Nucleopolyhedrovirus Genome Research Articles

The DEAD/H-box helicase DHX9 contributes to suppression of Bombyx mori nucleopolyhedrovirus propagation in B. mori cells

Antiviral immune responses are mainly triggered through the recognition of virus-derived nucleic acids by host-specific pattern recognition receptors (PRRs). Here, we identified and characterized homologs of human PRRs for virus-derived DNA in Bombyx mori upon infection with a nucleopolyhedrovirus (NPV), a member of the family Baculoviridae. We found that progeny virus production of B. mori NPV was promoted in B. mori cells silenced with B. mori homolog of DEAD/H box polypeptide 9 gene (Bm-DHX9), but not in cells silenced with the other examined genes. Silencing of Bm-DHX9 expression has no effect on apoptosis induction, one of the major antiviral responses in B. mori cells. We also showed that Bm-DHX9 has the ability to bind DNA containing unmethylated C-phosphate-G-motif, which are characteristic of microbial pathogens and contained in the NPV genome with high frequency. Our findings suggest that Bm-DHX9 has the potential for sensing NPV-derived DNA to induce antiviral immune responses.

Improvement of protein production in baculovirus expression vector system by removing a total of 10 kb of nonessential fragments from Autographa californica multiple nucleopolyhedrovirus genome.

Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.

Identification and characterization of coiled-coil motifs across Autographa californica multiple nucleopolyhedrovirus genome

Coiled coils (CCs) are protein structural motifs universally found in proteins and mediate a plethora of biological interactions, and thus their reliable annotation is crucial for studies of protein structure and function. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus and encodes 154 proteins. In this study, genome-wide scans of previously uncharacterized CC motifs throughout AcMNPV was conducted using CC prediction software. In total, 24 CC motifs in 19 CC proteins with high confidence were identified. The characteristic of viral CC motifs were analyzed. The CC proteins could be divided into 12 viral structural proteins and 7 non-structural proteins, including viral membrane fusion proteins, enzymes, and transcription factors. Moreover, CC motifs are conserved in the baculoviral orthologs of 14 of the 19 proteins. It is noted that five CC proteins, including Ac51, Ac66, Exon0, Ac13, and GP16, were previously identified to function in the nuclear egress of nucleocapsids, and Ac66 contains multiple CC motifs, the longest of which comprises 252 amino acids, suggesting a role of CC motifs in this process. Taken together, the CC motifs identified in this study are valuable resource for studying protein function and protein interaction networks during virus replication.

Identification and Characterization of Coiled-Coil Motifs Across Autographa Californica Multiple Nucleopolyhedrovirus Genome

Identification and Characterization of Coiled-Coil Motifs Across Autographa Californica Multiple Nucleopolyhedrovirus Genome

Loss of p24 from the Bombyx mori nucleopolyhedrovirus genome results in the formation of cuboidal occlusion bodies

Some insect viruses produce the occlusion body (OB), a large crystalline particle comprising a viral protein that occludes virions to protect them from harsh environments. The shapes and sizes of OBs are diverse depending on baculovirus species, but the detailed molecular mechanism determining them has yet to be totally clarified yet. Here we generated Bombyx mori nucleopolyhedrovirus (BmNPV) mutants of the p24 gene that encodes a viral capsid protein and found that p24-mutated BmNPVs produced cuboidal OBs with a slightly larger size than typical truncated octahedral OBs produced by wild-type BmNPVs. Meanwhile, p24 disruption has no significant impact on progeny virus production and viral pathogenicity. In addition, we experimentally demonstrated that a single amino acid substitution found in the P24 protein of the BmNPV Cubic isolate caused cuboidal OB production. These results suggest that p24 has a crucial role in generating the typical shape of OBs.

Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHORTM DNA Labeling Technology.

Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues.

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.

Transgenic expression of Dendrolimus kikuchii nucleopolyhedrovirus enhancin in tobacco enhances the mortality and affects the development of Spodoptera exigua larvae

ABSTRACTA novel 2551-bp enhancin gene designated En-Dk was isolated from the Dendrolimus kikuchii nucleopolyhedrovirus genome. Sequence analysis revealed that En-Dk encodes a polypeptide chain of 792 amino acid residues containing a conserved enhancin domain and an α-helical transmembrane domain, but no signal peptide. Compared with wild-type tobacco (Nicotiana tabacum L.), transgenic tobacco showed significant phenotypic changes following En-Dk overexpression with slower growth and delayed initial flowering. An interesting finding is that the transgenic tobacco exerted a toxic effect on newly hatched Spodoptera exigua larvae, with a significant inhibitory effect on third-instar larvae in terms of body weight, pupal weight, pupation and the emergence process.

Complete Sequence, Analysis and Organization of the Orgyia leucostigma Nucleopolyhedrovirus Genome

The complete genome of the Orgyia leucostigma nucleopolyhedrovirus (OrleNPV) isolated from the whitemarked tussock moth (Orgyia leucostigma, Lymantridae: Lepidoptera) was sequenced, analyzed, and compared to other baculovirus genomes. The size of the OrleNPV genome was 156,179 base pairs (bp) and had a G+C content of 39%. The genome encoded 135 putative open reading frames (ORFs), which occupied 79% of the entire genome sequence. Three inhibitor of apoptosis (ORFs 16, 43 and 63), and five baculovirus repeated ORFs (bro-a through bro-e) were interspersed in the OrleNPV genome. In addition to six direct repeat (drs), a common feature shared among most baculoviruses, OrleNPV genome contained three homologous regions (hrs) that are located in the latter half of the genome. The presence of an F-protein homologue and the results from phylogenetic analyses placed OrleNPV in the genus Alphabaculovirus, group II. Overall, OrleNPV appears to be most closely related to group II alphabaculoviruses Ectropis obliqua (EcobNPV), Apocheima cinerarium (ApciNPV), Euproctis pseudoconspersa (EupsNPV), and Clanis bilineata (ClbiNPV).

Mass identification of transcriptional units expressed from the Bombyx mori nucleopolyhedrovirus genome

In order to identify the transcriptional units expressed from an entire nucleopolyhedrovirus (NPV) genome during infection, we constructed a full-length-enriched cDNA library from Bombyx mori NPV (BmNPV)-infected BmN cells. We randomly sequenced 11,520 clones from both ends to obtain a total of 4679 BmNPV-derived transcriptional units. The data revealed a number of novel transcripts, including putative non-coding RNAs, most of which are expressed from recognized baculovirus early or late promoter motifs. These findings provide new insights into the complex transcriptional regulation of an NPV genome and suggest roles for as-yet-uncharacterized transcripts.

Hycu-hr6, A large homologous region of the Hyphantria cunea nucleopolyhedrovirus genome, as a powerful and versatile enhancer in insect expression systems

We previously identified a large homologous region (hr), hycu-hr6, in the genome of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) and suggested that hycu-hr6 was the largest baculovirus promoter enhancer hr identified so far. In this study, we examined the enhancement activity of hycu-hr6 against two promoters from constitutive baculovirus immediate early genes, the HycuNPV ie1 (hycu-ie1) and the Orgyia pseudotsugata multicapsid (M) NPV ie2 (op-ie2), and against a promoter from the inducible Drosophila heat shock protein 70 gene (hsp70) in five lepidopteran (BmN-4, Ld652Y, Sf9, SpIm, and TN368) and one dipteran (S2) insect cell lines. Comparative analyses of transient expression assays using the firefly luciferase gene (luc) as a reporter showed that hycu-hr6 enhanced the activity of all three promoters in all tested cell lines. Comparison of the enhancement efficiency of hycu-hr6 with that of Autographa californica MNPV (AcMNPV) hr5, an enhancer commonly used to improve the activity of AcMNPV ie1 promoter in many insect expression vectors, established that hycu-hr6 was a more efficient enhancer. These results indicate that hycu-hr6 is a versatile and superior promoter enhancer in several insect cell lines and favor the incorporation of hycu-hr6 into insect expression vectors to maximize promoter activity.

Characterization of theHelicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene region

A local strain of Helicoverpa assulta nucleopolyhedrovirus (HasNPV) was isolated from infected H.assulta larvae in Korea. Restriction endonuclease fragment analysis, using 4 restriction enzymes, estimated that the total genome size of HasNPV is about 138 kb. A degenerate polymerase chain reaction (PCR) primer set for the polyhedrin gene successfully amplified the partial polyhedrin gene of HasNPV. The sequencing results showed that the about 430 bp PCR product was a fragment of the corresponding polyhedrin gene. Using HasNPV partial predicted polyhedrin to probe the Southern blots, we identified the location of the polyhedrin gene within the 6 kb Eco RI, 15 kb Nco I, 20 kb Xho I, 17 kb Bgl II and 3 kb Cla I fragments, respectively. The 3 kb Cla I fragment was cloned and the nucleotide sequences of the polyhedrin coding region and its flaking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame of 735 nucleotides which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of HasNPV polyhedrin shared 73.7% identity with the polyhedrin gene from Autographa californica NPV but were most closely related to Helicoverpa and Heliothis species NPVs with over 99% sequence identity.

Gene organization and complete sequence of the Hyphantria cunea nucleopolyhedrovirus genome

The whole-genome sequence of the Hyphantria cunea nucleopolyhedrovirus (HycuNPV) was analysed. The entire nucleotide sequence of the HycuNPV genome was 132 959 bp long, with a G+C content of 45.1 mol%. A total of 148 open reading frames (ORFs) consisting of more than 50 aa were encoded by the genome. HycuNPV shares more than 122 ORFs with other lepidopteran group I NPVs, including Autographa californica MNPV, Bombyx mori NPV, Choristoneura fumiferana MNPV (CfMNPV), Choristoneura fumiferana defective NPV, Epiphyas postvittana MNPV and Orgyia pseudotsugata MNPV (OpMNPV). Six ORFs are identified as being unique to HycuNPV. Most of the HycuNPV ORFs showed higher similarity to CfMNPV and OpMNPV ORFs than to those of the other group I NPVs. HycuNPV encodes two conotoxin-like homologues (ctls), which are observed only in OpMNPV in group I NPVs. HycuNPV encodes three inhibitors of apoptosis (iaps), hycu-iap-1, hycu-iap-2 and hycu-iap-3, a feature that it shares only with CfMNPV. In addition, six homologous regions (hrs) are identified in the HycuNPV genome. These hrs are located in regions similar to those of the OpMNPV hrs, but different from most of the CfMNPV hrs. Based on the close phylogenetic relationship and conservation of group I NPV-specific genes, such as gp64, ie-2 and ptp-1, it is concluded that HycuNPV belongs to the group I NPVs and is most similar to CfMNPV or OpMNPV.

Gene organization and sequencing of the Choristoneura fumiferana defective nucleopolyhedrovirus genome

Two distinct nucleopolyhedrovirus species of the eastern spruce budworm, Choristoneura fumiferana, exist in a symbiont-like relationship. C. fumiferana defective nucleopolyhedrovirus (CfDEFNPV) only infects C. fumiferana larvae per os in the presence of C. fumiferana nucleopolyhedrovirus Ireland strain (CfMNPV), but is infective when injected into the haemolymph. CfDEFNPV synergizes CfMNPV in per os infections and CfMNPV is always the predominant progeny. This study was undertaken to report the genomic makeup and organization of CfDEFNPV in an attempt to identify its defect and understand its synergistic role. The genome was mapped, sequenced, characterized and compared to other baculoviruses. The CfDEFNPV genome was 131,160 nt long with 149 putative open reading frames (ORFs) and a G + C content of 45.8 mol%. Homologues of all 62 conserved lepidopteran baculovirus genes were found including those implicated in per os infectivity, p74, per os infectivity factor (pif) and pif-2. Although no obvious deletions were observed to explain the defect, two ORFs, Cfdef79 and Cfdef99 (inhibitor of apoptosis-4), contained potential deletions. Cfdef50 (late expression factor-10)/Cfdef51 (vp1054) and Cfdef76/Cfdef77 (telokin-like protein) had large overlaps and a potential homologue to ac105/he65 was split. Four baculovirus repeat ORFs were present, as were two unique genes, but no enhancins were identified. CfDEFNPV contained 13 homologous regions, each with one to five palindromes. Comparison with fully sequenced baculovirus genomes identified CfDEFNPV as a group I NPV with the closest average amino acid identity to Epiphyas postvittana NPV, followed by Orgyia pseudotsugata MNPV and CfMNPV, with its closest matches being to individual Anticarsia gemmatalis MNPV gene sequences.

Sequence and Organization of the Mamestra configurata Nucleopolyhedrovirus Genome

The nucleotide sequence of the genome of the nucleopolyhedrovirus (NPV) from Mamestra configurata (MacoNPV, isolate 90/2), a group II NPV, was determined and analyzed. The MacoNPV DNA genome consists of 155,060 bp and has an overall G+C content of 41.7%. Computer-assisted analysis predicted 169 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. BLAST searches and comparisons with completely sequenced baculoviruses indicated that there were 66 ORFs conserved among the nine baculoviruses compared and an additional 17 ORFs were conserved among the NPVs. The gene content and gene arrangement in MacoNPV were most similar to those of SeMNPV, including two putative odv-e66 and p26 gene homologues. However, in contrast to SeMNPV, 8 ORFs with homology to baculovirus repeat ORFs (bro) and single copies of enhancin and conotoxin-like protein ORFs were found in MacoNPV. The MacoNPV genome contained four homologous regions, each with 10 to 17 repeated sequences. Each repeat was 60 to 86 nucleotides in length and contained an approximately 43-bp-long imperfect palindrome. There were 13 ORFs unique to MacoNPV, ranging from a small ORF of 196 bp to larger ORFs of up to 1047 bp, and many of these contained typical early and late baculovirus consensus promoters.

Physical and genetic map of the Wiseana nucleopolyhedrovirus genome.

Wiseana nucleopolyhedrovirus (NPV) is the major pathogen of the New Zealand endemic pasture pest, Wiseana spp. To characterize this potential biological control agent, the genome of a virus isolated from Wiseana signata was purified and cloned. The complete genome was cloned as BamHI or HindIII restriction fragments, which were mapped by Southern hybridization and restriction analysis. To verify the physical map, the junctions between all HindIII fragments were confirmed by sequencing. The viral genome was estimated to be 128 kbp. Sequence data generated at the termini of cloned restriction fragments were compared to sequence databases to identify putative gene homologues. Seventeen putative ORFs, which were homologous to other baculoviral sequences, were identified. These putative ORFs were located on the Wiseana NPV physical map and their distribution was compared to genetic maps of NPVs isolated from Autographa californica, Orgyia pseudotsugata and Lymantria dispar. Although the virus from W. signata was significantly different from these other NPVs, a core region of the viral genome was conserved.

Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143).

LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays. LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases. In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses. Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165. Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.

Mapping and polyhedrin gene analysis of the Epiphyas postvittana nucleopolyhedrovirus genome.

The light brown apple moth, Epiphyas postvittana, is a major insect pest of a variety of fruit crops grown in New Zealand and we are studying a nucleopolyhedrovirus, EppoNPV, isolated from this insect. Restriction endonuclease analysis of EppoNPV DNA shows that this is a single strain of virus with a genome size of approximately 119 kbp and a complete library of the EppoNPV genome has been cloned. A strategy of single-stranded sequencing of the termini of REN fragment clones was employed to map the virus genome. Sequence homologies to NPV gene sequences present in the GenBank database allowed a nearly complete restriction map of the EppoNPV genome to be constructed. The mapping was completed with Southern blotting and restriction analysis. Fifty-five open reading frames (ORFs) with similarity to genes from other NPVs have been identified and placement of these on the restriction map shows that EppoNPV has a nearly identical genome organization to Orgyia pseudotsugata (Op)MNPV. The polyhedrin gene of EppoNPV has been fully sequenced and an ORF of 738 bp encodes a predicted protein of 28.8 kDa. The conserved 12 bp consensus sequence typical of very late baculovirus gene promoters, AATAAGTAATTT, has been located upstream of the ATG initiation codon. An ORF located downstream of the polyhedrin gene shows homology to the 1629-capsid protein from OpMNPV. Phylogenetic comparison to polyhedrin gene sequences from 23 other NPVs shows EppoNPV to be a group I NPV closely related to OpMNPV.