scholarly journals The N-Terminal Region of an Entomopoxvirus Fusolin Is Essential for the Enhancement of Peroral Infection, whereas the C-Terminal Region Is Eliminated in Digestive Juice

2008 ◽  
Vol 82 (24) ◽  
pp. 12406-12415 ◽  
Author(s):  
Yutaka Takemoto ◽  
Wataru Mitsuhashi ◽  
Ritsuko Murakami ◽  
Hirosato Konishi ◽  
Kazuhisa Miyamoto
Keyword(s):  
Carbohydrate Chain ◽  
Amino Acid Replacement ◽  
Terminal Region ◽  
Peritrophic Membrane ◽  
Deletion Mutants ◽  
Binding Ability ◽  
Digestive Juice ◽  
Chitin Binding Domain ◽  
Anomala Cuprea ◽  
Baculovirus System

ABSTRACT The spindles of Anomala cuprea entomopoxvirus (AncuEPV), which are composed of glycoprotein fusolin, are known to enhance the peroral infectivity of AncuEPV itself and of nucleopolyhedroviruses. This has been demonstrated to involve the disruption of intestinal peritrophic membrane (PM), composed of chitin matrix, glycosaminoglycans, and proteins. To identify essential and nonessential regions for this enhancement activity, AncuEPV fusolin and its deletion mutants were expressed in Sf21 cells using a baculovirus system, and their enhancement abilities were analyzed. The recombinant fusolin enhanced the peroral infectivity of Bombyx mori nucleopolyhedrovirus up to 320-fold and facilitated the infection of host insect with AncuEPV. Deletion mutagenesis revealed that the N-terminal region (amino acids 1 to 253), a possible chitin-binding domain, is essential for the enhancement of infection, whereas the C-terminal region is entirely dispensable. The glycosylation-defective mutants N191Q, whose Asn191 is replaced with Gln, and ΔSIG, whose signal peptide is deleted, showed considerably reduced and abolished enhancing activities, respectively, indicating that the carbohydrate chain is important in the enhancing activity. Interestingly, the C-terminal dispensable region was digested by a serine protease(s) in insect digestive juice. Moreover, both the N-terminal conserved region and the carbohydrate chain were necessary not only for chitin binding but also for stability in digestive juice. A triple amino acid replacement mutant, IHE (Ile-His-Glu161 to Ala-Ala-Ala), was stable in digestive juice and had chitin-binding ability but did not retain its enhancing activity. These results suggest that the enhancement of infectivity involves more than the tolerance to digestive juice and chitin-binding ability.

scholarly journals The Amino-Terminal Region of Major Capsid Protein P3 Is Essential for Self-Assembly of Single-Shelled Core-Like Particles of Rice Dwarf Virus

2004 ◽  
Vol 78 (6) ◽  
pp. 3145-3148 ◽  
Author(s):  
Kyoji Hagiwara ◽  
Takahiko Higashi ◽  
Naoyuki Miyazaki ◽  
Hisashi Naitow ◽  
R. Holland Cheng ◽  
...  
Keyword(s):  
Self Assembly ◽  
Major Capsid Protein ◽  
Core Protein ◽  
Terminal Region ◽  
Dwarf Virus ◽  
Rice Dwarf Virus ◽  
Amino Terminal ◽  
The Core ◽  
Baculovirus System ◽  
Terminal Amino

ABSTRACT The core protein P3 of Rice dwarf virus constructs asymmetric dimers, one of which is inserted by the amino-terminal region of another P3 protein. The P3 proteins with serial amino-terminal deletions, expressed in a baculovirus system, formed particles with gradually decreasing stability. The capacity for self-assembly disappeared when 52 of the amino-terminal amino acids had been deleted. These results demonstrated that insertion of the amino-terminal arm of one P3 protein into another appears to play an important role in stabilizing the core particles.

Calcium binding to abnormal fibrinogens with a single amino acid replacement in the NH2-terminal region of fibrin α - or β-chain

1992 ◽  
Vol 67 (5) ◽  
pp. 613-618 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Hajime Hirata ◽  
Shinji Asakura ◽  
Kensuke Yamazumi ◽  
Michio Matsuda ◽  
...  
Keyword(s):  
Amino Acid ◽  
Calcium Binding ◽  
Amino Acid Replacement ◽  
Terminal Region ◽  
Single Amino Acid ◽  
Β Chain

scholarly journals Binding-Site Interactions between Epstein-Barr Virus Fusion Proteins gp42 and gH/gL Reveal a Peptide That Inhibits both Epithelial and B-Cell Membrane Fusion

2007 ◽  
Vol 81 (17) ◽  
pp. 9216-9229 ◽  
Author(s):  
Austin N. Kirschner ◽  
Amanda S. Lowrey ◽  
Richard Longnecker ◽  
Theodore S. Jardetzky
Keyword(s):  
Epithelial Cell ◽  
Cell Membrane ◽  
B Cell ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Terminal Region ◽  
Deletion Mutants ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.

scholarly journals Human beta2-glycoprotein I as an anticardiolipin cofactor determined using mutants expressed by a baculovirus system

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3262-3270 ◽  
Author(s):  
M Igarashi ◽  
E Matsuura ◽  
Y Igarashi ◽  
H Nagae ◽  
K Ichikawa ◽  
...  
Keyword(s):  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Terminal Region ◽  
Mutant Protein ◽  
Mutant Proteins ◽  
Glycoprotein I ◽  
Baculovirus System ◽  
Domain V ◽  
Mutant Genes

beta2-Glycoprotein I (beta2-GPI) consists of five repeats of a homologous domain. We designed a series of human beta2-GPI mutant genes, ie, three mutant genes lacking the domain(s) present in the NH2- terminal region and two of those present in the COOH-terminal region. These mutant genes were expressed in Spodoptera frugiperda insect cells (Sf9) infected with recombinant baculoviruses and the mutant proteins were secreted into the culture medium. The molecular mass of the purified mutant proteins, estimated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, was fairly consistent with the size calculated from their nucleotide sequences. Binding of beta2-GPI to solid-phase cardiolipin (CL) was diminished by the deletion of the fifth domain (domain V) from its complete structure. Thus, the phospholipid binding site of beta2-GPI is located on its domain V. Monoclonal anti-CL antibodies (aCL) derived either from NZW x BXSB (WB) F1 mice or from patients with antiphospholipid syndrome bound directly to the domain V-deleted mutant protein (DI-IV) absorbed not only on an oxygenated but also on a plain polystyrene surface. We conclude from this study that the epitope for aCL is exposed on a conformationally changed structure of beta2-GPI by interacting with negatively charged phospholipid or on the mutant protein, DI-IV.

scholarly journals Downregulation of a Chitin Deacetylase-Like Protein in Response to Baculovirus Infection and Its Application for Improving Baculovirus Infectivity

2009 ◽  
Vol 84 (5) ◽  
pp. 2547-2555 ◽  
Author(s):  
Agata K. Jakubowska ◽  
Silvia Caccia ◽  
Karl H. Gordon ◽  
Juan Ferré ◽  
Salvador Herrero
Keyword(s):  
Spodoptera Frugiperda ◽  
Expressed Sequence Tags ◽  
Spodoptera Exigua ◽  
Peritrophic Membrane ◽  
Dependent Manner ◽  
Chitin Deacetylase ◽  
Concentration Dependent Manner ◽  
Chitin Binding Domain ◽  
Baculovirus Infection ◽  
Expressed Sequence

ABSTRACT Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability, in a concentration-dependent manner. Bioassays on Spodoptera frugiperda and Spodoptera exigua larvae revealed that NPV expressing HaCDA5a was more infective than its parental virus. However, no difference in virulence was observed when the viruses were injected intrahemocoelically. These findings support the downregulation of a midgut-specific CDA-like protein as a possible mechanism used by H. armigera to reduce susceptibility to baculovirus by decreasing PM permeability.

scholarly journals Spindles of an Entomopoxvirus Facilitate Its Infection of the Host Insect by Disrupting the Peritrophic Membrane

2007 ◽  
Vol 81 (8) ◽  
pp. 4235-4243 ◽  
Author(s):  
Wataru Mitsuhashi ◽  
Hiromu Kawakita ◽  
Ritsuko Murakami ◽  
Yutaka Takemoto ◽  
Tomoaki Saiki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Peritrophic Membrane ◽  
Infection Site ◽  
Initial Infection ◽  
Host Insect ◽  
Anomala Cuprea ◽  
Enhancing Factor ◽  
Pseudaletia Separata ◽  
Scanning Electron

ABSTRACT The mode of action by which entomopoxvirus (EPV) spindles, proteinaceous crystalline bodies produced by EPVs, enhance EPV infection has not been clarified. We fed Anomala cuprea EPV (AcEPV) spindles to host insects; subsequent scanning electron microscopy revealed the disruption of the peritrophic membranes (PMs) of these insects. The PM is reportedly a barrier against the infection of some insects by viruses. Quantitative PCR of AcEPV DNA in the ectoperitrophic area revealed that PM disruption facilitated the passage of EPVs through the PM toward the initial infection site, the midgut epithelium. These results indicate that EPV spindles enhance infection by EPVs by disrupting the PM in the host insects. Fusolin is almost exclusively the constituent protein of the spindles and is the enhancing factor of the infectivity of nucleopolyhedroviruses (NPVs) and possibly that of EPVs. Spheroid is another type of proteinaceous crystalline structure produced by EPVs. Pseudaletia separata EPV (PsEPV) spheroids reportedly contain considerable amounts of fusolin and enhance NPV infection. We assessed the ability of AcEPV spheroids to enhance EPV infectivity and their effect on the PM and carried out immunological experiments; these experiments showed that AcEPV spheroids contain little or no fusolin and are biologically inactive, in contrasts to the situation in PsEPV.

Sequence analysis of two deletion mutants in the hisC gene of Salmonella typhimurium

1994 ◽  
Vol 40 (2) ◽  
pp. 158-160
Author(s):  
W. L. Albritton ◽  
L. Chui
Keyword(s):  
Salmonella Typhimurium ◽  
Sequence Data ◽  
Polymerase Chain Reaction Amplification ◽  
Terminal Region ◽  
Phenotypic Characterization ◽  
Deletion Mutants ◽  
Base Pair Deletion ◽  
P2 Promoter ◽  
Reaction Amplification

Limited sequencing of the terminal region of the hisC gene in two deletion mutants involving the hisC gene of Salmonella typhimurium was carried out after polymerase chain reaction amplification of the appropriate region, using oligonucleotide primers selected from the published sequence of the histidine operon from this organism. his2648 was shown to have a 34 base pair deletion in the terminal region of the hisC gene between the P2 promoter and the Shine–Delgamo sequence of the hisB gene. hisHB22 was shown to have a 1.4 kilobase deletion extending from the TGA termination codon of the hisC gene to the middle of the hisH gene. The sequence data were consistent with previous genetic and phenotypic characterization of these strains.Key words: Salmonella typhimurium, hisC gene, deletion mutants.

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