukaryotic PP2C proteins. Boldfaced letters represent metal-coordinating residues (28), asterisks mark highly conserved residues, shaded residues represent amino acids with identity to MsEPV, and overlined residues mark the Prosite signature (PS00142). Abbreviations: Human, Homo sapiens, accession no. P35813; Param, Paramecium tetraurelia, accession no. Z36985; Sacch, Saccharomyces cerevisiae, accession no. U72346; Arabid, Arabidopsis thaliana, accession no. U78721.

FIG. 4

Multiple amino acid sequence alignments of MsEPV ORFs with protein modification enzymes. (A) Alignment of MSV175, MSV176, and MSV179 with the catalytic or zinc-binding regions of zinc-dependent proteases. Boldfaced letters represent amino acids which are either histidine zinc ligands or glutamic acid catalytic residues, and shaded residues represent amino acids with identity to the corresponding MsEPV ORF. The consensus for the metzincin (Metzn) subfamily (81) is exhibited underneath. (where b is any bulky hydrophobic amino acid and x is any amino acid). Abbreviations: Xenop, Xenopus laevis, accession no. L49412; Human, Homo sapiens, accession no. P39900; Mus, Mus musculus, accession no. L36244; Gmax, Glycine max, accession no. U63725; Bfrag, Bacteroides fragilis, accession no. U90931. (B) Alignment of MSV081 and MSV135 with eukaryotic PP2C proteins. Boldfaced letters represent metal-coordinating residues (28), asterisks mark highly conserved residues, shaded residues represent amino acids with identity to MsEPV, and overlined residues mark the Prosite signature (PS00142). Abbreviations: Human, Homo sapiens, accession no. P35813; Param, Paramecium tetraurelia, accession no. Z36985; Sacch, Saccharomyces cerevisiae, accession no. U72346; Arabid, Arabidopsis thaliana, accession no. U78721.

FIG. 5

Multiple amino acid sequence alignments of MsEPV ORFs with cellular and viral homologues. Boldfaced letters represent active site residues, asterisks mark residues from a Prosite signature (when indicated) or those exhibiting ≥85% conservation, and shaded residues represents amino acids with identity to MsEPV. Amino acid positions are indicated on the right. (A) Alignment of MSV048 at the active site of triacylglycerol lipases. Abbreviations: Rhizopus, Rhizopus niveus, accession no. D12680; Synecho, Synechocystis sp., accession no. D64004; Celegans, Caenorhabditis elegans, accession no. U97001; Ipomoea, Ipomoea nill, accession no. U55867; and Cowpox, CPV putative lipase, accession no. X94355. The Prosite signature is PS00120. (B) Alignment of MSV097 with two EF-hand motifs from calcium-binding proteins. Abbreviations: Tvaginalis, Trichomonas vaginalis, accession no. U38786; Calbicans, Candida albicans, accession no. P23286; Atriplex, Atriplex nummularia, accession no. PRF: 1906390A; Smansoni, Schistosoma mansoni, accession no. P15845; Brassica, Brassica napus, accession no. D63152. The Prosite signature is PS00018. (C) Alignment of MSV206 with bacterial glycosyltransferase genes. Abbreviations: Nmening, Neisseria meningitidis, accession no. U65788; Hinfluen, Haemophilus influenzae, accession no. U36398; Hpylori, Helicobacter pylori, accession no. AE000592; Phaemol, Pasteurella haemolytica, accession no. U15958; Hsomnus, Haemophilus somnus, accession no. U94833; Hducreyi, Haemophilus ducreyi, accession no. U58147. (D) Alignment of MSV087 with thioredoxin genes. Abbreviations: Strept, Streptomyces aureofaciens, accession no. P33791; Coryne, Corynebacterium nephridii, accession no. P00275; Ecoli, E. coli, accession no. M54881; Eubact, Eubacterium acidaminophilum, accession no. P21610; Neuros, Neurospora crassa, accession no. D45892. The Prosite signature is PS00194.

FIG. 5

Multiple amino acid sequence alignments of MsEPV ORFs with cellular and viral homologues. Boldfaced letters represent active site residues, asterisks mark residues from a Prosite signature (when indicated) or those exhibiting ≥85% conservation, and shaded residues represents amino acids with identity to MsEPV. Amino acid positions are indicated on the right. (A) Alignment of MSV048 at the active site of triacylglycerol lipases. Abbreviations: Rhizopus, Rhizopus niveus, accession no. D12680; Synecho, Synechocystis sp., accession no. D64004; Celegans, Caenorhabditis elegans, accession no. U97001; Ipomoea, Ipomoea nill, accession no. U55867; and Cowpox, CPV putative lipase, accession no. X94355. The Prosite signature is PS00120. (B) Alignment of MSV097 with two EF-hand motifs from calcium-binding proteins. Abbreviations: Tvaginalis, Trichomonas vaginalis, accession no. U38786; Calbicans, Candida albicans, accession no. P23286; Atriplex, Atriplex nummularia, accession no. PRF: 1906390A; Smansoni, Schistosoma mansoni, accession no. P15845; Brassica, Brassica napus, accession no. D63152. The Prosite signature is PS00018. (C) Alignment of MSV206 with bacterial glycosyltransferase genes. Abbreviations: Nmening, Neisseria meningitidis, accession no. U65788; Hinfluen, Haemophilus influenzae, accession no. U36398; Hpylori, Helicobacter pylori, accession no. AE000592; Phaemol, Pasteurella haemolytica, accession no. U15958; Hsomnus, Haemophilus somnus, accession no. U94833; Hducreyi, Haemophilus ducreyi, accession no. U58147. (D) Alignment of MSV087 with thioredoxin genes. Abbreviations: Strept, Streptomyces aureofaciens, accession no. P33791; Coryne, Corynebacterium nephridii, accession no. P00275; Ecoli, E. coli, accession no. M54881; Eubact, Eubacterium acidaminophilum, accession no. P21610; Neuros, Neurospora crassa, accession no. D45892. The Prosite signature is PS00194.

FIG. 5

Multiple amino acid sequence alignments of MsEPV ORFs with cellular and viral homologues. Boldfaced letters represent active site residues, asterisks mark residues from a Prosite signature (when indicated) or those exhibiting ≥85% conservation, and shaded residues represents amino acids with identity to MsEPV. Amino acid positions are indicated on the right. (A) Alignment of MSV048 at the active site of triacylglycerol lipases. Abbreviations: Rhizopus, Rhizopus niveus, accession no. D12680; Synecho, Synechocystis sp., accession no. D64004; Celegans, Caenorhabditis elegans, accession no. U97001; Ipomoea, Ipomoea nill, accession no. U55867; and Cowpox, CPV putative lipase, accession no. X94355. The Prosite signature is PS00120. (B) Alignment of MSV097 with two EF-hand motifs from calcium-binding proteins. Abbreviations: Tvaginalis, Trichomonas vaginalis, accession no. U38786; Calbicans, Candida albicans, accession no. P23286; Atriplex, Atriplex nummularia, accession no. PRF: 1906390A; Smansoni, Schistosoma mansoni, accession no. P15845; Brassica, Brassica napus, accession no. D63152. The Prosite signature is PS00018. (C) Alignment of MSV206 with bacterial glycosyltransferase genes. Abbreviations: Nmening, Neisseria meningitidis, accession no. U65788; Hinfluen, Haemophilus influenzae, accession no. U36398; Hpylori, Helicobacter pylori, accession no. AE000592; Phaemol, Pasteurella haemolytica, accession no. U15958; Hsomnus, Haemophilus somnus, accession no. U94833; Hducreyi, Haemophilus ducreyi, accession no. U58147. (D) Alignment of MSV087 with thioredoxin genes. Abbreviations: Strept, Streptomyces aureofaciens, accession no. P33791; Coryne, Corynebacterium nephridii, accession no. P00275; Ecoli, E. coli, accession no. M54881; Eubact, Eubacterium acidaminophilum, accession no. P21610; Neuros, Neurospora crassa, accession no. D45892. The Prosite signature is PS00194.

FIG. 5

Multiple amino acid sequence alignments of MsEPV ORFs with cellular and viral homologues. Boldfaced letters represent active site residues, asterisks mark residues from a Prosite signature (when indicated) or those exhibiting ≥85% conservation, and shaded residues represents amino acids with identity to MsEPV. Amino acid positions are indicated on the right. (A) Alignment of MSV048 at the active site of triacylglycerol lipases. Abbreviations: Rhizopus, Rhizopus niveus, accession no. D12680; Synecho, Synechocystis sp., accession no. D64004; Celegans, Caenorhabditis elegans, accession no. U97001; Ipomoea, Ipomoea nill, accession no. U55867; and Cowpox, CPV putative lipase, accession no. X94355. The Prosite signature is PS00120. (B) Alignment of MSV097 with two EF-hand motifs from calcium-binding proteins. Abbreviations: Tvaginalis, Trichomonas vaginalis, accession no. U38786; Calbicans, Candida albicans, accession no. P23286; Atriplex, Atriplex nummularia, accession no. PRF: 1906390A; Smansoni, Schistosoma mansoni, accession no. P15845; Brassica, Brassica napus, accession no. D63152. The Prosite signature is PS00018. (C) Alignment of MSV206 with bacterial glycosyltransferase genes. Abbreviations: Nmening, Neisseria meningitidis, accession no. U65788; Hinfluen, Haemophilus influenzae, accession no. U36398; Hpylori, Helicobacter pylori, accession no. AE000592; Phaemol, Pasteurella haemolytica, accession no. U15958; Hsomnus, Haemophilus somnus, accession no. U94833; Hducreyi, Haemophilus ducreyi, accession no. U58147. (D) Alignment of MSV087 with thioredoxin genes. Abbreviations: Strept, Streptomyces aureofaciens, accession no. P33791; Coryne, Corynebacterium nephridii, accession no. P00275; Ecoli, E. coli, accession no. M54881; Eubact, Eubacterium acidaminophilum, accession no. P21610; Neuros, Neurospora crassa, accession no. D45892. The Prosite signature is PS00194.