scholarly journals Herpes Simplex Virus gE/gI Expressed in Epithelial Cells Interferes with Cell-to-Cell Spread

2003 ◽  
Vol 77 (4) ◽  
pp. 2686-2695 ◽  
Author(s):  
Wendy J. Collins ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Cell Types ◽  
Cell Junctions ◽  
Neuronal Tissues ◽  
Extensive Cell ◽  
Simplex Virus ◽  
Cellular Components ◽  
Cell Spread

ABSTRACT The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.

scholarly journals Herpes Simplex Virus gE/gI Must Accumulate in the trans-Golgi Network at Early Times and Then Redistribute to Cell Junctions To Promote Cell-Cell Spread

2006 ◽  
Vol 80 (7) ◽  
pp. 3167-3179 ◽  
Author(s):  
Aaron Farnsworth ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Junctions ◽  
Virus Spread ◽  
Neuronal Tissues ◽  
Mediate Cell ◽  
Simplex Virus ◽  
Golgi Network ◽  
Cell Spread ◽  
Cell Cell

ABSTRACT Herpes simplex virus (HSV) glycoprotein heterodimer gE/gI is necessary for virus spread in epithelial and neuronal tissues. Deletion of the relatively large gE cytoplasmic (CT) domain abrogates the ability of gE/gI to mediate HSV spread. The gE CT domain is required for the sorting of gE/gI to the trans-Golgi network (TGN) in early stages of virus infection, and there are several recognizable TGN sorting motifs grouped near the center of this domain. Late in HSV infection, gE/gI, other viral glycoproteins, and enveloped virions redistribute from the TGN to epithelial cell junctions, and the gE CT domain is also required for this process. Without the gE CT domain, newly enveloped virions are directed to apical surfaces instead of to cell junctions. We hypothesized that the gE CT domain promotes virus envelopment into TGN subdomains from which nascent enveloped virions are sorted to cell junctions, a process that enhances cell-to-cell spread. To characterize elements of the gE CT domain involved in intracellular trafficking and cell-to-cell spread, we constructed a panel of truncation mutants. Specifically, these mutants were used to address whether sorting to the TGN and redistribution to cell junctions are necessary, and sufficient, for gE/gI to promote cell-to-cell spread. gE-519, lacking 32 C-terminal residues, localized normally to the TGN early in infection and then trafficked to cell junctions at late times and mediated virus spread. By contrast, mutants gE-495 (lacking 56 C-terminal residues) and gE-470 (lacking 81 residues) accumulated in the TGN but did not traffic to cell junctions and did not mediate cell-to-cell spread. A fourth mutant, gE-448 (lacking most of the CT domain), did not localize to cell junctions and did not mediate virus spread. Therefore, the capacity of gE/gI to promote cell-cell spread requires early localization to the TGN, but this is not sufficient for virus spread. Additionally, gE CT sequences between residues 495 and 519, which contain no obvious cell sorting motifs, are required to promote gE/gI traffic to cell junctions and cell-to-cell spread.

scholarly journals The Extracellular Domain of Herpes Simplex Virus gE Is Indispensable for Efficient Cell-to-Cell Spread: Evidence for gE/gI Receptors

2005 ◽  
Vol 79 (18) ◽  
pp. 11990-12001 ◽  
Author(s):  
Katarina Polcicova ◽  
Kim Goldsmith ◽  
Barb L. Rainish ◽  
Todd W. Wisner ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Critical Role ◽  
Cell Junctions ◽  
Extracellular Virus ◽  
Spread Function ◽  
Neuronal Tissues ◽  
Simplex Virus ◽  
And Function ◽  
Cell Spread

ABSTRACT Herpes simplex virus (HSV) spreads rapidly and efficiently within epithelial and neuronal tissues. The HSV glycoprotein heterodimer gE/gI plays a critical role in promoting cell-to-cell spread but does not obviously function during entry of extracellular virus into cells. Thus, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. There was previous evidence that the large extracellular (ET) domains of gE/gI might be important in cell-to-cell spread. First, gE/gI extensively accumulates at cell junctions, consistent with being tethered there. Second, expression of gE/gI in trans interfered with HSV spread between epithelial cells. To directly test whether the gE ET domain was necessary for gE/gI to promote virus spread, a panel of gE mutants with small insertions in the ET domain was constructed. Cell-to-cell spread was reduced when insertions were made within either of two regions, residues 256 to 291 or 348 to 380. There was a strong correlation between loss of cell-to-cell spread function and binding of immunoglobulin. gE ET domain mutants 277, 291, and 348 bound gI, produced mature forms of gE that reached the cell surface, and were incorporated into virions yet produced plaques similar to gE null mutants. Moreover, all three mutants were highly restricted in spread within the corneal epithelium, in the case of mutant 277 to only 4 to 6% of the number of cells compared with wild-type HSV. Therefore, the ET domain of gE is indispensable for efficient cell-to-cell spread. These observations are consistent with our working hypothesis that gE/gI can bind extracellular ligands, so-called gE/gI receptors that are concentrated at epithelial cell junctions. This fits with similarities in structure and function of gE/gI and gD, which is a receptor binding protein.

scholarly journals Cytoplasmic Domain of Herpes Simplex Virus gE Causes Accumulation in the trans-Golgi Network, a Site of Virus Envelopment and Sorting of Virions to Cell Junctions

2001 ◽  
Vol 75 (4) ◽  
pp. 1928-1940 ◽  
Author(s):  
Tom N. McMillan ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Chimeric Protein ◽  
Cell Junctions ◽  
Cytoplasmic Vesicles ◽  
Simplex Virus ◽  
Golgi Network ◽  
Late Stages ◽  
Cell Spread

ABSTRACT Alphaherpesviruses express a heterodimeric glycoprotein, gE/gI, that facilitates cell-to-cell spread between epithelial cells and neurons. Herpes simplex virus (HSV) gE/gI accumulates at junctions formed between polarized epithelial cells at late times of infection. However, at earlier times after HSV infection, or when gE/gI is expressed using virus vectors, the glycoprotein localizes to thetrans-Golgi network (TGN). The cytoplasmic (CT) domains of gE and gI contain numerous TGN and endosomal sorting motifs and are essential for epithelial cell-to-cell spread. Here, we swapped the CT domains of HSV gE and gI onto another HSV glycoprotein, gD. When the gD-gICT chimeric protein was expressed using a replication-defective adenovirus (Ad) vector, the protein was found on both the apical and basolateral surfaces of epithelial cells, as was gD. By contrast, the gD-gECT chimeric protein, gE/gI, and gE, when expressed by using Ad vectors, localized exclusively to the TGN. However, gD-gECT, gE/gI, and TGN46, a cellular TGN protein, became redistributed largely to lateral surfaces and cell junctions during intermediate to late stages of HSV infection. Strikingly, gE and TGN46 remained sequestered in the TGN when cells were infected with a gI−HSV mutant. The redistribution of gE/gI to lateral cell surfaces did not involve widespread HSV inhibition of endocytosis because the transferrin receptor and gE were both internalized from the cell surface. Thus, gE/gI accumulates in the TGN in early phases of HSV infection then moves to lateral surfaces, to cell junctions, at late stages of infection, coincident with the redistribution of a TGN marker. These results are related to recent observations that gE/gI participates in the envelopment of nucleocapsids into cytoplasmic vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004–4016, 2000) and that gE/gI can sort nascent virions from cytoplasmic vesicles specifically to the lateral surfaces of epithelial cells (D. C. Johnson, M. Webb, T. W. Wisner, and C. Brunetti, J. Virol. 75:821–833, 2000). Therefore, gE/gI localizes to the TGN, through interactions between the CT domain of gE and cellular sorting machinery, and then participates in envelopment of cytosolic nucleocapsids there. Nascent virions are then sorted from the TGN to cell junctions.

scholarly journals The Extracellular Domain of Herpes Simplex Virus gE Is Sufficient for Accumulation at Cell Junctions but Not for Cell-to-Cell Spread

2000 ◽  
Vol 74 (5) ◽  
pp. 2278-2287 ◽  
Author(s):  
Todd Wisner ◽  
Craig Brunetti ◽  
Kevin Dingwell ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Phosphorylation Site ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Cell Junctions ◽  
Apical Surface ◽  
Membrane Glycoproteins ◽  
Simplex Virus ◽  
Cell Spread

ABSTRACT Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.

scholarly journals Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus 2 Release from Cells

2018 ◽  
Vol 92 (23) ◽  
Author(s):  
James Hopkins ◽  
Tejabhiram Yadavalli ◽  
Alex M. Agelidis ◽  
Deepak Shukla
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Cathepsin L ◽  
Cell Types ◽  
Vaginal Epithelial Cells ◽  
Simplex Virus ◽  
Herpes Simplex Virus 2

ABSTRACTHerpes simplex virus 2 (HSV-2) can productively infect many different cell types of human and nonhuman origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L, in HSV-2 release from cells. In vaginal epithelial cells, HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-κB nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface and thus facilitate virus release from cells.IMPORTANCEGenital infections by HSV-2 represent one of the most common sexually transmitted viral infections. The virus causes painful lesions and sores around the genitals or rectum. Intermittent release of the virus from infected tissues during sexual activities is the most common cause of transmission. At the molecular level, cell surface heparan sulfate (HS) is known to provide attachment sites for HSV-2. While the removal of HS during HSV-1 release has been shown, not much is known about the host factors and their regulators that contribute to HSV-2 release from natural target cell types. Here we suggest a role for the host enzyme heparanase in HSV-2 release. Our work reveals that in addition to the regulation of transcription by NF-κB, HPSE is also regulated posttranslationally by cathepsin L and that inhibition of heparanase activity directly affects HSV-2 release. We provide unique insights into the host mechanisms controlling HSV-2 egress and spread.

scholarly journals Differential Requirements for gE, gI, and UL16 among Herpes Simplex Virus 1 Syncytial Variants Suggest Unique Modes of Dysregulating the Mechanism of Cell-to-Cell Spread

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Jillian C. Carmichael ◽  
John W. Wills
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Neutralizing Antibodies ◽  
Cell Junctions ◽  
Accessory Protein ◽  
Accessory Proteins ◽  
Viral Fusion ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Cell Spread

ABSTRACTLike all the herpesviruses, herpes simplex virus encodes machinery that enables it to move through cell junctions to avoid neutralizing antibodies. This cell-to-cell spread mechanism requires the viral fusion machinery (gD, gH/gL, and gB) and numerous accessory proteins. Of all of these, minor alterations to only four proteins (gB, gK, UL20, or UL24) will dysregulate the fusion machinery, allowing the formation of syncytia. In contrast, removal of individual accessory proteins will block cell-to-cell spread, forcing the virus to transmit in a cell-free manner. In the context of a Syn variant, removal of a required accessory protein will block cell fusion, again forcing cell-free spread. This has been investigated most thoroughly for gBsyn variants, which lose their syncytial phenotype in the absence of several accessory proteins, including gE, gI, UL16, and UL21, which are known to physically interact. Recently it was found that UL21 is not needed for gKsyn-, UL20syn-, or UL24syn-induced cell fusion, and hence it was of interest to ascertain whether gE, gI, and UL16 are required for Syn variants other than gBsyn. Null mutants of these were each combined with seven syncytial variants distributed among gK, UL20, and UL24. Surprisingly, very different patterns of accessory protein requirements were revealed. Indeed, for the three gKsyn variants tested, two different patterns were found. Also, three mutants were able to replicate without causing cytopathic effects. These findings show that mutations that produce Syn variants dysregulate the cell-to-cell-spread machinery in unique ways and provide clues for elucidating how this virus moves between cells.IMPORTANCEApproximately 2/3 of adults worldwide are latently infected with herpes simplex virus 1. Upon reactivation, the virus has the ability to evade neutralizing antibodies by moving through cell junctions, but the mechanism of direct cell-to-cell spread is poorly understood. The machinery that assembles between cells includes the viral fusion proteins and various accessory proteins that prevent cells from fusing. Alterations in four proteins will dysregulate the machinery, allowing neighboring cells to fuse to make syncytia, but this can be prevented by removing various individual accessory proteins to further disable the machinery. Previously, the accessory protein UL21 was found to be important for the activity of some syncytial variants but not others. In this study, we discovered that UL16, gE, and gI all act differently in how they control the fusion machinery. A better understanding of the mechanism of cell-to-cell spread may enable the development of drugs that block it.

scholarly journals Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus-2 Release from Cells

2018 ◽  
Author(s):  
James Hopkins ◽  
Tejabhiram Yadavalli ◽  
Alex Agelidis ◽  
Deepak Shukla
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Heparan Sulfate ◽  
Cathepsin L ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Vaginal Epithelial Cells ◽  
Simplex Virus ◽  
Herpes Simplex Virus 2

Herpes simplex virus-2 (HSV-2) can productively infect many different cell types of human and non-human origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L in HSV-2 release from cells. In vaginal epithelial cells and other cell lines tested, HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-kB nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface, and thus facilitate virus release from cells.

scholarly journals Herpes Simplex Virus gE/gI Sorts Nascent Virions to Epithelial Cell Junctions, Promoting Virus Spread

2001 ◽  
Vol 75 (2) ◽  
pp. 821-833 ◽  
Author(s):  
David C. Johnson ◽  
Mike Webb ◽  
Todd W. Wisner ◽  
Craig Brunetti
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Pseudorabies Virus ◽  
Membrane Vesicles ◽  
Cell Junctions ◽  
Virus Spread ◽  
Varicella Zoster ◽  
Virus Particles ◽  
Simplex Virus

ABSTRACT Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, μ1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004–4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

scholarly journals The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

1998 ◽  
Vol 72 (11) ◽  
pp. 8933-8942 ◽  
Author(s):  
Kevin S. Dingwell ◽  
David C. Johnson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cell Contact ◽  
Junction Protein ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT The herpes simplex virus (HSV) glycoprotein complex gE-gI mediates the spread of viruses between adjacent cells, and this property is especially evident for cells that form extensive cell junctions, e.g., epithelial cells, fibroblasts, and neurons. Mutants lacking gE or gI are not compromised in their ability to enter cells as extracellular viruses. Therefore, gE-gI functions specifically in the movement of virus across cell-cell contacts and, as such, provides a molecular handle on this poorly understood process. We expressed gE-gI in human epithelial cells by using replication-defective adenovirus (Ad) vectors. gE-gI accumulated at lateral surfaces of the epithelial cells, colocalizing with the adherens junction protein β-catenin but was not found on either the apical or basal plasma membranes and did not colocalize with ZO-1, a component of tight junctions. In subconfluent monolayers, gE-gI was found at cell junctions but was absent from those lateral surfaces not in contact with another cell, as was the case for β-catenin. Similar localization of gE-gI to cell junctions was observed in HSV-infected epithelial cells. By contrast, HSV glycoprotein gD, expressed using a recombinant Ad vectors, was found primarily along the apical surfaces of cells, with little or no protein found on the basal or lateral surfaces. Expression of gE-gI without other HSV polypeptides did not cause redistribution of either ZO-1 or β-catenin or alter tight-junction functions. Together these results support a model in which gE-gI accumulates at sites of cell-cell contact by interacting with junctional components. We hypothesize that gE-gI mediates transfer of HSV across cell junctions by virtue of these interactions with cell junction components.

The effect of prostaglandins on the multiplication and cell-to-cell spread of herpes simplex virus type 2 in vitro

1982 ◽  
Vol 144 (3) ◽  
pp. 346-349 ◽  
Author(s):  
David A. Baker ◽  
Joanne Thomas ◽  
Judy Epstein ◽  
Dominic Possilico ◽  
Martin L. Stone
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Simplex Virus ◽  
Cell Spread
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