scholarly journals Compartmentalization of VP16 in Cells Infected with Recombinant Herpes Simplex Virus Expressing VP16-Green Fluorescent Protein Fusion Proteins

2004 ◽  
Vol 78 (15) ◽  
pp. 8002-8014 ◽  
Author(s):  
Sylvie La Boissière ◽  
Ander Izeta ◽  
Sophie Malcomber ◽  
Peter O'Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
De Novo ◽  
Structural Protein ◽  
Dynamic Features ◽  
Protein Fusion ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT VP16 is an essential structural protein of herpes simplex virus. It plays important roles in immediate-early transcriptional regulation, in the modulation of the activities of other viral components, and in the pathway of assembly and egress of infectious virions. To gain further insight into the compartmentalization of this multifunctional protein we constructed and characterized recombinant viruses expressing VP16 linked to the green fluorescent protein (GFP). These viruses replicate with virtually normal kinetics and yields and incorporate the fusion protein into the virion, resulting in autofluorescent particles. De novo-synthesized VP16-GFP was first detected in a diffuse pattern within the nucleus. Nuclear VP16-GFP was progressively recruited to replication compartments, which coalesced into large globular domains. By 10 to 12 h after infection additional distinct foci containing VP16-GFP could be seen, almost exclusively located at the periphery of the replication compartments. At the same time pronounced accumulation was observed in the cytoplasm, first in a diffuse pattern and then accumulating in vesicle-like compartments which were concentrated in an asymmetric fashion reminiscent of the Golgi. Inhibition of DNA replication resulted in prolonged diffuse nuclear distribution with minimal cytoplasmic accumulation. Treatment with brefeldin disrupted the cytoplasm vesicular pattern, resulting in redistributed large foci. Time-lapse microscopy demonstrated various dynamic features of infection, including the active induction of very long cellular projections (up to 100 μM). Vesicular clusters containing VP16 were transported within projections to the termini, which developed bulbous ends and appeared to embed into the membranes of adjacent uninfected cells.

scholarly journals Live-Cell Analysis of a Green Fluorescent Protein-Tagged Herpes Simplex Virus Infection

1999 ◽  
Vol 73 (5) ◽  
pp. 4110-4119 ◽  
Author(s):  
Gillian Elliott ◽  
Peter O’Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Live Cell ◽  
Cell Periphery ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.

scholarly journals Complexes between Herpes Simplex Virus Glycoproteins gD, gB, and gH Detected in Cells by Complementation of Split Enhanced Green Fluorescent Protein

2007 ◽  
Vol 81 (20) ◽  
pp. 11532-11537 ◽  
Author(s):  
Elisa Avitabile ◽  
Cristina Forghieri ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Split Egfp ◽  
Green Fluorescent ◽  
Simplex Virus ◽  
In Cells

ABSTRACT The interactions between herpes simplex virus gD and its nectin1 receptor or between gD, gB, and gH were analyzed by complementation of the N and C portions of split enhanced green fluorescent protein (EGFP) fused to the glycoproteins. The gDN-NectC complex was readily detected; the gDN-gCC complex was undetectable, highlighting the specificity of the assay. Split EGFP complementation was detected between proteins designated gDN+gHC, gDN+gBC, and gHN+gBC+wtgD (gB was deleted of endocytosis motifs), both in cells transfected with two-tree glycoproteins and in syncytia. The in situ assay provides evidence that gD interacts with gH and gB independently of each other and supports a model whereby gH and gB in complex exert their activities to gD.

scholarly journals Herpes Simplex Virus Capsids Are Transported in Neuronal Axons without an Envelope Containing the Viral Glycoproteins

2006 ◽  
Vol 80 (22) ◽  
pp. 11165-11177 ◽  
Author(s):  
Aleksandra Snyder ◽  
Todd W. Wisner ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Membrane Vesicles ◽  
Protein Fusion ◽  
Viral Glycoproteins ◽  
Glycoprotein Transport ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.

scholarly journals ATP-Dependent Localization of the Herpes Simplex Virus Capsid Protein VP26 to Sites of Procapsid Maturation

2000 ◽  
Vol 74 (3) ◽  
pp. 1468-1476 ◽  
Author(s):  
Jung Hee I. Chi ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Cold Sensitivity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Protein Fusion ◽  
Capsid Shell ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutanttsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.

Oncolytic Herpes Simplex Virus-1 Mutant Expressing Green Fluorescent Protein Can Detect and Treat Peritoneal Cancer

2004 ◽  
Vol 15 (6) ◽  
pp. 609-618 ◽  
Author(s):  
Stephen F. Stanziale ◽  
Brendon M. Stiles ◽  
Amit Bhargava ◽  
Scott A. Kerns ◽  
Nagesh Kalakonda ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Green Fluorescent Protein ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Herpes Simplex Virus 1 ◽  
Peritoneal Cancer ◽  
Oncolytic Herpes Simplex Virus ◽  
Green Fluorescent ◽  
Simplex Virus
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