scholarly journals Function of Dynein and Dynactin in Herpes Simplex Virus Capsid Transport

2002 ◽  
Vol 13 (8) ◽  
pp. 2795-2809 ◽  
Author(s):  
Katinka Döhner ◽  
André Wolfstein ◽  
Ute Prank ◽  
Christophe Echeverri ◽  
Denis Dujardin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cytoplasmic Dynein ◽  
Transport Processes ◽  
Viral Envelope ◽  
Cell Periphery ◽  
Virus Binding ◽  
Viral Protein Synthesis ◽  
Simplex Virus

After fusion of the viral envelope with the plasma membrane, herpes simplex virus type 1 (HSV1) capsids are transported along microtubules (MTs) from the cell periphery to the nucleus. The motor ATPase cytoplasmic dynein and its multisubunit cofactor dynactin mediate most transport processes directed toward the minus-ends of MTs. Immunofluorescence microscopy experiments demonstrated that HSV1 capsids colocalized with cytoplasmic dynein and dynactin. We blocked the function of dynein by overexpressing the dynactin subunit dynamitin, which leads to the disruption of the dynactin complex. We then infected such cells with HSV1 and measured the efficiency of particle binding, virus entry, capsid transport to the nucleus, and the expression of immediate-early viral genes. High concentrations of dynamitin and dynamitin-GFP reduced the number of viral capsids transported to the nucleus. Moreover, viral protein synthesis was inhibited, whereas virus binding to the plasma membrane, its internalization, and the organization of the MT network were not affected. We concluded that incoming HSV1 capsids are propelled along MTs by dynein and that dynein and dynactin are required for efficient viral capsid transport to the nucleus.

scholarly journals Evidence of a Role for Nonmuscle Myosin II in Herpes Simplex Virus Type 1 Egress

2002 ◽  
Vol 76 (7) ◽  
pp. 3471-3481 ◽  
Author(s):  
Hans van Leeuwen ◽  
Gill Elliott ◽  
Peter O'Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Specific Inhibitor ◽  
Transport Processes ◽  
Cell Periphery ◽  
Nonmuscle Myosin ◽  
Extracellular Medium ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT After cell entry, herpes simplex virus (HSV) particles are transported through the host cell cytoplasm to nuclear pores. Following replication, newly synthesized virus particles are transported back to the cell periphery via a complex pathway including a cytoplasmic phase involving some form of unenveloped particle. These various transport processes are likely to make use of one or more components of the cellular cytoskeletal systems and associated motor proteins. Here we report that the HSV type 1 (HSV-1) major tegument protein, VP22, interacts with the actin-associated motor protein nonmuscle myosin IIA (NMIIA). HSV-1 infection resulted in reorganization of NMIIA, inducing retraction of NMIIA from the cell periphery and condensation into a spoke-like distribution around the nucleus along with a second effect of accumulation in a perinuclear cluster. VP22 did not appear to colocalize with the reorganized cagelike distribution of NMIIA. However, VP22 has been previously reported to localize in a perinuclear vesicular pattern, and significant overlap was observed between this pattern and the perinuclear clusters of NMIIA. Inhibition of the ATPase activity of NMIIA with the myosin-specific inhibitor butanedione monoxime impaired the formation of the perinuclear vesicular VP22 accumulations and also the release of virus into the extracellular medium while having much less effect on the yield of cell-associated virus. Virus infection frequently results in the induction of highly extended processes emanating from the infected cell, and we observed that VP22-containing particles line up along NMIIA-containing filaments which run through these protrusions.

scholarly journals Herpes Simplex Virus ICP27 Activation of Stress Kinases JNK and p38

2005 ◽  
Vol 79 (13) ◽  
pp. 8348-8360 ◽  
Author(s):  
Danna Hargett ◽  
Tim McLean ◽  
Steven L. Bachenheimer
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Viral Gene ◽  
Functional Domain ◽  
Viral Protein Synthesis ◽  
Immediate Early Proteins ◽  
Simplex Virus

ABSTRACT We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0−/4−) and d103 (4−/22−/47−) mutants activated p38 and JNK, while the d106 (4−/22−/27−/47−) and d107 (4−/27−) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.

scholarly journals A Functional Interaction between Herpes Simplex Virus 1 Glycoprotein gH/gL Domains I and II and gD Is Defined by Using Alphaherpesvirus gH and gL Chimeras

2015 ◽  
Vol 89 (14) ◽  
pp. 7159-7169 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Host Cell ◽  
Current Model ◽  
Viral Envelope ◽  
Functional Interaction ◽  
Herpes Simplex Virus 1 ◽  
Homotypic Interaction ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTWhereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry, and thus communication among these proteins is required. For most alphaherpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast, neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions.IMPORTANCEThe first step of the herpesvirus life cycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two alphaherpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I and II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.

scholarly journals Surface Plasmon Resonance Reveals Direct Binding of Herpes Simplex Virus Glycoproteins gH/gL to gD and Locates a gH/gL Binding Site on gD

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Tina M. Cairns ◽  
Noah T. Ditto ◽  
Doina Atanasiu ◽  
Huan Lou ◽  
Benjamin D. Brooks ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Binding Site ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Geographical Area ◽  
Direct Interaction ◽  
Viral Envelope ◽  
Simplex Virus

ABSTRACTHerpes simplex virus (HSV) requires fusion between the viral envelope and host membrane. Four glycoproteins, gD, gH/gL, and gB, are essential for this process. To initiate fusion, gD binds its receptor and undergoes a conformational change that hypothetically leads to activation of gH/gL, which in turn triggers the fusion protein gB to undergo rearrangements leading to membrane fusion. Our model predicts that gD must interact with both its receptor and gH/gL to promote fusion. In support of this, we have shown that gD is structurally divided into two “faces”: one for the binding receptor and the other for its presumed interaction with gH/gL. However, until now, we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used surface plasmon resonance to show that the ectodomain of gH/gL binds directly to the ectodomain of gD when (i) gD is captured by certain anti-gD monoclonal antibodies (MAbs) that are bound to a biosensor chip, (ii) gD is bound to either one of its receptors on a chip, and (iii) gD is covalently bound to the chip surface. To localize the gH/gL binding site on gD, we used multiple anti-gD MAbs from six antigenic communities and determined which ones interfered with this interaction. MAbs from three separate communities block gD-gH/gL binding, and their epitopes encircle a geographical area on gD that we propose comprises the gH/gL binding domain. Together, our results show that gH/gL interacts directly with gD, supporting a role for this step in HSV entry.IMPORTANCEHSV entry is a multistep process that requires the actions of four glycoproteins, gD, gH/gL, and gB. Our current model predicts that gD must interact with both its receptor and gH/gL to promote viral entry. Although we know a great deal about how gD binds its receptors, until now we have been unable to demonstrate a direct interaction between gD and gH/gL. Here, we used a highly sensitive surface plasmon resonance technique to clearly demonstrate that gD and gH/gL interact. Furthermore, using multiple MAbs with defined epitopes, we have delineated a domain on gD that is independent of that used for receptor binding and which likely represents the gH/gL interaction domain. Targeting this interaction to prevent fusion may enhance both therapeutic and vaccine strategies.

scholarly journals The Herpes Simplex Virus 1 UL34 Protein Interacts with a Cytoplasmic Dynein Intermediate Chain and Targets Nuclear Membrane

2000 ◽  
Vol 74 (3) ◽  
pp. 1355-1363 ◽  
Author(s):  
Guo-Jie Ye ◽  
Kevin T. Vaughan ◽  
Richard B. Vallee ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Viral Proteins ◽  
Cytoplasmic Dynein ◽  
Dependent Manner ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Intermediate Chain

ABSTRACT To express the function encoded in its genome, the herpes simplex virus 1 capsid-tegument structure released by deenvelopment during entry into cells must be transported retrograde to the nuclear pore where viral DNA is released into the nucleus. This path is essential in the case of virus entering axons of dorsal root ganglia. The objective of the study was to identify the viral proteins that may be involved in the transport. We report the following findings. (i) The neuronal isoform of the intermediate chain (IC-1a) of the dynein complex pulled down, from lysates of [35S]methionine-labeled infected cells, two viral proteins identified as the products of UL34 and UL31 open reading frames, respectively. UL34 protein is a virion protein associated with cellular membranes and phosphorylated by the viral kinase US3. UL31 protein is a largely insoluble, evenly dispersed nuclear phosphoprotein required for optimal processing and packaging of viral DNA into preformed capsids. Reciprocal pulldown experiments verified the interaction of IC-1a and UL34 protein. In similar experiments, UL34 protein was found to interact with UL31 protein and the major capsid protein ICP5. (ii) To determine whether UL34 protein is transported to the nuclear membrane, a requirement if it is involved in transport, the UL34 protein was inserted into a baculovirus vector under the cytomegalovirus major early promoter. Cells infected with the recombinant baculovirus expressed UL34 protein in a dose-dependent manner, and the UL34 protein localized primarily in the nuclear membrane. An unexpected finding was that UL34-expressing cells showed a dissociation of the inner and outer nuclear membranes reminiscent of the morphologic changes seen in cells productively infected with herpes simplex virus 1. UL34, like many other viral proteins, may have multiple functions expressed both early and late in infection.

scholarly journals Packaging of the Virion Host Shutoff (Vhs) Protein of Herpes Simplex Virus: Two Forms of the Vhs Polypeptide Are Associated with Intranuclear B and C Capsids, but Only One Is Associated with Enveloped Virions

2007 ◽  
Vol 81 (3) ◽  
pp. 1148-1161 ◽  
Author(s):  
G. Sullivan Read ◽  
Mary Patterson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Viral Envelope ◽  
Translation Product ◽  
Primary Translation Product ◽  
Virion Host Shutoff ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.

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 A Herpes Simplex Virus 1 (McKrae) Mutant Lacking the Glycoprotein K Gene Is Unable To Infect via Neuronal Axons and Egress from Neuronal Cell Bodies

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Andrew T. David ◽  
Ahmad Saied ◽  
Anu Charles ◽  
Ramesh Subramanian ◽  
Vladimir N. Chouljenko ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Recombinant Virus ◽  
Neuronal Cell ◽  
Viral Envelope ◽  
Viral Glycoprotein ◽  
Herpes Simplex Virus 1 ◽  
Neuronal Cell Bodies ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTWe have shown that the herpes simplex virus 1 (HSV-1) gK gene is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglion neuroinvasion in mice (A. T. David, A. Baghian, T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, Curr. Eye Res. 33:455–467, 2008). To further investigate the role of gK in neuronal infection, we utilized a microfluidic chamber system separating neuronal cell bodies and axonal termini. HSV-1 (McKrae) engineered virus constitutively expressing enhanced green fluorescence protein (GFP) was efficiently transmitted in both a retrograde and an anterograde manner. These results were corroborated by expression of virion structural proteins in either chamber, as well as detection of viral genomes and infectious viruses. In contrast, efficient infection of either chamber with a gK-null virus did not result in infection of the apposed chamber. These results show that gK is an important determinant in virion axonal infection. Moreover, the inability of the gK-null virus to be transmitted in an anterograde manner suggests that virions acquire cytoplasmic envelopes prior to entering axons.IMPORTANCEHerpes simplex virus 1 (HSV-1) enters mucosal epithelial cells and neurons via fusion of the viral envelope with cellular membranes, mediated by viral glycoprotein B (gB) in cooperation with other viral glycoproteins. Retrograde transport of virions to neuronal cell bodies (somata) establishes lifelong latent infection in ganglionic neurons. We have previously reported that gK binds gB and is required for gB-mediated membrane fusion (Jambunatathan et al., J. Virol. 85:12910–12918, 2011; V. N. Chouljenko, A. V. Iyer, S. Chowdhury, J. Kim, and K. G. Kousoulas, J. Virol. 84:8596–8606, 2010). In the current study, we constructed a recombinant virus with the gK gene deleted in the highly virulent ocular HSV-1 strain McKrae. This recombinant virus failed to infect rat ganglionic neuronal axons alone or cocultured with Vero cells in microfluidic chambers. In addition, lack of gK expression prevented anterograde transmission of virions. These results suggest that gK is a critical determinant for neuronal infection and transmission.

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