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.     

粉纹夜蛾颗粒体病毒重组增效蛋白的增效作用

采用时间 剂量 死亡率模型 ,分析了粉纹夜蛾 (Trichoplusiani)颗粒体病毒重组增效蛋白P96对棉铃虫 (Helicoverpaarmigera)核型多角体病毒 (HaNPV)感染棉铃虫幼虫的增效作用。结果显示 :感染后 11d ,HaNPV P96组的LC50 值为 3.4 7× 10 3 多角体 /mL ,比HaNPV组 ( 3.89× 10 4 多角体 /mL)降低了 91.0 8% ;在 1.6× 10 4 ～ 1.6× 10 6多角体 /mL浓度范围内 ,HaNPV P96组的LT50 值较HaNPV组缩短 0 .3～ 1.8d。P96显著提高了HaNPV对棉铃虫幼虫的毒力     

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.     

Overexpression of δ-Opioid Receptors in Recombinant Baculovirus-Infected Trichoplusia ni"High 5" Insect Cells

Abstract: "High 5" cells derived from Trichoplusia ni ovaries were infected with baculovirus bearing the cDNA of the mouse δ-opioid receptor. The maximal binding capacity for the narcotic antagonist [³H]naltrindole was 1.4 pmol/mg of membrane protein, and that for the agonist [³H][ d -penicillamine², d -penicillamine⁵]enkephalin (DPDPE) was 0.3 pmol/mg. DPDPE proved highly potent in competing with its tritiated analogue at δ-receptors of NG108-15 hybrid cells and of High 5 and Sf9 insect cells. However, in insect cells the opioid was more than 100-fold less effective in competing with [³H]naltrindole as compared with the mammalian cells. This decline in potency was counteracted in a dose-dependent manner by exposure of High 5 membranes to the exogenous G protein G_o, which increased the binding capacity for DPDPE. Functional studies revealed a dose-dependent inhibition (up to 30%) by opioids on forskolin-stimulated cyclic AMP synthesis, and this effect was potentiated by G_o. Quantification of Gα_o and Gα_i disclosed striking differences between Sf9 and High 5 insect cells, both of which overexpressed the cloned δ-opioid receptor. Although no inhibitory G proteins were detected in membranes of Sf9 cells, High 5 cells contained 0.5 pmol of Gα_o/mg of membrane protein, and a 20-fold higher concentration for Gα_i. The distinct G-protein expression in insect cells may be considered an advantage for studying functions of G protein-coupled receptors.     

A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.     

Vairimorpha necatrix in Adipose Cells of Trichoplusia ni

Vairimorpha necatrix infected adipose ceiis of the fat body organ of Trichoplusia ni larvae 3–31/2 days after exposure of the larvae to infective spores. During the subsequent 4–6 days, the parasitized adipose cells were hypertrophied in part due to the rapid propagation of V. necatrix schizonts. A calcium-sensitive tubule network developed at the interface of the schizonts and the adipose ceil cytoplasm. The paired nuclei of V. necatrix have pores at the nuclear interface. The pores for each nucleus at this interface are spatially positioned so that they are in conjunction; hence, there is the potential for a channel system between the 2 nuclei.     

异源病毒感染对银纹夜蛾血淋巴酯酶的影响

对染病昆虫酯酶同工酶（简称酯酶）的变化进行分析测定,已成为了解病毒进入虫体靶器官后的病理生化变化以及病毒复制与虫体新陈代谢之间关系的重要途径之一,这方面的报道“J侧重于同源病毒——寄主系统的研究,本研究则针对银纹夜蛾（AWrammaagnata）幼虫感染异源粉纹夜蛾核型多角体病毒（TnNPV）后血淋巴酯酶的变化进行了探讨。现将结果报告如下：材料和方法1材料11虫源实验室内用半人工饲料饲养三代的健康银纹夜蛾五龄村幼虫。l．2毒源由中山大学昆虫研究所生物工程室提供的已纯化的TnNPV病毒株。1．3幼虫血淋巴样品的制备挑选工龄…     

Recombinant protein synthesis in Trichoplusia ni BTI-Tn-5B1-4 insect cell aggregates.

The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 cell aggregates and cells selected to grow predominantly as individual cells. No significant difference was found between cell aggregates and cells growing predominantly individually in regard to cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 insect cell line.     

Dynamics of the Multiplicity of Cellular Infection in a Plant Virus

Recombination, complementation and competition profoundly influence virus evolution and epidemiology. Since viruses are intracellular parasites, the basic parameter determining the potential for such interactions is the multiplicity of cellular infection (cellular MOI), i.e. the number of viral genome units that effectively infect a cell. The cellular MOI values that prevail in host organisms have rarely been investigated, and whether they remain constant or change widely during host invasion is totally unknown. Here, we fill this experimental gap by presenting the first detailed analysis of the dynamics of the cellular MOI during colonization of a host plant by a virus. Our results reveal ample variations between different leaf levels during the course of infection, with values starting close to 2 and increasing up to 13 before decreasing to initial levels in the latest infection stages. By revealing wide dynamic changes throughout a single infection, we here illustrate the existence of complex scenarios where the opportunity for recombination, complementation and competition among viral genomes changes greatly at different infection phases and at different locations within a multi-cellular host.     

Trichoplusia ni gloverin,an inducible immune gene encoding an antibacterial insect protein

By using differential display PCR, we obtained a cDNA clone encoding a gloverin homologue from the cabbage looper, Trichoplusia ni. The expression of the gene was induced by bacterial infections. The gene codes for a 174 amino acid residue protein, including a signal sequence and a prosegment. The deduced mature protein is 14 kDa and shows 58% and 49% identity to P2 from Helicoverpa armigera and to Hyalophora gloveri gloverin, respectively. The protein was detected in hemolymph and hemocytes from bacteria-immunized animals. We expressed gloverin using the baculovirus expression system. N-terminal amino acid sequence analysis showed that the purified protein contained a propart. This progloverin inhibited the growth of E. coli and the activity is comparable to that of H. gloveri mature gloverin. Processing of progloverin was possible in vitro, using human furin.     

Expression and functional analysis of an inhibitor of apoptosis protein from Trichoplusia ni

An inhibitor of the apoptosis protein (IAP) family gene from Trichoplusia ni, Tn-IAP1v, a variant of lepidopteran Tn-IAP1, was cloned by RT-PCR. There are six single nucleotide polymorphisms between the two Tn-IAP1 variants, resulting in three predicted single amino acid polymorphisms. With the GST fusion expression system, soluble recombinant Tn-IAP1v was highly expressed in Escherichia coli and then purified by affinity chromatography. Caspase inhibition assays indicated that recombinant Tn-IAP1v could specifically inhibit human caspase-9 in vitro instead of caspase-3, -7, and -8, which was further confirmed by the observation that recombinant Tn-IAP1v can directly bind caspase-9 in the protein pull-down assay. These results suggested that Tn-IAP1v might serve as an initiator caspase inhibitor in vivo in the conserved mitochondria apoptotic pathway.     

Short-hairpin RNA-mediated gene expression interference in Trichoplusia ni cells

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and beta-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and beta-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.     

Development of the polyembryonic parasitoid Copidosoma floridanum in Trichoplusia ni

Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) parasitized by the polyembryonic egg-larval parasitoid Copidosoma floridanum (Ashmead) (Hymenoptera: Encyrtidae) attained significantly larger final weights and head capsule widths than unparasitized controls. The difference in weight between parasitized and unparasitized hosts was not entirely accounted for by the weight of the C. floridanum brood. The head capsule widths of all parasitized and unparasitized fifth instars used in the study exceeded the critical threshold of 1.66 mm previously established for T. ni metamorphosis. The critical ratios associated with each T. ni instar of: 1) maximum weight within the instar:head capsule width and 2) maximum weight within the instar:weight at the beginning of the instar differed between parasitized and unparasitized larvae. Development of C. floridanum was synchronized with that of its host. Germ band formation and gastrulation of morulae destined to produce reproductive larvae invariably coincided with the host molt to the ultimate, fifth instar. Reproductive larvae had two instars. Eclosion from the egg to the first instar occurred during day 2 of the host's fifth instar, and ecdysis from the first to the second instar was synchronized with host cocoon spinning. Conversely, embryogenesis of morulae destined to produce precocious larvae began during the host first instar, continued through the second and third instar and ceased during the penultimate, fourth instar. Precocious larvae never molted and died when the host was consumed by the reproductive larvae.

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Résumé T. ni Hübner parasité par le parasitoïde ovo-larvaire C. floridanum Ashmead à développement polyembryonnaire atteint un poids final signficativement plus élevé avec une capsule céphalique plus grosse que les témoins non parasités, sans subir de mues surnuméraires. La différence de poids entre noctuelles parasitées ou non ne correspondait pas entièrement au poids des C. floridanum. Les largeurs des capsules céphaliques de tous les T. ni du cinquième stade dépassaient toutes le seuil critique de 1,66 mm lié à la métamorphose, mais les seuils critiques de taille du corps:largeur de la capsule céphalique et/ou taille et corps, taille initiale du corps au début du stade associé à la mue, différaient chez T. ni parasités ou non. Les développements de T. ni et de C. floridanum étaient synchrones. La formation de la bande germinative et la gastrulation de la morula produisant la multiplication des larves ont coïncidé invariablement avec la mue de l'hôte donnant le dernier stade. Les larves polyembryonnaires ont présenté deux stades. L'éclosion des oeufs s'est produite le deuxième jour du cinquième stade de T. ni, et le passage du premier au second était synchrone de la formation du cocon de l'hôte. Réciproquement, l'embryogenèse de la morula qui donnait des larves précoces commençait pendant le premier stade de l'hôte et se poursuivait à travers les second et troisième, pour cesser pendant le quatrième et pénultième stade.

     

Identification and characterization of Trichoplusia ni furin truncated mutant

We report a new prohormone convertase gene of insect origin, Trichoplusia ni furin, which was cloned from BTI-Tn-5B-4 (Tn5) insect cells. We constructed a truncated mutant that lacked Cys-rich repeated segments. Using baculovirus expression system and standard enzymatic assay, we obtained recombinant Tn furin and evaluated aspects of its function.