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.

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

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.

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