scholarly journals Herpes Simplex Virus Capsid Localization to ESCRT-VPS4 Complexes in the Presence and Absence of the Large Tegument Protein UL36p

2016 ◽  
Vol 90 (16) ◽  
pp. 7257-7267 ◽  
Author(s):  
Himanshu Kharkwal ◽  
Caitlin G. Smith ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Herpes Simplex Virus 1 ◽  
Absolute Requirement ◽  
Innermost Layer ◽  
Complex Protein ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACTUL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of tegument, the complex protein layer between the capsid and envelope. UL36p performs multiple functions in the HSV life cycle, including a critical but unknown role in capsid cytoplasmic envelopment. We tested whether UL36p is essential for envelopment because it is required to engage capsids with the cellular ESCRT/Vps4 apparatus. A green fluorescent protein (GFP)-fused form of the dominant negative ATPase Vps4-EQ was used to irreversibly tag ESCRT envelopment sites during infection by UL36p-expressing and UL36-null HSV strains. Using fluorescence microscopy and scanning electron microscopy, we quantitated capsid/Vps4-EQ colocalization and examined the ultrastructure of the corresponding viral assembly intermediates. We found that loss of UL36p resulted in a two-thirds reduction in the efficiency of capsid/Vps4-EQ association but that the remaining UL36p-null capsids were still able to engage the ESCRT envelopment apparatus. It appears that although UL36p helps to couple HSV capsids to the ESCRT pathway, this is likely not the sole reason for its absolute requirement for envelopment.IMPORTANCEEnvelopment of the HSV capsid is essential for the assembly of an infectious virion and requires the complex interplay of a large number of viral and cellular proteins. Critical to envelope assembly is the virally encoded protein UL36p, whose function is unknown. Here we test the hypothesis that UL36p is essential for the recruitment of cellular ESCRT complexes, which are also known to be required for envelopment.

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 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 A novel role for phagocytosis-like uptake in herpes simplex virus entry

2006 ◽  
Vol 174 (7) ◽  
pp. 1009-1021 ◽  
Author(s):  
Christian Clement ◽  
Vaibhav Tiwari ◽  
Perry M. Scanlan ◽  
Tibor Valyi-Nagy ◽  
Beatrice Y.J.T. Yue ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Herpes Simplex Virus 1 ◽  
Mode Of Entry ◽  
Virion Envelope ◽  
Membrane Protrusions ◽  
Simplex Virus ◽  
Hsv 1

It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defined. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and trafficking of the virions in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type–specific. Clathrin-coated vesicles had no detectable role in virion trafficking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.

scholarly journals Relaxed Repression of Herpes Simplex Virus Type 1 Genomes in Murine Trigeminal Neurons

2007 ◽  
Vol 81 (22) ◽  
pp. 12394-12405 ◽  
Author(s):  
Tracy Terry-Allison ◽  
Colton A. Smith ◽  
Neal A. DeLuca
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Trichostatin A ◽  
Cell Types ◽  
Immediate Early ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT The expression of herpes simplex virus (HSV) genomes in the absence of viral regulatory proteins in sensory neurons is poorly understood. Previously, our group reported an HSV immediate early (IE) mutant (d109) unable to express any of the five IE genes and encoding a model human cytomegalovirus immediate early promoter-green fluorescent protein (GFP) transgene. In cultured cells, GFP expressed from this mutant was observed in only a subset of infected cells. The subset exhibited cell type dependence, as the fractions of GFP-expressing cells varied widely among the cell types examined. Herein, we characterize this mutant in murine embryonic trigeminal ganglion (TG) cultures. We found that d109 was nontoxic to neural cultures and persisted in the cultures throughout their life spans. Unlike with some of the cultured cell lines and strains, expression of the GFP transgene was observed in a surprisingly large subset of neurons. However, very few nonneuronal cells expressed GFP. The abilities of ICP0 and an inhibitor of histone deacetylase, trichostatin A (TSA), to activate GFP expression from nonexpressing cells were also compared. The provision of ICP0 by infection with d105 reactivated quiescent genomes in nearly every cell, whereas reactivation by TSA was much more limited and restricted to the previously nonexpressing neurons. Moreover, we found that d109, which does not express ICP0, consistently reactivated HSV type 1 (KOS) in latently infected adult TG cultures. These results suggest that the state of persisting HSV genomes in some TG neurons may be more dynamic and more easily activated than has been observed with nonneuronal cells.

scholarly journals The Amino Terminus of the Herpes Simplex Virus 1 Protein Vhs Mediates Membrane Association and Tegument Incorporation

2006 ◽  
Vol 80 (20) ◽  
pp. 10117-10127 ◽  
Author(s):  
Aparna Mukhopadhyay ◽  
Grace E. Lee ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Amino Terminus ◽  
Membrane Association ◽  
Herpes Simplex Virus 1 ◽  
Herpes Simplex Viruses ◽  
Amino Terminal ◽  
Virion Host Shutoff ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT Assembly of herpes simplex viruses (HSV) is a poorly understood process involving multiple redundant interactions between large number of tegument and envelope proteins. We have previously shown (G. E. Lee, G. A. Church, and D. W. Wilson, J. Virol. 77:2038-2045, 2003) that the virion host shutoff (Vhs) tegument protein is largely insoluble in HSV-infected cells and is also stably associated with membranes. Here we demonstrate that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore, we have found that the amino-terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a green fluorescent protein (GFP) reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. We conclude that the amino terminus of Vhs mediates targeting to sites of HSV assembly and to the viral tegument.

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