The complete genome of Rachiplusia nu nucleopolyhedrovirus (RanuNPV) and the identification of a baculoviral CPD-photolyase homolog.

We described a novel baculovirus isolated from the polyphagous insect pest Rachiplusia nu. The virus presented pyramidal-shaped occlusion bodies (OBs) with singly-embed nucleocapsids and a dose mortality response of 6.9 × 103 OBs/ml to third-instar larvae of R. nu. The virus genome is 128,587 bp long with a G + C content of 37.9% and 134 predicted ORFs. The virus is an alphabaculovirus closely related to Trichoplusia ni single nucleopolyhedrovirus, Chrysodeixis chalcites nucleopolyhedrovirus, and Chrysodeixis includens single nucleopolyhedrovirus and may constitute a new species. Surprisingly, we found co-evolution among the related viruses and their hosts at species level. Besides, auxiliary genes with homologs in other baculoviruses were found, e.g. a CPD-photolyase. The gene seemed to be result of a single event of horizontal transfer from lepidopterans to alphabaculovirus, followed by a transference from alpha to betabaculovirus. The predicted protein appears to be an active enzyme that ensures likely DNA protection from sunlight.

The deficiency in nuclear localization signal of Neodiprion lecontei nucleopolyhedrovirus DNA polymerase prevents rescue of viral DNA replication and virus production in dnapol-null Autographa californica multiple nucleopolyhedrovirus.

DNA polymerase (DNApol) is highly conserved in baculovirus and is required for viral DNA replication. However, little is known about gammabaculovirus DNApol. Here DNApol of the gammabaculovirus Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) was cloned into a dnapol-null alphabaculovirus AcMNPV bacmid, creating Bac-GFP-AcΔPol-NlPol. The resulting recombinant bacmid did not spread to neighboring cells, virus growth curve and real-time PCR revealed that NeleNPV dnapol substitution did not rescue AcMNPV DNA replication and virus production. Immunofluorescence microscopy revealed that NeleNPV DNApol was expressed but could not localize to the nucleus. Subsequently NeleNPV DNApol was fused to SpltNPV DNApol nuclear localization signal (NLS) and the fused DNApol could import into nucleus. The NLS-fusing NeleNPV DNApol was further transposed into the dnapol-null AcMNPV bacmid, creating Bac-GFP-AcΔPol-HA:NlPolNLS. The recombinant virus could replicate and produce infectious virus in Sf9 cells, albeit at reduced levels compared to wild type AcMNPV. Taken together, our results suggested that the NLS deficiency of NeleNPV DNApol blocked viral DNA replication and production of infectious virus in dnapol-null AcMNPV bacmid.

The structure of viral cathepsin from Bombyx mori Nuclear Polyhedrosis Virus as a target against grasserie: docking and molecular dynamics simulations.

The viral cathepsin from Bombyx mori Nuclear Polyhedrosis Virus (BmNPV-Cath) is a broad-spectrum protease that participates in the horizontal transmission of this virus in silkworm by facilitating solubilization of the integument of infected caterpillars. When a B. mori farm is attacked by BmNPV, there are significant sericultural losses because no drugs or therapies are available. In this work, the structure of viral cathepsin BmNPV-Cath was used as a target for virtual screening simulations, aiming to identify potential molecules that could be used to treat the infection. Virtual screening of the Natural Products library from the Zinc Database selected four molecules. Theoretical calculations of ΔGbinding by the molecular mechanics Poisson-Boltzmann surface analysis (MM-PBSA) method indicated that the molecule Zinc12888007 (Bm5) would have high affinity for the enzyme. The in vivo infection models of B. mori caterpillars with BmNPV showed that treatment with a dose of 100 µg Bm5 dissolved in Pluronic-F127 0.02% was able to reduce the mortality of caterpillars in 22.6%, however, it did not impede the liquefaction of dead bodies. Our results suggest a role of BmNPV-Cath in generating a pool of amino acids necessary for viral replication and indicate a mechanism to be exploited in the search for treatments for grasserie disease of the silkworm.

Analysis of synonymous codon usage bias in helicase gene from Autographa californica multiple nucleopolyhedrovirus.

The helicase gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not only involved in viral DNA replication, but also plays a role in viral host range. To identify the codon usage bias of helicase of AcMNPV, the codon usage bias of helicase was especially studies in AcMNPV and 41 reference strains of baculoviruses by calculating the codon adaptation index (CAI), effective number of codon (ENc), relative synonymous codon usage (RSCU), and other indices. The helicase of baculovirus is less biased (mean ENc = 50.539 > 40; mean CAI = 0.246). AcMNPV helicase has a strong bias toward the synonymous codons with G and C at the third codon position (GC3s = 53.6%). The plot of GC3s against ENc values revealed that GC compositional constraints are the main factor that determines the codon usage bias of major of helicase. Several indicators supported that the codon usage pattern of helicase is mainly subject to mutation pressure. Analysis of variation in codon usage and amino acid composition indicated AcMNPV helicase shows the significant preference for one or more postulated codons for each amino acid. A cluster analysis based on RSCU values suggested that AcMNPV is evolutionarily closer to members of group I alphabaculovirus. Comparison of the codon usage pattern among E. coli, yeast, mouse, human and AcMNPV showed that yeast is a suitable expression system for AcMNPV helicase. AcMNPV helicase shows weak codon usage bias. This study may help in elucidating the functional mechanism of AcMNPV helicase and the evolution of baculovirus helicases.

Significant differences in the intra-host genetic diversity of Helicoverpa armigera nucleopolyhedrovirus dnapol after serial in vivo passages in the same insect population.

A denaturing gradient gel electrophoresis assay was used to assess the genetic diversity within a region of the DNA polymerase gene (dnapol) in Helicoverpa armigera nucleopolyhedrovirus (HearNPV) populations over serial in vivo passages. There was no evidence of movement towards a consensus dnapol variant composition in the different host larvae after multiple per os passages. The study showed that the HearNPV variant structure after in vivo passages in the same host population is not necessarily convergent, and that it may be reasonable to expect significant differences in intra-host HearNPV genetic diversity after inoculation of larvae with a genotypically-diverse HearNPV inoculum.

Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function.

Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity.IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

A betabaculovirus DNA polymerase cannot substitute for the DNA polymerase of the alphabaculovirus Autographa californica nucleopolyhedrovirus.

DNA polymerase (DNApol) is present in all baculoviruses and plays a crucial role in viral DNA replication. Previously we showed that the DNApol of the alphabaculovirus group II Spodoptera litura nucleopolyhedrovirus (SpltNPV) could partially substitute for the DNApol of a group I alphabaculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, it is not known if a betabaculovirus DNApol could subsititute for the alphabaculovirus DNApol in AcMNPV. In this report, DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) was inserted into a dnapol-null AcMNPV bacmid, creating Bac-AcΔpol:PrPol. The repair virus did not spread to neighboring cells; virus growth curve and real-time PCR revealed that the PiraGV dnapol substitution abrogated AcMNPV DNA replication and virus production. Immunofluorescence microscopy showed that PiraGV DNApol could be expressed and localized to the nucleus. Collectively, our results suggested that the alphabaculovirus AcMNPV DNApol could not be replaced by a DNApol from the betabaculovirus, PiraGV.

Molecular insights into RNA-binding properties of Escherichia coli-expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.

Genome-Wide Analysis of Differentially Expressed microRNA in Bombyx mori Infected with Nucleopolyhedrosis Virus.

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV.

Rescue of dnapol-null Autographa californica multiple nucleopolyhedrovirus with DNA polymerase (DNApol) of Spodoptera litura nucleopolyhedrovirus (SpltNPV) and identification of a nuclear localization signal in SpltNPV DNApol.

DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. It determines the fidelity of baculovirus DNA replication by inserting the correct nucleotides into the primer terminus and proofreading any mispaired nucleotides. DNApols of groups I and II of the genus Alphabaculovirus in the family Baculoviridae share many common structural features. However, it is not clear whether a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNApol can be substituted by a group II NPV DNApol. Here we report the successful generation of AcMNPV dnapol-null virus being rescued by a group II Spodoptera litura NPV (SpltNPV) dnapol (Bac-AcΔPol : Slpol). Viral growth curves and quantitative real-time PCR showed that the dnapol replacement reduced the level of viral production and DNA replication of Bac-AcΔPol : SlPol compared with WTrep, a native dnapol insertion in an AcMNPV dnapol-null virus. Light microscopy showed that production of occlusion bodies for Bac-AcΔPol : Slpol was reduced. We also identified a nuclear localization signal (NLS) for the SpltNPV DNApol C terminus at residues 827-838 by mutational analysis and confocal microscopy. Multiple point substitution of SpltNPV DNApol NLS abrogated virus production and viral DNA replication. Overall, these data suggested that the NLS plays an important role in SpltNPV DNApol nuclear localization and that SpltNPV DNApol cannot efficiently substitute the AcMNPV DNApol in AcMNPV.

Expression of the Cydia pomonella granulovirus matrix metalloprotease enhances Autographa californica multiple nucleopolyhedrovirus virulence and can partially substitute for viral cathepsin.

The Cydia pomonella granulovirus open reading frame 46 (CpGV-ORF46) contains predicted domains found in matrix metalloproteases (MMPs), a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. We showed that CpGV-MMP was active in vitro. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing CpGV-ORF46 replicated similarly to a control virus lacking CpGV-ORF46 in cultured cells. The effects of AcMNPV expressing CpGV-MMP on virus infection in cultured cells and Trichoplusia ni larvae in the presence or absence of other viral degradative enzymes, cathepsin and chitinase, were evaluated. In the absence of cathepsin and chitinase or cathepsin alone, larval time of death was significantly delayed. This delay was compensated by the expression of CpGV-MMP. CpGV-MMP was also able to promote larvae melanization in the absence of cathepsin and chitinase. In addition, CpGV-MMP partially substituted for cathepsin in larvae liquefaction when chitinase, which is usually retained in the endoplasmic reticulum, was engineered to be secreted.

Baculovirus-induced tree-top disease: how extended is the role of egt as a gene for the extended phenotype?

Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a 'gene for an extended phenotype' is therefore not generally applicable for all baculovirus-host interactions. We hypothesize that in some baculovirus-host systems (including LdMNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour.

The Trichoplusia ni single nucleopolyhedrovirus tn79 gene encodes a functional sulfhydryl oxidase enzyme that is able to support the replication of Autographa californica multiple nucleopolyhedrovirus lacking the sulfhydryl oxidase ac92 gene.

The Autographa californica multiple nucleopolyhedrovirus ac92 is a conserved baculovirus gene with homology to flavin adenine dinucleotide-linked sulfhydryl oxidases. Its product, Ac92, is a functional sulfhydryl oxidase. Deletion of ac92 results in almost negligible levels of budded virus (BV) production, defects in occlusion-derived virus (ODV) co-envelopment and their inefficient incorporation into occlusion bodies. To determine the role of sulfhydryl oxidation in the production of BV, envelopment of nucleocapsids, and nucleocapsid incorporation into occlusion bodies, the Trichoplusia ni single nucleopolyhedrovirus ortholog, tn79, was substituted for ac92. Tn79 was found to be an active sulfhydryl oxidase that substituted for Ac92, resulting in the production of infectious BV, albeit about 10-fold less than an ac92-containing virus. Tn79 rescued defects in ODV morphogenesis caused by a lack of ac92. Active Tn79 sulfhydryl oxidase activity is required for efficient BV production, ODV envelopment, and their subsequent incorporation into occlusion bodies in the absence of ac92.

Autographa californica multiple nucleopolyhedrovirus DNA polymerase C terminus is required for nuclear localization and viral DNA replication.

UNLABELLED: The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE: The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication.

Genome segment 5 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes a bona fide guanylyltransferase.

BACKGROUND: Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized. RESULTS: In this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed K(m) of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol). CONCLUSION: Viral capping enzymes play a critical role in the post transcriptional or post replication modification in case of RNA virus. Our results of cloning, sequencing and functional analysis of AmCPV S5 indicates that S5 encoded p65 through its guanylyltransferase activity can transfer guanine residue to the 5' end of viral RNA for capping. Further studies will help to understand complete capping process of cypoviral RNA during viral replication within the viral capsid.

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus.

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.

Codon optimization enhances protein expression of Bombyx mori nucleopolyhedrovirus DNA polymerase in E. coli.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.

Expression and functional validation of Bombyx mori nucleopolyhedrovirus ORF29, a conserved Nudix motif protein.

Our previous study showed that Bombyx mori nucleopolyhedrovirus (BmNPV) orf29 encodes a 26 kDa protein expressed in the early stage of infection cycle. BmNPV ORF29, contains a conserved motif of Nudix (nucleotide diphosphate X) superfamily. It has the highest homology with ADP-ribose pyrophosphatase (ADPRase), a subfamily of Nudix pyrophosphatase. In this work, we purified the recombinant BmNPV ORF29 in Escherichia coli by metal chelating affinity chromatography. The amino acid sequence of recombinant protein was confirmed by mass spectroscopic analysis and found that the purified protein could be able to catalyze the breakdown of ADP-ribose to AMP and ribose 5-phosphate, with Km and Kcat values of 182 µmol/l and 5.3 s-1 respectively. The optimal activity was at alkaline pH (8.5) with Mg2+ (0.5-mmol/l) ions as the cofactor.

The baculovirus sulfhydryl oxidase Ac92 (P33) interacts with the Spodoptera frugiperda P53 protein and oxidizes it in vitro.

The Autographa californica M nucleopolyhedrovirus (AcMNPV) sulfhydryl oxidase Ac92 is essential for production of infectious virions. Ac92 also interacts with human p53 and enhances human p53-induced apoptosis in insect cells, but it is not known whether any relationship exists between Ac92 and native p53 homologs from insect hosts of AcMNPV. We found that Ac92 interacted with SfP53 from Spodoptera frugiperda in infected cells and oxidized SfP53 in vitro. However, Ac92 did not interact with or oxidize a mutant of SfP53 predicted to lack DNA binding. Silencing Sfp53 expression did not rescue the ability of an ac92-knockout virus to produce infectious virus. Similarly, ac92 expression did not affect SfP53-stimulated caspase activity or the localization of SfP53. Thus, although Ac92 binds to SfP53 during AcMNPV replication and oxidizes SfP53 in vitro, we could not detect any effects of this interaction on AcMNPV replication in cultured cells.

Characterization of a novel chitinase, DkChi, from Dendrolimus kikuchii nucleopolyhedrovirus.

Dendrolimus kikuchii Matsumura nucleopolyhedrovirus (DkNPV) is a novel nucleopolyhedrovirus strain that has exhibited high potential as biological control agent against D. kikuchii. In this work, a 1755-bp DkChi gene with sequence homology to a chitinase gene was cloned from the genomic DNA of DkNPV using a DNA fragment library. The DkChi gene, encoding 558 residues protein with a predicted mass of 61.6 kDa, was expressed at high levels in Escherichia coli and purified by affinity chromatography. We confirmed that the prepared protein was the DkChi protein by mass spectrometry analysis. Enzyme activity analysis showed that DkChi had both endo- and exo-chitinase activities. Interestingly, the DkChi protein displayed a strong insecticidal activity against Spodoptera exigua, Hyphantria cunea, Helicoverpa armigera and Lymantria dispar. The results suggest that DkChi is a good candidate protein for significantly contributing to pest control.