scholarly journals Autographa californica multiple nucleopolyhedrovirus ORF 23 null mutant produces occlusion-derived virions with fewer nucleocapsids

2009 ◽  
Vol 90 (6) ◽  
pp. 1499-1504 ◽  
Author(s):  
Ian-Ling Yu ◽  
Doug Bray ◽  
Ying-Chu Lin ◽  
Oliver Lung
Keyword(s):  
Fluorescent Protein ◽  
Autographa Californica ◽  
Dapi Staining ◽  
Recombinant Viruses ◽  
Multiple Nucleopolyhedrovirus ◽  
Gfp Fluorescence ◽  
Occlusion Bodies ◽  
Green Fluorescent ◽  
And Control ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

Two envelope fusion protein gene homologues have been identified in the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV GP64 protein is fusogenic and essential for propagation and pathogenicity. The F homologue (Ac23) is not essential, is fusion-incompetent in standard assays, but contributes to faster host death. Here, we show that occlusion bodies (OBs) from Ac23null mutants and control viruses do not differ significantly in size and the number of occlusion-derived virions (ODVs) contained; however, Ac23null OBs had a much higher percentage of ODVs with a single nucleocapsid (44.6 %) than the near-isogenic control (11.3 %). Infection of Sf9 cells with Ac23–green fluorescent protein (gfp)-expressing recombinant viruses showed Ac23–gfp fluorescence overlapping perinuclear DAPI staining at later times, a pattern not observed with GP64. These results suggest that F proteins have evolved functions beyond envelope fusion and play a different role from that of GP64 in viruses that contain both proteins.

scholarly journals A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag

2001 ◽  
Vol 82 (9) ◽  
pp. 2279-2287 ◽  
Author(s):  
Jeffrey M. Slack ◽  
Edward M. Dougherty ◽  
Susan D. Lawrence
Keyword(s):  
Spodoptera Frugiperda ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Chimeric Proteins ◽  
Ring Zone ◽  
Multiple Nucleopolyhedrovirus ◽  
C Terminus ◽  
Green Fluorescent ◽  
Autographa Californica Multiple Nucleopolyhedrovirus ◽  
Gfp Tag

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74–GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74–GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74–GFP chimeras in recombinant baculoviruses. When C-terminal region S580–F645 was deleted from p74, p74–GFP chimera localization became non-specific and chimeras became soluble. p74 region S580–F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.

scholarly journals Characterization of a virion occlusion-defective Autographa californica multiple nucleopolyhedrovirus mutant lacking the p26, p10 and p74 genes

2009 ◽  
Vol 90 (7) ◽  
pp. 1641-1648 ◽  
Author(s):  
Lihua Wang ◽  
Tamer Z. Salem ◽  
Dean J. Campbell ◽  
Colin M. Turney ◽  
C. M. Senthil Kumar ◽  
...  
Keyword(s):  
Insect Pests ◽  
Horizontal Transmission ◽  
Autographa Californica ◽  
Individual Gene ◽  
Dna Deletion ◽  
Multiple Nucleopolyhedrovirus ◽  
Occlusion Bodies ◽  
Autographa Californica Multiple Nucleopolyhedrovirus ◽  
Control Insect

Nucleopolyhedroviruses (NPVs), family Baculoviridae, are insect-specific viruses with the potential to control insect pests in agriculture and forestry. NPVs are occluded in polyhedral occlusion bodies. Polyhedra protect virions from inactivation in the environment as well as assisting virions in horizontal transmission in the insect population. The process of virion occlusion in the polyhedra is undefined and the genes that regulate the virion occlusion process have not been well investigated yet. An Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant (AcDef) that has a 2136 bp DNA deletion, including p26, p10 and p74 genes, has been isolated. No virions were detected in the polyhedra of AcDef. Restoration of all the missing sequences into AcDef led to proper virion occlusion. Individual gene deletion of either p10 or p26 could not abolish virion occlusion in the polyhedra of AcMNPV, but p10 deletion reduced virion occlusion efficiency more than threefold compared with the wild-type AcMNPV. Previous studies by other research groups on deletion of AcMNPV gene p74 suggested that p74 is a per os infectivity factor, and deletion of the p74 gene did not eliminate virion occlusion. Collectively, the three genes (p26, p10 and p74) may act in concert to regulate the virion occlusion process. Therefore, p26, p10 and p74 are all required for proper virion occlusion in the polyhedra of AcMNPV.

scholarly journals Autographa californica multiple nucleopolyhedrovirus and Choristoneura fumiferana multiple nucleopolyhedrovirus v-cath genes are expressed as pre-proenzymes

2009 ◽  
Vol 90 (4) ◽  
pp. 995-1000 ◽  
Author(s):  
Jeffrey J. Hodgson ◽  
Basil M. Arif ◽  
Peter J. Krell
Keyword(s):  
Fluorescent Protein ◽  
Choristoneura Fumiferana ◽  
Immunoblot Analysis ◽  
Autographa Californica ◽  
Proteolytic Processing ◽  
Post Translational Modifications ◽  
Peptide Cleavage ◽  
Multiple Nucleopolyhedrovirus ◽  
Group I ◽  
Green Fluorescent

Intracellular processing and trafficking of the baculovirus v-cath expressed cathepsin (V-CATH), which lacks canonical targeting signals, are poorly understood. The cathepsins of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and most other alphabaculovirus group I nucleopolyhedroviruses have well-conserved N-termini containing overlapping chymotrypsin-cleavage (Y11) and myristoylation (G12) motifs, which are suggestive of proteolytic signal-peptide cleavage to generate proV-CATH and subsequent acylation. To determine proteolytic N-terminal processing of V-CATH, haemagglutinin epitope-coding tags were fused to the 5′ and/or 3′ ends of AcMNPV and CfMNPV v-cath. Immunoblot analysis suggested that a small N-terminal peptide is cleaved for both viruses, indicating that v-cath is expressed as a pre-proenzyme. The two viral homologues undergo similar proteolytic processing, but have different glycosylation or other post-translational modifications. An AcMNPV V-CATH–DsRED fusion protein co-localized to the endoplasmic reticulum with an HDEL motif-containing green fluorescent protein. Based on these findings, pre-proV-CATH processing and trafficking mechanisms are postulated.

scholarly journals Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

2009 ◽  
Vol 83 (23) ◽  
pp. 12569-12578 ◽  
Author(s):  
Minggang Fang ◽  
Yingchao Nie ◽  
Stephanie Harris ◽  
Martin A. Erlandson ◽  
David A. Theilmann
Keyword(s):  
Trichoplusia Ni ◽  
Core Gene ◽  
Autographa Californica ◽  
Immunofluorescence Analysis ◽  
Viral Dna Replication ◽  
Multiple Nucleopolyhedrovirus ◽  
Confocal Immunofluorescence ◽  
Budded Virus ◽  
And Control ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc 96 null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc 96 null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc 96 null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R

2007 ◽  
Vol 292 (5) ◽  
pp. F1303-F1313 ◽  
Author(s):  
Xianhua Yi ◽  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Shaliha Bechoua ◽  
Tian-xiao Sun ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Human Kidney ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Parathyroid Hormone Receptor ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
G Protein Coupled ◽  
Regulatory Event

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK1 epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

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