scholarly journals Bacmid Expression of Granulovirus Enhancin En3 Accumulates in Cell Soluble Fraction to Potentiate Nucleopolyhedrovirus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1233
Author(s):  
Adriana Ricarte-Bermejo ◽  
Oihane Simón ◽  
Ana Beatriz Fernández ◽  
Trevor Williams ◽  
Primitivo Caballero
Keyword(s):  
Trichoplusia Ni ◽  
Recombinant Virus ◽  
Spodoptera Exigua ◽  
Insect Cells ◽  
Soluble Fraction ◽  
Autographa Californica ◽  
Insect Midgut ◽  
Baculovirus Infection ◽  
Occlusion Bodies ◽  
Infected Cells

Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy.

scholarly journals Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection

FEBS Letters ◽  
1998 ◽  
Vol 441 (1) ◽  
pp. 49-52 ◽  
Author(s):  
Kathrin Marheineke ◽  
Sylvia Grünewald ◽  
William Christie ◽  
Helmut Reiländer
Keyword(s):  
Spodoptera Frugiperda ◽  
Lipid Composition ◽  
Trichoplusia Ni ◽  
Insect Cells ◽  
Baculovirus Infection

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

2003 ◽  
Vol 69 (4) ◽  
pp. 2052-2057 ◽  
Author(s):  
James C. Bull ◽  
H. C. J. Godfray ◽  
David R. O'Reilly
Keyword(s):  
Cell Culture ◽  
Population Genetics ◽  
Trichoplusia Ni ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Cell Infection ◽  
Occlusion Bodies ◽  
Budded Virus

ABSTRACT 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.

scholarly journals Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

2016 ◽  
Vol 90 (7) ◽  
pp. 3480-3495 ◽  
Author(s):  
Siddhartha Biswas ◽  
Gary W. Blissard ◽  
David A. Theilmann
Keyword(s):  
Plasma Membrane ◽  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Nucleocapsid Proteins ◽  
Content Type ◽  
Multiple Nucleopolyhedrovirus ◽  
Microtubule Transport ◽  
Infected Cells ◽  
Budded Virus

ABSTRACTThe mechanism by which nucleocapsids ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with theDrosophila melanogasterkinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteranTrichoplusia niKLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated withT. niKLC. Direct analysis of the role ofT. nikinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation.IMPORTANCEIn two key processes of the replication cycle of the baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.

scholarly journals A Mathematical Model of Baculovirus Infection on Insect Cells at Low Multiplicity of Infection

2004 ◽  
Vol 36 (11) ◽  
pp. 729-740 ◽  
Author(s):  
You-Hong Zhang ◽  
José C. Merchuk
Keyword(s):  
Mathematical Model ◽  
Insect Cell ◽  
Insect Cells ◽  
Virus Production ◽  
Infection Process ◽  
Multiplicity Of Infection ◽  
Cell Infection ◽  
Baculovirus Infection ◽  
Infected Cells ◽  
Foreign Genes

Abstract The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of infection (MOI), which is based on the multi-infection cycles of insect cell infection at low MOI. A mathematical model for the amount of viruses released from primary infected cells and the amount of free viruses before secondary infected cells release viruses has been developed. Comparison of the simulation results with the experimental data confirms qualitatively that this model is highly reasonable before secondary infected cells release viruses. This model is considered as a base for further modeling the entire complicated infection process.

scholarly journals A new ascovirus from Spodoptera exigua and its relatedness to the isolate from Spodoptera frugiperda

2000 ◽  
Vol 81 (12) ◽  
pp. 3083-3092 ◽  
Author(s):  
Xiao-Wen Cheng ◽  
Gerald R. Carner ◽  
Basil M. Arif
Keyword(s):  
Spodoptera Frugiperda ◽  
Heliothis Virescens ◽  
Trichoplusia Ni ◽  
Spodoptera Exigua ◽  
Southern Hybridization ◽  
Fat Body ◽  
Occlusion Body ◽  
Occlusion Bodies ◽  
Dna Restriction ◽  
A New Species

A new ascovirus was isolated from Spodoptera exigua in Indonesia and was tentatively assigned as a new species, Spodoptera exigua ascovirus 5a (SeAV-5a) according to the present ICTV ascovirus naming scheme based on DNA restriction fragment length polymorphism (RFLP), hybridization, formation of occlusion body, tissue tropism and host spectrum. SeAV-5a replicated primarily in the fat body of susceptible hosts. SeAV-5a could be transmitted to S. frugiperda, Pseudoplusia includens and Trichoplusia ni, but not to Heliothis virescens. Infection with SeAV-5a arrested growth of the hosts, but prolonged their survival, which continued up to 33 days. Clusters of virions were seen inside the characteristic vesicles. Occasionally, virions were contained within vacuoles (one to five per vacuole) and some virions were embedded in occlusion bodies. The size of the SeAV-5a virion was 347×134 nm; however, aberrant long secondary viral products were also seen. The presence of occlusion body and Southern hybridization and Western immunoblot analyses suggest that SeAV-5a is more closely related to S. frugiperda ascovirus 1a (SfAV-1a) than to Trichoplusia ni ascovirus 2 (TnAV-2). Certain regions of the 182 kb genome of SeAV-5a showed hybridization to that of SfAV-1a. Two fragments in each of the SfAV-1a EcoRI and HindIII digests hybridized to the SeAV-5a genomic DNA probe. Five to eight HindIII and EcoRI fragments in SeAV-5a DNA hybridized to the SfAV-1a genomic probe.

scholarly journals A Virus-Encoded RNA Polymerase Purified from Baculovirus-Infected Cells

1998 ◽  
Vol 72 (10) ◽  
pp. 7985-7991 ◽  
Author(s):  
Linda A. Guarino ◽  
Bin Xu ◽  
Jianping Jin ◽  
Wen Dong
Keyword(s):  
Rna Polymerase ◽  
Insect Cells ◽  
Integral Function ◽  
Autographa Californica ◽  
Protein Sequencing ◽  
Amino Terminal ◽  
Promoter Recognition ◽  
Infected Cells ◽  
Nuclear Polyhedrosis ◽  
Rna Polymerase Subunits

ABSTRACT A DNA-dependent RNA polymerase was purified to homogeneity, starting from insect cells infected with the baculovirusAutographa californica nuclear polyhedrosis virus (AcNPV). The purified polymerase supported accurate and specific transcription from late and very late promoters but was not active on viral early promoters. Thus, promoter recognition is an integral function of the purified enzyme. The purified RNA polymerase was composed of only four equimolar subunits, which makes it the simplest DNA-directed RNA polymerase from a eukaryotic source described so far. Amino-terminal protein sequencing, peptide fingerprinting, and immunochemical analyses were used to identify the four subunits, all of which are virus encoded. Overexpression of the four viral proteins (LEF-8, LEF-4, LEF-9, and p47) in baculovirus-infected cells resulted in a significant increase in the levels of RNA polymerase produced in the infected cells. Thus, the overexpression data are consistent with our identification of the RNA polymerase subunits.

scholarly journals Localization of a Baculovirus-Induced Chitinase in the Insect Cell Endoplasmic Reticulum

1998 ◽  
Vol 72 (12) ◽  
pp. 10207-10212 ◽  
Author(s):  
Carole J. Thomas ◽  
Helen L. Brown ◽  
Chris R. Hawes ◽  
Bum Yong Lee ◽  
Mi-Kyung Min ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Trichoplusia Ni ◽  
Immunofluorescence Microscopy ◽  
Chitinase Activity ◽  
Autographa Californica ◽  
Insect Larvae ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Infected Insect ◽  
Infected Cells

ABSTRACT Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion ofp10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infectedTrichoplusia ni larvae by the same period.

scholarly journals Insect Virus Proteins (FALPE and p10) Self-Associate To Form Filaments in Infected Cells

1998 ◽  
Vol 72 (3) ◽  
pp. 2213-2223 ◽  
Author(s):  
Moulay Hicham Alaoui-Ismaili ◽  
Christopher D. Richardson
Keyword(s):  
Insect Cells ◽  
Interaction Analysis ◽  
Recombinant Baculovirus ◽  
Host Cells ◽  
Filament Assembly ◽  
Late Protein ◽  
Structural And Functional Properties ◽  
Occlusion Bodies ◽  
Infected Insect ◽  
Infected Cells

ABSTRACT Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta mooreientomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.

scholarly journals Caspase inhibitor P35 is required for the production of robust baculovirus virions in Trichoplusia ni TN-368 cells

2009 ◽  
Vol 90 (3) ◽  
pp. 654-661 ◽  
Author(s):  
Bart Bryant ◽  
Rollie J. Clem
Keyword(s):  
Spodoptera Frugiperda ◽  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Mutant Virus ◽  
Progeny Virus ◽  
Caspase Inhibitor ◽  
Wild Type ◽  
Lepidopteran Insects ◽  
Baculovirus Infection

Apoptosis can protect lepidopteran insects against baculovirus infection by limiting viral replication. Baculoviruses counter this response by expressing anti-apoptotic proteins such as the caspase inhibitor P35, which is expressed by several baculoviruses including Autographa californica mutiple nucleopolyhedrovirus (AcMNPV). Mutants of AcMNPV that lack the p35 gene induce apoptosis in Spodoptera frugiperda cells, and replication of these mutants is severely curtailed in S. frugiperda cell lines and larvae. However, cells from another lepidopteran species, Trichoplusia ni, do not undergo apoptosis when infected with AcMNPV mutants lacking p35, and p35 mutant and wild-type viruses replicate to equivalent levels in the T. ni cell line TN-368 and have equivalent infectivity in T. ni larvae by either oral or intrahaemocoelic injection. This has led to the conclusion that p35 is not required for AcMNPV replication in T. ni. However, in this study it was found that stocks of p35 mutant virus produced in TN-368 cells had defects in virion stability and infectivity. TN-368 cells infected with p35 mutant AcMNPV exhibited caspase activity, despite a lack of apoptosis, and propagation of the mutant virus in the presence of a chemical caspase inhibitor restored the normal infection phenotype to the progeny virus. These results suggest that caspases can directly or indirectly damage baculovirus virions, and reveal a novel aspect of the role of apoptosis in antiviral defence.

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