Melanoplus Sanguinipes Entomopoxvirus Research Articles

Reannotation of protein‐coding genes based on an improved graphical representation of DNA sequence

Over annotation of protein coding genes is common phenomenon in microbial genomes, the genome of Amsacta moorei entomopoxvirus (AmEPV) is a typical case, because more than 63% of its annotated ORFs are hypothetical. In this article, we propose an improved graphical representation titled I-TN (improved curve based on trinucleotides) curve, which allows direct inspection of composition and distribution of codons and asymmetric gene structure. This improved graphical representation can also provide convenient tools for genome analysis. From this presentation, 18 variables are exploited as numerical descriptors to represent the specific features of protein coding genes quantitatively, with which we reannotate the protein coding genes in several viral genomes. Using the parameters trained on the experimentally validated genes, all of the 30 experimentally validated genes and 63 putative genes in AmEPV genome are recognized correctly as protein coding, the accuracies of the present method for self-test and cross-validation are 100%, respectively. Twenty-eight annotated hypothetical genes are predicted as noncoding, and then the number of reannotated protein coding genes in AmEPV should be 266 instead of 294 reported in the original annotations. Extending the present method trained in AmEPV to other entomopoxvirus genomes directly, such as Melanoplus sanguinipes entomopoxvirus (MsEPV), all of the 123 annotated function-known and putative genes are recognized correctly as protein coding, and 17 hypothetical genes are recognized as noncoding. The present method could also be extended to other genomes with or without adaptation of training sets with high accuracy.

Identification and expression of a conserved ubiquitin gene homologue of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I)

An ORF having a potential to code for a polypeptide of 79 amino acids has been identified within 993 nt sequence of 2 kb EcoRI-W fragment of Spodoptera litura nucleopolyhedrovirus (SpltNPV-I). Nucleotide and deduced amino acid sequence analyses showed its identity with the ubiquitin homologue of eukaryotes (79-80%), Melanoplus sanguinipes entomopoxvirus (76%) and other baculoviruses (72-89%). The ORF is under baculovirus late promoter motif RTAAG but unlike other baculoviruses, three such motifs at -6, -10 and -27 position are present in SpltNPV. The ORF expresses as a 10 kDa proteinin E. coli and the purified recombinant protein showed crossreactivity with the rabbit anti-ubiquitin antibodies.

Unique ligation properties of eukaryotic NAD +-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus

The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD +)-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD + ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD +-dependent fashion, while adenosine 5′-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg 2+ or Mn 2+. MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD +-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD +-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD + DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3′ side of the nick but substantial 5′ T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3′ side of the nick more frequently than at the 5′ side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.

Characterization of a novel ubiquitin-fusion gene Uba256 from Spodoptera litura nucleopolyhedrovirus

The complete nucleotide sequence of Spodoptera litura nucleopolyhedrovirus (SpltMNPV) Uba256 gene, encoding ubiquitin fused to GP37 protein of 256 amino acids was determined. The first 76 amino acids of the SpltMNPV ubiquitin showed 78–88, 77 and 81–84% amino acid sequence identity to baculovirus, Melanoplus sanguinipes entomopoxvirus and eukaryotes ubiquitins, respectively. The deduced amino acid sequence of SpltMNPV GP37 protein was similar to other baculovirus GP37 proteins and to entomopoxvirus fusolin proteins. The GP37 protein also showed a distant similarity to Pseudaletia separata entomopoxvirus enhancing factor, bacterial chitinase B and chitin-binding protein 1, but the significance of this is unclear. The mRNA start site of Uba256 fusion gene was mapped within a consensus baculovirus late promoter sequence (ATAAG), commonly found for baculovirus late genes. Uba256 transcripts were present from 48 h p.i. and remained detectable until 72 h p.i. Western blot analysis of SpltMNPV-infected Sl-zsu-1 cells revealed that the intact Uba256 was processed to free ubiquitin and GP37 protein. Whereas expression Uba256 gene in Escherichia coli did not result in processing of the fusion protein. Tunicamycin treatment of SpltMNPV-infected cells confirmed that SpltMNPV GP37 protein is N-glycosylated. These findings provide additional information on the evolution of ubi genes and insight into genomic variation in baculoviruses.

Purification and Partial Characterization of an Entomopoxvirus (DlEPV) from a Parasitic Wasp of Tephritid Fruit Flies.

An insect poxvirus [entomopoxvirus (EPV)] occurs in the poison gland apparatus of female Diachasmimorpha longicaudata, a parasitic wasp of the Caribbean fruit fly, Anastrepha suspensa and other tephritid fruit flies. The DlEPV virion is 250–300 nm in diameter, has a “bumpy” appearance and a unipartite double stranded DNA genome of 290–300 kb. DlEPV DNA restriction fragment profiles differed from those reported for Amsacta moorei EPV (AmEPV) and Melanoplus sanguinipes EPV (MsEPV), the only two EPVs whose genomes have been sequenced, and from those reported for vaccinia (Vac), a vertebrate poxvirus (chordopoxvirus, ChPV). Blast search and ClustalW alignment of the amino acids deduced from the 2316 nucleotides of a DlEPV DNA fragment cloned from an EcoR1 genomic library revealed 75–78% homology with the putative DNA-directed RNA polymerases of AmEPV, MsEPV, and two ChPV homologs of the Vac J6R gene. Of the deduced 772 amino acids in the DlEPV sequence, 28.4% are conserved/substituted among the four poxviruses aligned, 12.9% occur in at least one EPV, 6.5% in at least one ChPV, 3.1% in at least one EPV and one ChPV, and 49.1% occur only in DlEPV. Although the RI-36-1 fragment represents a portion of the gene, it contains nucleotides that encode the NADFDGDE consensus sequence of known DNA-directed RNA polymerases. Western blots using a mouse polyclonal anti-DlEPV serum recognized six major protein bands in combined fractions of sucrose-purified DlEPV, at least one band in homogenates of male and female wasps, and at least two bands in host hemolymph that contained DlEPV virions. A digoxigenin-labeled DlEPV genomic DNA probe recognized DNA in dot-blots of male and female wasps. These results confirm that DlEPV is a true EPV and probably a member of the Group C EPVs. Unlike other EPVs, DlEPV does not express the spheroidin protein. Since it also replicates in both the wasp and fly, members of two different insect Orders, DlEPV may represent a new EPV Group, or a subgroup of the Group C viruses.

Melanoplus sanguinipes Entomopoxvirus DNA Topoisomerase: Site-Specific DNA Transesterification and Effects of 5′-Bridging Phosphorothiolates

Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at ∼0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were kcl = 0.3 s−1 and Kcl = 0.25. The introduction of a 5′-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to ≥30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a ∼10−4 decrement in the rate of topoisomerase-catalyzed attack of 5′-SH DNA on the DNA-(3′-phosphotyrosyl)-enzyme intermediate. 5′-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on kcl.

The Complete Genome Sequence of Shope (Rabbit) Fibroma Virus

We have determined the complete DNA sequence of the Leporipoxvirus Shope fibroma virus (SFV). The SFV genome spans 159.8 kb and encodes 165 putative genes of which 13 are duplicated in the 12.4-kb terminal inverted repeats. Although most SFV genes have homologs encoded by other Chordopoxvirinae, the SFV genome lacks a key gene required for the production of extracellular enveloped virus. SFV also encodes only the smaller ribonucleotide reductase subunit and has a limited nucleotide biosynthetic capacity. SFV preserves the Chordopoxvirinae gene order from S012L near the left end of the chromosome through to S142R (homologs of vaccinia F2L and B1R, respectively). The unique right end of SFV appears to be genetically unstable because when the sequence is compared with that of myxoma virus, five myxoma homologs have been deleted (C. Cameron, S. Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay, D. Willer, D. Evans, and G. McFadden, 1999, Virology 264, 298–318). Most other differences between these two Leporipoxviruses are located in the telomeres. Leporipoxviruses encode several genes not found in other poxviruses including four small hydrophobic proteins of unknown function (S023R, S119L, S125R, and S132L), an α 2,3-sialyltransferase (S143R), a protein belonging to the Ig-like protein superfamily (S141R), and a protein resembling the DNA-binding domain of proteins belonging to the HIN-200 protein family S013L). SFV also encodes a type II DNA photolyase (S127L). Melanoplus sanguinipes entomopoxvirus encodes a similar protein, but SFV is the first mammalian virus potentially capable of photoreactivating ultraviolet DNA damage.

Changes in hemolymph of the migratory grasshopper, Melanoplus sanguinipes, infected with an entomopoxvirus

Hemolymph changes in response to infection with Melanoplus sanguinipes entomopoxvirus (MsEPV) were studied in the migratory grasshopper, M. sanguinipes. Secondinstar grasshoppers were inoculated orally with MsEPV (5.0 × 10 3 occlusion bodies (OBs)/individual) and hemolymph was collected at specified intervals from 0 to 32 days post-inoculation (p.i.). Mature OBs were detected in hemolymph samples from Day 21 p.i. onward. Total hemocyte counts decreased in infected versus control grasshoppers after 10–15 days p.i. Phenoloxidase activity initially peaked at Day 4 p.i. in infected grasshoppers and then declined to control levels until late in infection. Hemocytes from infected individuals on Day 28 p.i. showed a five- to sixfold increase in binding of fluorescein-isothiocyanate-labeled wheat germ agglutinin. Hemocyte morphological changes were observed late in infection including a small percentage of enlarged hemocytes and cells with extensive pseudopodia. MsEPV OBs were also observed in the cytoplasm of hemocytes late in infection. Whether the changes noted in M. sanguinipes hemolymph were a direct result of virus infection or a physiological response secondary to infection is discussed.

Wheat Foliage Consumption by Grasshoppers (Orthoptera: Acrididae) Infected with Melanoplus sanguinipes Entomopoxvirus

First-instar grasshoppers infected with Melanoplus sanguinipes entomopoxvirus consumed 25% less food within 5 d (75 degree-days (DD), base 10°C) after inoculation with 5 × 10 3 virus occlusion bodies. Grasshoppers ate 25 and 40% less food by the end of the second week (200 DD, base 100°C) after being infected as first instars with 5 × 10 2 and 5 × 10 3 occlusion bodies per individual, respectively. When infected as third instars with 1 × l0 4 and 1 × 10 5 occlusion bodies per individual, grasshoppers ate 20 and 35% less food by the second week of the study, respectively. The mean amount of food consumed by infected grasshoppers over the course of the study was 36, 31, 76, and 57% that of noninfected grasshoppers at dosages of 5 × 10 2 or 5 × 10 3 occlusion bodies per first instar, and 1 × 10 4 or 1 × 10 5 occlusion bodies per third instar. This study suggests that an application of entomopoxvirus to first-instar grasshoppers will reduce their damage potential by causing initial mortality, by reducing the amount of plant material consumed by the surviving grasshoppers, and by preventing grasshopper maturation and reproduction (at higher doses). The negative influence of entomopoxvirus on grasshoppers in the interim between infection and death is integral to future evaluations of its potential as a crop protection measure.

Protease activity associated with occlusion body preparations of an entomopoxvirus from Melanoplus sanguinipes

An alkaline protease, associated with Melanoplus sanguinipes entomopoxvirus occlusion body preparations, was characterized. The protease had a pH optimum between 8 and 9 and a temperature optimum between 45 and 50°C. Protease activity was inhibited by phenylmethylsulfonyl fluoride, an inhibitor of serine proteases. The protease was shown to have trypsin-like activity based on the hydrolysis of a trypsin-specific synthetic substrate and inhibition by a trypsin-specific inhibitor. The protease was inactivated by heat treatment above 60°C. Heat treatment (>70°C) inhibited complete dissolution of the occlusion bodies and the release of virions in alkaline dissolution buffer. In dissolution buffer, the occlusion body matrix protein, spheroidin (108.8 kDa), was degraded into smaller polypeptides.

Cross infection of three grasshopper species with the Melanoplus sanguinipes entomopoxvirus

Cross infection of three grasshopper species with the Melanoplus sanguinipes entomopoxvirus

Molecular weight and base composition of DNA isolated from Melanoplus sanguinipes entomopoxvirus

The DNA genome of the orthopteran entomopoxvirus (EPV) isolated from Melanoplus sanguinipes was released from the virus by treatment with proteinase K and sodium dodecyl sulfate (SDS). The average length of the virus DNA molecule was determined by electron microscopy to be 62.8 μm, corresponding to a molecular weight of 124.3 × 10 6 daltons (80 kb). The buoyant density of Melanoplus EPV DNA in cesium chloride was calculated to be 1.678 g/cm 3, which corresponds to a base ratio of 18.6 mole% guanine + cytosine.