Plutella xylostella granulovirus late gene promoter activity in the context of the Autographa californica multiple nucleopolyhedrovirus genome.

Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus (PlxyGV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, and compared with homologous late gene promoters of AcMNPV in Sf9 cells. In transient expression assays, all PlxyGV late promoters were activated in cells transfected with the individual reporter plasmids together with an AcMNPV bacmid. In infected cells, reporter gene expression levels with the promoters of PlxyGV e18 and AcMNPV vp39 and gp41 were significantly higher than those of the corresponding AcMNPV or PlxyGV promoters, which had fewer late promoter motifs. Observed expression levels were lower for the PlxyGV p6.9, pk1, gran, p10a, and p10b promoters than for the corresponding AcMNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the AcMNPV polh, p10, and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of PlxyGV gran, p10c, and pk1. The results of this study demonstrated that PlxyGV late gene promoters could be effectively activated by the RNA polymerase from AcMNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.

[Optimized expression and functional analysis of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps)].

Objective: To explore the feasibility of using enhancin as synergist to Bacillus thuringiensis (Bt), the truncated fragments of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps) were optimized and the enhancing effects were studied. Methods: Based on bioinformational analysis of the function domain of PuGV-Ps enhancin, the prokaryotic expression vectors were constructed, and the protein expression levels as well as their enhancing effects on the degradation of peritrophic membrane (PM) proteins were analyzed, and the function domains of PuGV-Ps enhancin were confirmed. Results: Three domains were found in the enhancin of PuGV-Ps, including M60-like domain, Zincins catalytic domain and putative mucin or carbohydrate-binding domain. Thirteen predicted N-glycosylation sites were also identified. Based on the sequences of truncated M60-like domain (P69) and carbohydrate-binding domain (P77), two expression vectors, pET15b-P69 and pET15b-P77, were constructed. The expressed P69 and P77 abundance were higher than that of full length enhancing (P104). The degradation activity of purified P69 on the PM proteins of Spodoptera litura was higher than that of purified P77, but both showed lower degradation activities than P104. Both P69 and P77 improved the toxicity of Bt against larvae of Plutella xylostella. However, their synergistic effects were significantly lower than that of P104. Conclusion: The results revealed that the M60-like domain in N-terminus and carbohydrate-binding domain in C-terminus of PuGV-Ps enhancin all contributed to the enhancing effects of enhancin as well as the maintenance of its native conformation. The truncated P69 fragment may function in keeping the activity of enhacin and improving prokaryotic expression levels. These results provide some useful guidance for the industrialized production of enhancin.

Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38.

The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth.

[Functional analysis of the late expression factor genes of plutella xylostella granulovirus].

Plutella xylostella granulovirus (PlxyGV) contains homologs of 15 Autographa californica MNPV (AcMNPV) late expression factor (lef) genes. The prospective products of 14 PlxyGV lef genes (ie-0 is not included) share 13%-53% amino acid similarity with their corresponding homologs of AcMNPV, among which LEF-9, LEF-8 and P47, three subunits of the virus-encoded RNA polymerase, share 49%, 53% and 46% sequence identity, respectively. In this study, an established transient expression system was used to test the ability of the PlxyGV LEFs to activate an AcMNPV vp39 promoter-driven reporter gene in SF9 cells. It was shown that PlxyGV le f-2 replaced the corresponding AcMNPV gene and exhibited partial activity in the context of the remaining set of AcMNPV le fs. PlxyGV LEF-2 was found to contain additional 100aa and 70aa at the C-terminus in comparison with the LEF-2 of other GVs and lepidopteran NPVs respectively.

ODV-associated proteins of the Pieris rapae granulovirus.

Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus--host interaction studies.

Interaction of Xestia c-nigrum granulovirus with peritrophic matrix and Spodoptera litura nucleopolyhedrovirus in Spodoptera litura.

Xestia c-nigrum granulovirus (XcGV) was tested for its ability to increase Spodoptera litura nucleopolyhedrovirus (SINPV) infection in larvae of S. litura (F.). The interaction of XcGV with peritrophic matrix and SINPV in S. litura also was studied to account for the synergism. In dose-response bioassays with a constant XcGV concentration of 5-mg/ ml capsules and SINPV concentration that varied from 10(3) to 10(7) polyhedral inclusion bodies (PIB) per larva, XcGV increased the virulence of SINPV infection in fifth instars of S. litura. The lethal concentration of 50% individuals (LC50) of SINPV combined with XcGV was 3.35 x 10(5)PIB/ml, which was significantly lower than that of SINPV alone (2.17 x 10(6)). Compared with 10(7) PIB/ml SINPV alone, the lethal time of 50% individuals (LT50) of 10(7) PIB/ml SINPV combined with XcGV was not significantly shortened. In addition, no significant improvement in the activity and killing speed of SINPV progeny was noted after propagation with XcGV, indicating that native characters of SINPV associated with viral potency were not altered by XcGV. Investigation via environmental scanning electronic microscopy showed that the peritrophic matrix (PM) of S. litura exposed to XcGV or XcGV enhancin, or the combination treatment, was markedly disrupted. The outer surface of the PM was loose, or ruptured, which potentially facilitated the passage of virions through the PM. Therefore, it is reasonable to conclude that the synergy between XcGV and SINPV was closely associated with the disruption of the PM in S. litura.

Choristoneura fumiferana Granulovirus pk-1: a baculoviral protein kinase.

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk- 1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80 % amino acid identity.

Transcription, translation, and immunolocalization of ODVP-6E/ODV-E56 and p74 proteins: two highly conserved ODV-associated envelope proteins of Choristoneura fumiferana Granulovirus.

Choristoneura fumiferana granulovirus (ChfuGV) infection results two types of enveloped virions: Occlusion-derived virus (ODV) and budded virus (BV). Structural proteins ODVP-6E/ODV-E56 and p74 are two major conserved ODV-associated proteins that may be involved in the initiation of viral infection cycle in susceptible host insect larvae. This study presents the characterization of ChfuGV odvp-6e/odv-e56 and p74 transcription and translation as well as immunolocalization of these proteins in the occluded ChfuGV virion. Our results revealed that the transcription of odvp-6e/odv-e56 and p74 genes, both, start at 24 hours post infection (h p.i.). Using monospecific polyclonal antibodies made against ODVP-6E/ODV-E56 and p74 we demonstrated that these proteins are both expressed late in infection (24 h p.i.). Immunogold labeling using antisera against ODVP-6E/ODV-E56 and p74 proteins demonstrated that ODVP-6E/ODV-E56 and p74 proteins are both associated with the ODV envelop of ChfuGV.

Identification, characterization and phylogenic analysis of conserved genes within the p74 gene region of Choristoneura fumiferana granulovirus genome.

The genes located within the p74 gene region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing an 8.9 kb BamHI restriction fragment on the ChfuGV genome. The global guanine-cytosine (GC) content of this region of the genome was 33.02%. This paper presents the ORFs within the p74 gene region along with their transcriptional orientations. This region contains a total of 15 open reading frames (ORFs). Among those, 8 ORFs were found to be homologues to the baculoviral ORFs: Cf-i-p , Cf-vi, Cf-vii, Cf-viii (ubiquitin), Cf-xi (pp31), Cf-xii (lef-11), Cf-xiii (sod) and Cf-xv-p (p74). To date, no specific function has been assigned to the ORFs: Cf-i, Cf-ii, Cf-iii, Cf-iv, Cf-v, Cf-vi, Cf-vii, Cf-ix and Cf-x. The most noticeable ORFs located in this region of the ChfuGV genome were ubiquitin, lef-11, sod, fibrillin and p74. The phylogenetic trees (constructed using conceptual products of major conserved ORFs) and gene arrangement in this region were used to further examine the classification of the members of the granulovirus genus. Comparative studies demonstrated that ChfuGV along with the Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Adoxophyes orana granulovirus (AoGV) and Cryptophlebia leucotreta granulovirus (ClGV) share a high degree of amino acids sequence and gene arrangement preservation within the studied region. These results support a previous report, which classified a granuloviruses into 2 distinct groups: Group I: ChfuGV, CpGV, PhopGV and AoGV and Group II: Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The phylogenetic and gene arrangement studies also placed ClGV as a novel member of the Group I granuloviruses.

Identification and characterization of a putative baculoviral transcriptional factor IE-1 from Choristoneura fumiferana granulovirus.

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.

Identification and characterization of a conserved baculoviral structural protein ODVP-6E/ODV-E56 from Choristoneura fumiferana granulovirus.

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).

Expression of the Cydia pomonella granulovirus iap3 gene.

The IAP3 protein of Cydia pomonella granulovirus (CpGV) was the first identified member of the baculovirus IAP family of proteins, which have been shown to block apoptosis in diverse systems. However, little is known of the expression and subcellular localisation of CpGV IAP3 during a viral infection. This study examined IAP3 in cells infected by CpGV and in cells infected by an Autographa californica nucleopolyhedrovirus (AcMNPV) recombinant that carried the CpGV iap3 gene. The levels of iap3 specific transcripts were monitored and production of the protein was assessed using an IAP3-specific antiserum. The data showed that iap3 is expressed during both early and late phases of infection, with a switch occurring from distal early transcription start sites to proximal late start sites. Protein levels are highest after DNA replication. IAP3 is localised exclusively in the cytoplasm. Subcellular fractionation experiments demonstrated that the protein is present in both soluble and membrane-bound cytosolic fractions. The membrane-bound fraction includes IAP3 that is associated with the mitochondria. However, the data do not support the hypothesis that release of cytochrome C from the mitochondria is involved in baculovirus-induced apoptosis.