Homologous recombination between the inverted terminal repeats of defective transposon TCp3.2 causes an inversion in the genome of Cydia pomonella granulovirus.
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In this study, a new mutant of the Cydia pomonella granulovirus (CpGV), which shows spontaneous inversion of a transposable element during in vivo replication, is described. CpGV-MCp4 is a natural mutant of CpGV-M, containing the transposable element TCp3.2, which originated from the genome of the host C. pomonella. During in vivo cloning studies of CpGV-MCp4, a mutant called CpGV-MCp4inv was isolated. CpGV-MCp4inv shows heterogeneity in the genome area of transposon insertion. Restriction mapping, PCR analysis and subsequent sequence analysis gave strong evidence that an inversion of TCp3.2 is caused by homologous recombination between the long inverted terminal repeats (ITRs) of the transposon. This finding demonstrated that extensive homologous repeat regions such as the ITRs of transposons cause inversions by homologous recombination during in vivo replication. The observed in vivo inversion between the ITRs can be considered as a model for the contribution of repeated sequences in the genome rearrangement of baculoviruses and a source for genetic heterogeneity among different baculoviruses and baculovirus genotypes. (+info)
The expansion of a hypervariable, non-hr ori-like region in the genome of Cryptophlebia leucotreta granulovirus provides in vivo evidence for the utilization of baculovirus non-hr oris during replication.
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In this report a naturally occurring hypervariable region within the genome of different Cryptophlebia leucotreta granulovirus genotypes is characterized. The region consists of a stretch of direct repeats, short palindromes and an unusual AT-rich region. Although the organization of these repeat sequences is unique to baculoviruses, it has the structural features of a 'non-hr' origin of DNA replication (ori). Restriction analysis and Southern hybridization revealed that this region is expanded during virus replication. Sequence comparison of different isolated genotypes indicated that the expansion is caused by concatenation of short repeats within the region or by concatenation of the complete region. These findings indicate that the expansion of non-hr origin-like regions is not restricted to defective-interfering particles, as was found previously for baculoviruses propagated in cell culture. Moreover, it appears that non-hr complexity contributes to the natural heterogeneity and genetic plasticity of baculovirus genomes. Also, circumstantial evidence is discussed that hr oris might have developed from internal rearrangement and multiplication of a non-hr ori during baculovirus evolution. (+info)
Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129.
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Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55% amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1.1 kb. 5'-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT-PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S. exigua larvae, neither in concentrated BV and ODV preparations. These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts. Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed. (+info)
Identification and characterization of a putative baculoviral transcriptional factor IE-1 from Choristoneura fumiferana granulovirus.
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A gene that encodes a protein homologue to baculoviral IE-1 was identified and sequenced in the genome of the Choristoneura fumiferana granulovirus (ChfuGV). The gene has an 1278 nucleotide (nt) open-reading frame (ORF) that encodes 426 amino acids with an estimated molecular weight of 50.33 kDa. At the nucleotide level, several cis-acting regulatory elements were detected within the promoter region of the ie-1 gene of ChfuGV along with other studied granuloviruses (GVs). Two putative CCAAT elements were detected within the noncoding leader region of this gene; one was located on the opposite strand at -92 and the other at -420 nt from the putative start triplet. Two baculoviral late promoter motifs (TAAG) were also detected within the promoter region of the ie-1 gene of ChfuGV. A single polyadenylation signal, AATAAA, was located 18nt downstream of the putative translational stop codon of ie-1 from ChfuGV. At the protein level, the amino acid sequence data that was derived from the nucleotide sequence in ChfuGV IE-1 was compared to those of the Cydia pomonella granulovirus (CpGV), Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The C-terminal regions of the granuloviral IE-1 sequences appeared to be more conserved when compared to the N-terminal regions. A domain, similar to the basic helix-loop-helix like (bHLH-like) domain in NPVs, was detected at the C-terminal region of IE-1 from ChfuGV (residues 387 to 414). A phylogenetic tree for baculoviral IE-1 was constructed using a maximum parsimony analysis. A phylogenetic estimation demonstrates that ChfuGV IE-1 is most closely related to that of CpGV. (+info)
Identification and characterization of a conserved baculoviral structural protein ODVP-6E/ODV-E56 from Choristoneura fumiferana granulovirus.
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A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicted molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with other baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N- and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the ChfuGV odvp-6e/odv-e56 gene. A slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV). (+info)
The complete sequence of the Adoxophyes orana granulovirus genome.
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The nucleotide sequence of the Adoxophyes orana granulovirus (AdorGV) DNA genome was determined and analysed. The genome contains 99,657 bp and has an A + T content of 65.5%. The analysis predicted 119 ORFs of 150 nucleotides or larger that showed minimal overlap. Of these putative genes, 104 (87%) were homologous to genes identified previously in other baculoviruses. The mean overall amino acid identity of AdorGV ORFs was highest with CpGV ORFs at 48%. Sixty-three ORFs were conserved among all lepidopteran baculoviruses and are considered to be common baculoviral genes. Several genes reported to have major roles in baculovirus biology were not found in the AdorGV genome. These included chitinase and cathepsin, which are involved in the liquefaction of the host, which explains why AdorGV-infected insects do not degrade in a typical manner. The AdorGV genome encoded two inhibitor of apoptosis (iap) genes iap-3 and iap-5. Among all of the granuloviruses genomes there was a very high level of gene collinearity. The genes shared by AdorGV and CpGV had exactly the same order along the genome with the exception of one gene, iap-3. The AdorGV genome did not contain typical homologous region (hr) sequences. However, it contained nine repetitive regions in the genome. (+info)
A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV). (+info)
The genome of the Cryptophlebia leucotreta granulovirus.
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The genome of the Cryptophlebia leucotreta granulovirus (CrleGV) was sequenced and analyzed. The double-stranded circular genome contains 110907 bp and potentially encodes 129 predicted open reading frames (ORFs), 124 of which were similar to other baculovirus ORFs. Five ORFs were CrleGV specific and 26 ORFs were common to other granulovirus genomes. One ORF showed a significant similarity to a nonstructural protein of Bombyx mori densovirus-2. A baculovirus chitinase gene was identified, which is most likely not functional, because its central coding region including the conserved chitinase active site signature is deleted. Three gene copies (Crle20, 23, and 24) containing the Baculo PEP N domain of the polyhedron envelope protein were identified in CrleGV and other GV genomes. One of them (Crle23) appeared also to contain a p10-like sequence encoding of a number of leucine-rich heptad repeats and a proline-rich domain. Another striking feature of the genome is the presence of a hypervariable non-hr ori-like region of about 1800 bp consisting of different kinds of repeats and palindromes. Three other repeat-rich regions were identified within the genome and are considered as homologous regions (hrs). CrleGV is most closely related to the Cydia pomonella granulovirus (CpGV) as revealed by genome order comparisons and phylogenetic analyses. However, the AT content of the CrleGV genome, which is 67.6% and the highest found so far in baculoviruses, differed by 12.8% from the AT content of CpGV. This resulted in a major difference in the codon usage of both viruses and may reflect adaptive selection constraints to their particular hosts. (+info)