Virus-host coevolutionary analyses of an Alphabaculovirus with a wide host range.

Baculoviruses are highly host specific, and their host range is usually restricted to a single or a few closely related insect species, except for few virus species, e.g. Alphabaculovirus aucalifonicae and Alphabaculovirus mabrassicae. In this study, two new alphabaculovirus isolates were isolated from the larvae of Mamestra brassicae and Mythimna separata, which were named as Mamestra brassicae multiple nucleopolyhedrovirus isolate QD (MbMNPV-QD) and Mythimna separata multiple nucleopolyhedrovirus isolate Hb (MyseMNPV-Hb), respectively. The Kimura two-parameter values based on the concatenated 38 core genes of baculovirus revealed that MbMNPV (isolates QD/CHb1/K1/CTa), MyseMNPV-Hb, Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) and Mamestra configurata nucleopolyhedrovirus B (MacoNPV-B) were different isolates of a same virus species. A phylogenetic tree of baculoviruses and nudiviruses constructed from their 20 homologous gene sequences, and that of their isolated hosts constructed from 13 protein-coding genes of the insect mitochondrial genomes, were used to analyse the coevolution of baculoviruses with their isolated hosts. The results showed that M. brassicae was the most likely ancestral host of these virus isolates, included MbMNPV isolates, MyseMNPV-Hb, HearMNPV, and MacoNPV-B. Therefore, we concluded that these virus isolates belong to the existing virus species - Alphabaculovirus mabrassicae with M. brassicae as their ancestral host.

Phosphorylation of the Autographa californica multiple nucleopolyhedrovirus polyhedron envelope protein plays an important role in the formation of the occlusion body envelope.

During the life cycle of a baculovirus, a crystallized protein matrix, formed by polyhedrin (POLH), is produced. The protein matrix is surrounded by a multilayered protein/carbohydrate envelope, and matrix and envelope together form a mature occlusion body (OB). The polyhedron envelope plays an important role in resistance against adverse external environments. The polyhedron envelope protein (PEP) is the main protein that forms the polyhedron envelope, but the mechanism of formation of the polyhedron envelope is unclear. Here, through immunofluorescence localization observations, we found that PEP interacted with both POLH and P10 during formation of the polyhedron envelope in the late stages of infection, and PEP was also required for P10 incorporation on the surface of OBs. In this process, the phosphorylation of PEP played an important role. PEP was determined to be a phosphorylated protein using the Phos-tag technique, and PK1 was determined to be the phosphokinase of PEP by co-immunoprecipitation and in vitro phosphorylation. Immunofluorescence localization revealed that PEP was continuously phosphorylated by PK1 after PEP entered the nucleus until PEP was correctly packaged on the OB surface. Multi-point mutations of PEP conservative potential phosphorylation sites showed that the simultaneous mutation of S85, T86 and Y92 caused changes in the location of PEP and P10 in the late stages of infection, and resulted in an OB surface that lacked the polyhedron envelope. These data suggested that the phosphorylation of PEP at particular sites, i.e. S85, T86 and Y92, plays an important role in the formation of the polyhedron envelope.

Genomic analysis of two Chinese isolates of hyphantria cunea nucleopolyhedrovirus reveals a novel species of alphabaculovirus that infects hyphantria cunea drury (lepidoptera: arctiidae).

BACKGROUND: Baculoviruses act as effective biological control agents against the invasive pest Hyphantria cunea Drury. In this study, two Chinese Hyphantria cunea nucleopolyhedrovirus (HycuNPV) isolates, HycuNPV-BJ and HycuNPV-HB, were deep sequenced and compared with the Japanese isolate, HycuNPV-N9, to determine whole-genome level diversity and evolutionary history. RESULTS: The divergence of the phylogenetic tree and the K2P distances based on 38 core-gene concatenated alignment revealed that two Chinese HycuNPV isolates were a novel species of Alphabaculovirus that infected Hyphantria cunea in China. The gene contents indicated significant differences in the HycuNPV genomes between the Chinese and Japanese isolates. The differences included gene deletions, acquisitions and structural transversions, but the main difference was the high number of single nucleotide polymorphisms (SNPs). In total, 10,393 SNPs, corresponding to approximately 8% of the entire HycuNPV-N9 genome sequence, were detected in the aligned reads. By analyzing non-synonymous variants, we found that hotspot mutation-containing genes had mainly unknown functions and most were early expressing genes. We found that the hycu78 gene which had early and late promoter was under positive selection. Biological activity assays revealed that the infectivity of HycuNPV-HB was greater than that of HycuNPV-BJ, and the killing speed of HycuNPV-HB was faster than that of HycuNPV-BJ. A comparison of molecular genetic characteristics indicated that the virulence differences between the two isolates were affected by SNP and structural variants, especially the homologous repeat regions. CONCLUSIONS: The genomes of the two Chinese HycuNPV isolates were characterized, they belonged to a novel species of Alphabaculovirus that infected Hyphantria cunea in China. We inferred that the loss or gain of genetic material in the HycuNPV-HB and HycuNPV-BJ genomes resulted in new important adaptive capabilities to the H. cunea host. These results extend the current understanding of the genetic diversity of HycuNPV and will be useful for improving the applicability of this virus as a biological control agent.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

A Conserved Phenylalanine Residue of Autographa Californica Multiple Nucleopolyhedrovirus AC75 Protein Is Required for Occlusion Body Formation.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene that is essential for AcMNPV propagation. However, the key domains or residues of the AC75 protein that play a role in viral propagation have not been identified. In this study, sequence alignment revealed that residues Phe-54 and Gln-81 of AC75 were highly conserved among alphabaculoviruses and betabaculoviurses. Thus, Phe-54 and Gln-81 AC75 mutation bacmids were constructed. We found that Gln-81 was not required for viral propagation, whereas mutating Phe-54 reduced budded virus production by 10-fold and impaired occlusion body formation when compared with that of the wild-type AcMNPV. Electron microscopy observations showed that the Phe-54 mutation affected polyhedrin assembly and also occlusion-derived virus embedding, whereas western blot analysis revealed that mutating Phe-54 reduced the amount of AC75 but did not affect the localization of AC75 in infected cells. A protein stability assay showed that the Phe-54 mutation affected AC75 stability. Taken together, Phe-54 was identified as an important residue of AC75, and ac75 is a pivotal gene in budding virus production and occlusion body formation.

Comparison of Lethal Doses Calculated Using Logit/Probit-Log(Dose) Regressions With Arbitrary Slopes Using R.

The median lethal dose (LD50) is commonly used to indicate acute toxicity of an insecticide to an insect species. Approximate confidence intervals for LD50s are often calculated using the Fieller and delta methods. It is often necessary to compare the relative potencies of several insecticides with a population or of one insecticide with different populations. Comparing the LD50s using probit/logit-log(dose) regressions with parallel slopes can be implemented in many software packages, but for the cases with arbitrary slopes are not generally available. We used the glm function in R to calculate and compare lethal doses without assuming equal slopes. Bioassay datasets from the literature fitted using the logit model gave the 95% confidence limits (95% CLs) for the lethal doses using Fieller's theorem and incorporating a heterogeneity factor identical to the 95% CLs determined using the PoloPlus software. The delta method gave 95% CLs identical to the 95% CLs determined using the R drc package. The same datasets fitted using the probit model gave 95% CLs similar to the 95% CLs determined using PoloPlus and the drc package. The natural response rates for the control group were included using Abbott's equation. When the potency ratio method and the z-test were used to identify differences between two lethal doses, and when the χ2 and log likelihood ratio tests were used to determine whether the regression lines were parallel, the conclusions were the same as those gave by PoloPlus and the drc package.

Autographa Californica Multiple Nucleopolyhedrovirus orf13 Is Required for Efficient Nuclear Egress of Nucleocapsids.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study, we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif, colocalized with the nuclear lamina. Deletion of ac13 did not affect viral genome replication, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the OB morphogenesis was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.

Molecular and Biological Characterization of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Field Isolate and Genotypes from China.

The fall armyworm, Spodoptera frugiperda, is a new invading pest in China. The baculovirus Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a pathogenic agent of the fall armyworm and a potential agent for its control in integrated pest management strategies. In this work, we analyze the molecular and biological characteristics of an SfMNPV isolate collected from maize in China (SfMNPV-Hub). Two genotypes were further isolated from SfMNPV-Hub by an in vivo cloning method. The PstI profile of one genotype (SfHub-A) was similar to genotype A of the SfMNPV Colombian isolate, and the other (SfHub-E) was similar to genotype E of the Colombian isolate. The bioactivity of SfHub-A against second-instar S. frugiperda larvae was not significantly different from that of SfMNPV-Hub, whereas SfHub-E was 2.7-5.5 fold less potent than SfMNPV-Hub. The speed of kill of SfHub-E was quicker than SfMNPV-Hub, while SfHub-A acted slower than SfMNPV-Hub. Occlusion body (OB) production of SfHub-A in an S. frugiperda cadaver was significantly higher than that of SfMNPV-Hub, while SfHub-E yielded far fewer occlusion bodies (OBs) in the host larvae. These results provide basic information for developing a virus-based pesticide against the invading pest S. frugiperda.

Increased Excess Intracellular Cyclic di-AMP Levels Impair Growth and Virulence of Bacillus anthracis.

Cyclic di-AMP (c-di-AMP) is a recently identified bacterial second messenger that regulates biological processes. In this study, we found that inactivation of two c-di-AMP phosphodiesterases (PDEs), GdpP and PgpH, resulted in accumulation of 3.8-fold higher c-di-AMP levels than in the parental strain Sterne in Bacillus anthracis and inhibited bacterial growth. Moreover, excess c-di-AMP accumulation decreased bacterial toxin expression, increased sensitivity to osmotic stress and detergent, and attenuated virulence in both C57BL/6J and A/J mice. Complementation of the PDE mutant with a plasmid carrying gdpP or pgpH in trans from a Pspac promoter restored bacterial growth, virulence factor expression, and resistance to detergent. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in B. anthracis that is important for host-pathogen interaction.IMPORTANCE Anthrax is an ancient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis Vegetative cells of this species produce anthrax toxin proteins and S-layer components during infection of mammalian hosts. So far, how the expression of these virulence factors is regulated remains largely unknown. Our results suggest that excess elevated c-di-AMP levels inhibit bacterial growth and reduce expression of S-layer components and anthracis toxins as well as reduce virulence in a mouse model of disease. These results indicate that c-di-AMP signaling plays crucial roles in B. anthracis biology and disease.

Displaying enhancing factors on the surface of occlusion bodies improves the insecticidal efficacy of a baculovirus.

BACKGROUND: Baculoviruses provide long-lasting control of crop pests and are harmless to humans and non-target animals, making them attractive bioinsecticides. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has a wide-host range and is one such commercial bioinsecticide, but its low infectivity to older larvae and less-sensitive species precludes its large-scale application. We sought to improve the infectivity of AcMNPV. RESULTS: Two enhancing factors, the truncated enhancin from Agrotis segetum granulovirus and GP37 from Cydia pomonella granulovirus, were expressed in fusion with the N-terminal and middle domain of the polyhedrin envelope protein of AcMNPV. Western blotting and immunoelectron microscopy analysis indicated that the enhancing factors were expressed on the occlusion bodies of the resulting AcMNPV variants. Bioassays showed that the median lethal doses of the recombinant viruses were 3.9-fold to 7.4-fold lower than those of the wild-type virus against the second and fourth instar of Spodoptera exigua larvae. The yields of occlusion bodies from the two recombinants in S. exigua larvae were comparable with those of the wild-type virus both in vitro and in vivo. Further bioassays showed that the AcMNPV variants fusing the enhancing factors were incapable of infecting the second instar larvae of S. litura, Helicoverpa armigera, and Pyrausta nubilalis, which were not sensitive to the wild-type AcMNPV. CONCLUSION: These genetically modified AcMNPV variants exhibited an enhanced infectivity and may offer better baculovirus control of crop pests. © 2019 Society of Chemical Industry.

NSP2 forms viroplasms during Dendrolimus punctatus cypovirus infection.

Reoviruses are thought to replicate and assemble in special cytoplasmic structures called 'viroplasms'. However, little is known about the viroplasms of the insect reoviruses, the cypoviruses. To investigate the viroplasm of Dendrolimus punctatus cypovirus (DpCPV), all proteins encoded by the 10 genomic segments of DpCPV were expressed in Sf9 cells using the Bac-to-Bac system. The viral nonstructural protein NSP2 formed viroplasm-like dots which showed close apposition with the endoplasmic reticulum and were surrounded by intracellular membranes during transfection. Colocalization and coimmunoprecipitation assays showed that NSP2 interacts with 4 of 6 structural proteins and another 2 nonstructural proteins, while NSP1 only colocalized with VP4, and NSP3 did not colocalize with any structural protein. Immunoelectron microscopy revealed that NSP2 were nearby the endoplasmic reticulum and mitochondria, and viral particles were present in the electron-dense inclusions formed by NSP2. We proposed that NSP2 is responsible for forming the viroplasms structures of DpCPV.

A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase.

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5' and 3' untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

Granulovirus GP37 Facilitated ODVs Cross Insect Peritrophic Membranes and Fuse with Epithelia.

The Cydia pomonella granulovirus (CpGV) GP37 has synergistic effects on the infectivity of nucleopolyhedroviruses (NPVs), however, the mechanism employed is unclear. In this study, in vitro and in vivo binding assays indicated that GP37 efficiently bound to the midgut peritrophic membrane (PM) of Spodoptera exigua larvae. Treatment with GP37 led to the damage of the PM's compacted structure and the generation of the PM perforations, and the enhancement of the PM's permeability. qPCR results further demonstrated that GP37 increased the ability of occlusion-derived virions (ODV) to cross the PM. R18-labeling experiments exhibited that GP37 also promoted the fusion of ODVs and insect midgut epithelia. Altogether, our present results revealed that the synergistic mechanism of GP37 to the infectivity of NPV might involve two parts. GP37 damaged the integrity of the PM after binding, which enhanced the PM's permeability and increased the ability of ODVs to cross the PM, finally facilitating the ODVs reaching the midgut. In addition, GP37 promoted the fusion of ODVs and insect midgut epithelia. Our data expand the understanding of the mechanism used by baculovirus synergistic factors and provide a foundation for the development of high-efficiency baculoviral insecticides.

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis.

The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

Autographa Californica Multiple Nucleopolyhedrovirus Enters Host Cells via Clathrin-Mediated Endocytosis and Direct Fusion with the Plasma Membrane.

The cell entry mechanism of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not fully understood. Previous studies showed that AcMNPV entered host cells primarily through clathrin-mediated endocytosis, and could efficiently infect cells via fusion with the plasma membrane after a low-pH trigger. However, whether AcMNPV enters cells via these two pathways simultaneously, and the exact manner in which AcMNPV particles are internalized into cells remains unclear. In this study, using single-virus tracking, we observed that AcMNPV particles were first captured by pre-existing clathrin-coated pits (CCP), and were then delivered to early endosomes. Population-based analysis of single-virus tracking and quantitative electron microscopy demonstrated that the majority of particles were captured by CCPs and internalized via invagination. In contrast, a minority of virus particles were not delivered to CCPs, and were internalized through direct fusion with the plasma membrane without invagination. Quantitative electron microscopy also showed that, while inhibition of CCP assembly significantly impaired viral internalization, inhibition of endosomal acidification blocked virus particles out of vesicles. Collectively, these findings demonstrated that approximately 90% of AcMNPV particles entered cells through clathrin-mediated endocytosis and 10% entered via direct fusion with the plasma membrane. This study will lead toward a better understanding of AcMNPV infection.

Comparing lethal dose ratios using probit regression with arbitrary slopes.

BACKGROUND: Evaluating the toxicity or effectiveness of two or more toxicants in a specific population often requires specialized statistical software to calculate and compare median lethal doses (LD50s). Tests for equality of LD50s using probit regression with parallel slopes have been implemented in many software packages, while tests for cases of arbitrary slopes are not generally available. METHODS: In this study, we established probit-log(dose) regression models and solved them by the maximum likelihood method using Microsoft Excel. The z- and χ2-tests were used to assess significance and goodness of fit to the probit regression models, respectively. We calculated the lethal doses (LDs) of the toxicants at different significance levels and their 95% confidence limits (CLs) based on an accurate estimation of log(LD) variances. We further calculated lethal dose ratios and their 95% CLs for two examples without assuming parallel slopes following the method described by Robertson, et al., 2017. RESULTS: We selected representative toxicology datasets from the literature as case studies. For datasets without natural responses in the control group, the slopes, intercepts, χ2 statistics and LDs calculated using our method were identical to those calculated using Polo-Plus and SPSS software, and the 95% CLs of the lethal dose ratios between toxicants were close to those calculated using Polo-Plus. For datasets that included natural responses in the control group, our results were also close to those calculated using Polo-Plus and SPSS. CONCLUSION: This procedure yielded accurate estimates of lethal doses and 95% CLs at different significance levels as well as the lethal dose ratios and 95% CLs between two examples. The procedure could be used to assess differences in the toxicities of two examples without the assumption of parallelism between probit-log(dose) regression lines.

Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies.

The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity.IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

MTase Domain of Dendrolimus punctatus cypovirus VP3 Mediates Virion Attachment and Interacts with Host ALP Protein.

Dendrolimus punctatus cypovirus (DpCPV) is an important pathogen of D. punctatus, but little is known about the mechanisms of DpCPV infection. Here, we investigated the effects of VP3, VP4 and VP5 structural proteins on the viral invasion. Both the C-terminal of VP3 (methyltransferase (MTase) domain) and VP4 (A-spike) bound to Spodoptera exigua midgut brush border membrane vesicles (BBMVs) in a dose-dependent manner, and the binding was inhibited by purified DpCPV virions. Importantly, anti-MTase and anti-VP4 antibodies inhibited viral binding to S. exigua BBMVs. Using far-Western blots, a 65 kDa protein in Bombyx mori BBMVs, identified as alkaline phosphatase protein (BmALP) by mass spectrometry, specifically interacted with DpCPV MTase. The interaction between MTase and BmALP was verified by co-immunoprecipitation in vitro. Pretreatment of B. mori BBMVs with an anti-ALP antibody or incubation of DpCPV virions with prokaryotically expressed BmALP reduced viral attachment. Additionally, BmALP inhibited DpCPV infection in S. exigua larvae. Our data provide evidence that the MTase domain and A-spike function as viral attachment proteins during the DpCPV infection process, and ALP is the ligand that interacts with DpCPV via the MTase domain. These results augment our understanding of the mechanisms used by cypoviruses to enter their hosts.