Impact of viral enhancin genes on potency of Lymantria dispar multiple nucleopolyhedrovirus in L. dispar following disruption of the peritrophic matrix

Enhancins are metalloproteases found in many betabaculoviruses and several alphabaculoviruses, which enhance alphabaculovirus potency by degrading a protein component of the peritrophic matrix (PM), facilitating passage of virions through this structure. Earlier studies on betabaculovirus enhancins within heterologous systems suggested that enhancins facilitate virion binding to midgut cells. We compared the potency of wild-type Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) with that of single and double enhancin deletion viruses in L. dispar in the presence and absence of an intact PM. Compared to wild-type virus, the double enhancin deletion virus was 6-fold and 14-fold less potent, respectively, indicating that within this homologous system the LdMNPV enhancin genes have a function beyond PM degradation.     

昆虫病毒的增强蛋白

在某些昆虫病毒的包涵体中相继发现了增强蛋白．这类蛋白可以增强核型多角体病毒对昆虫幼虫及体外培养的昆虫细胞的感染力,缩短杀虫时间,提高杀虫效率;还可以增强其他生物杀虫剂（如：Bt）的毒力．关于增强蛋白的作用机制有两种观点：降解围食膜以利于病毒粒子侵染细胞或直接作用于细胞质膜促进病毒粒子的套膜与之融合．该蛋白是由病毒编码的,现已克隆出第一个增强蛋白基因,该基因含有一个2703bP的开放阅读框,其核苷酸序列分析业已完成．增强蛋白的发现有望对病毒杀虫剂的推广产生积极影响．     

Proteomics analysis of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus identified two new occlusion-derived virus-associated proteins, HA44 and HA100

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.     

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.     

Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.     

Identification of the lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae

The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein.     

核型多角体病毒侵染及其与宿主免疫系统互作的研究进展

核型多角体病毒(Nucleopolyhedrovirus,NPV)应用广泛,已被开发成微生物杀虫剂和用于重组蛋白表达等.NPV具有两种病毒颗粒:包埋型病毒粒子(occlusion-derived virus,ODV)和芽生型病毒粒子(budded virus,BV),两者的构成和组装存在差异.病毒包涵体在肠道中溶解后释放出ODV进行初级感染,子代核衣壳通过质膜出芽产生BV引发全身性的次级感染.病毒感染昆虫后能引发宿主的免疫反应,同时NPV病毒已进化出多种策略抑制或逃避宿主的免疫反应,如免疫信号通路、黑化和凋亡等过程.本文将总结鳞翅目特异性NPV与宿主的互作研究,着重介绍NPV的侵染机制、NPV与宿主免疫系统互作的研究进展.     

Enhancement of nucleopolyhedrovirus infectivity against Mamestra brassicae (Lepidoptera: Noctuidae) by proteins derived from granulovirus and a fluorescent brightener

The synergistic enhancement of nucleopolyhedrovirus (NPV) infection by granuloviruses (GVs) is well documented; and a GV granule protein, named viral enhancin, has been identified as an active contributor to this effect. We detected the presence of two proteins with molecular mass of 93 and 108 kDa in granules of a GV isolated from Xestia c-nigrum (L.) (XecnGV) as candidates for enhancin, and we confirmed that at least the 108-kDa protein enhances the infectivity of Mamestra brassicae nucleopolyhedrovirus (MabrNPV). We tested the effect of virion-free proteins obtained from XecnGV granules (GVPs) on MabrNPV infection, and we made a comparison with an enhancing chemical, the stilbene-derived fluorescent brightener Tinopal. Bioassay was performed employing the diet contamination method, by using second instars of Mamestra brassicae (L.) (Lepidoptera: Noctuidae). The enhancing effects of GVPs (0.1 mg/g diet) and Tinopal (1 mg/g diet) were estimated to be 70.7-81.5-fold and 26.9-33.7-fold, respectively, as calculated from the LC50 values of MabrNPV with or without the additives. The additives reduced the lethal time of MabrNPV-infected larvae and they caused death at a younger instar. These results suggest that GVPs can enhance MabrNPV infection as effectively as Tinopal.     

Helicoverpa armigera nucleopolyhedrovirus ORF80 encodes a late, nonstructural protein

The Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ORF80 (ha80) has 765 bp encoding a protein with approximately 254 amino acids and a predicted molecular weight of 30.8 kDa. Homologues of ha80 are found in most baculovirus sequences, including those from lepidopteran NPVs, lepidopteran granuloviruses (GVs), hymenopteran baculoviruses, and one dipteran baculovirus, yet their functions remain unclear. In this study we characterized ha80, and showed that it was transcribed late in infected host cells (HzAM1). The product of ha80 was a 31 kDa protein that was not a structural protein of budded virus (BV) or occlusion-derived virus (ODV) particles. Ha80 was first detected in the cytoplasm of infected HzAM1 cells at 12 h p.i., and was observed in the nucleus at later stages of infection, suggesting that it may be involved in transporting viral proteins into the host cell nucleus or play its roles in the nucleus.     

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).     

TnGV增强蛋白在AcMNPV中的表达和活性分析

为了研究杆状病毒增强蛋白的活性基团 ,本文构建了分别表达三种N端部分缺失的粉纹夜蛾颗粒体病毒增强蛋白的重组杆状病毒 ,这三种蛋白在N端分别缺失了 15 0 ,186和 2 5 0个氨基酸。用重组病毒感染Tn 5B1 4细胞 ,成功地表达了这三种蛋白 ,并得到了纯化的蛋白质。通过体外降解围食膜的方法检测这些部分缺失的增强蛋白的活性 ,结果证实这三种蛋白均失去了增强蛋白的降解围食膜粘蛋白的活性。这一结果表明 ,增强蛋白的N端对其降解围食膜粘蛋白的功能是必需的     

Identification of a novel apoptosis suppressor gene from the baculovirus Lymantria dispar multicapsid nucleopolyhedrovirus

Ld652Y cells from Lymantria dispar readily undergo apoptosis upon infection with a variety of nucleopolyhedroviruses (NPVs), while L. dispar multicapsid NPV (LdMNPV) infection of Ld652Y cells results in the production of a high titer of progeny viruses. Here, we identify a novel LdMNPV apoptosis suppressor gene, apsup, which functions to suppress apoptosis induced in Ld652Y cells by infection with vAcΔp35, a p35-defective recombinant Autographa californica MNPV. apsup also suppresses apoptosis of Ld652Y cells induced by actinomycin D and UV exposure. Apsup is expressed in LdMNPV-infected Ld652Y cells late in infection, and RNA interference-mediated apsup ablation induces apoptosis of LdMNPV-infected Ld652Y cells.     

Effect of two granulosis viruses on the activity of the gypsy moth (Lepidoptera: Lymantriidae) nuclear polyhedrosis virus

Two granulosis viruses (GV) were tested as enhancers for the gypsy moth nuclear polyhedrosis virus (LdMNPV). Helicoverpa armigera (Hübner) CV (HaGV) had no detrimental effect upon larval growth and development, but in combination with LdMNPV it reduced both the LC50 and the LT50 for the NPV. In addition, the combination also adversely affected the growth and development of gypsy moth larvae. The LC50 of LdMNPV was reduced by as much as 300-fold (HaGV at 10(-2) dilution) and the LT50 was reduced by as much as 18% (HaGV at 10(-2) dilution). Spodoptera frugiperda (J. E. Smith) GV reduced the LC50 of LdMNPV by as much as 13-fold but had no effect upon the LT50.     

Autographa californica multiple nucleopolyhedrovirus 38K is a novel nucleocapsid protein that interacts with VP1054, VP39, VP80, and itself

It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusion-derived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.     

Multiple Nucleocapsid Packaging of Autographa californica Nucleopolyhedrovirus Accelerates the Onset of Systemic Infection in Trichoplusia ni

Among the nucleopolyhedroviruses (Baculoviridae), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) containing a β-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar Trichoplusia ni were challenged with doses of ODV-S or ODV-M that yielded the same final mortality (~70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of AcMNPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.     

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

We prepared mouse hybrid cell lines which produced antibodies against herpes simplex virus type 1 and 2 nucleocapsids. Cell lines 1D4 and 3E1, respectively, secreted immunoglobulin G1 herpes simplex virus type 1 and immunoglobulin G1 herpes simplex virus type 2 antibodies which immunoprecipitated proteins designated p40 and p45 from homologous nucleocapsid preparations but precipitated no proteins from heterologous preparations. In contrast, guinea pig antisera prepared against either herpes simplex virus type 1 or 2 p40 precipitated p40 and p45 from both homologous and heterologous preparations. These findings suggest that p40 and p45 possess similar antigenic determinants and that the monoclonal antibodies that were tested reacted preferentially with the homologous determinants.     

Development of a nuclear polyhedrosis virus in midgut cells and penetration of the virus into the hemocoel of the armyworm,Pseudaletia unipuncta

The development of a nuclear polyhedrosis virus (NPV) in larval midgut cells of the armyworm, Pseudaletia unipuncta, is similar to that of other NPV. In the nucleus, the envelopes around the nucleocapsids seem to be derived de novo or from the inner layer of the nuclear envelope wich forms cisternae, blebs, or infoldings. The nucleocapsids are also enveloped by synhymenosis during passage through the nuclear membrane, the cell membrane, or the endoplasmic reticulum membrane. Both enveloped and unenveloped nucleocapsids may enter the cytoplasm through the nuclear pore or budding through the nuclear membrane. From the cytoplasm the virions may enter the hemocoel through the basal cell and basement membranes or through the endoplasmic reticulum, intercellular space, and the basement membrane.     

Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25K FP gene may be caused by DNA replication errors.

We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J. M. Slavicek, N. Hayes-Plazolles, and M. E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants exhibited the traits of few polyhedra (FP) mutants; however, no large DNA insertions or deletions that correlated with the appearance of the FP phenotype were found. In this study, we have characterized several of the PF mutants at the phenotypic and genetic levels. Genetic techniques were used to group the mutations in the LdMNPV PF mutants to the same or closely linked genes. Wild-type viruses were recovered after coinfection of L. dispar 652Y cells with certain combinations of PF mutants. These viruses were analyzed by restriction endonuclease analysis and found to be chimeras of the original PF mutants used in the coinfections. Marker rescue experiments localized the mutations in one group of PF isolates to the region containing the LdMNPV 25K FP gene. The mutations in these PF mutants were identified. Four of five of the LdMNPV FP mutants contain small insertions or deletions within the 25K FP gene. The fifth LdMNPV FP mutant analyzed contained a large deletion that truncated the C terminus of the 25K FP gene product. All of the deletions occurred within the same potential hairpin loop structure, which had the lowest free energy value (most stable hairpin) of the five potential hairpin loop structures present in the 25K FP gene. One of the insertion mutants contained an extra base within a repetitive sequence. These types of mutations are likely caused by errors that occur during DNA replication. The relationship between the types of mutations found within the LdMNPV 25K FP gene and DNA replication-based mutagenesis is discussed.