Effects of UV-B Irradiation on IL-1.ALPHA. Production from Herpes Simplex Virus Infected-Cultured Human Keratinocytes.

ABSTRACT The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

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Endocytic Internalization of Herpes Simplex Virus 1 in Human Keratinocytes at Low Temperature

Journal of Virology ◽

10.1128/jvi.02195-20 ◽

2020 ◽

Author(s):

Nydia De La Cruz ◽

Dagmar Knebel-Mörsdorf

Keyword(s):

Plasma Membrane ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Low Temperature ◽

Human Keratinocytes ◽

Herpes Simplex Virus 1 ◽

Successful Infection ◽

Simplex Virus ◽

The Impact ◽

Hsv 1

Herpes simplex virus 1 (HSV-1) can adopt a variety of pathways to accomplish cellular internalization. In human keratinocytes representing the natural target cell of HSV-1, both direct plasma membrane fusion and endocytic uptake have been found. The impact of either pathway in successful infection, however, remains to be fully understood. To address the role of each internalization mode, we performed infection studies at low temperature as a tool to interfere with endocytic pathways. Interestingly, successful HSV-1 entry in primary human keratinocytes and HaCaT cells was observed even at 7°C, although delayed compared to infection at 37°C. Moreover, ex vivo infection of murine epidermis demonstrated that virus entry at 7°C is not only accomplished in cultured cells but also in tissue. Control experiments with cholera toxin B confirmed a block of endocytic uptake at 7°C. In addition, uptake of dextran by macropinosomes and phagocytic uptake of latex beads was also inhibited at 7°C. Infection of nectin-1-deficient murine keratinocytes affirmed that the entry at 7°C was receptor-dependent. Strikingly, the lysosomotropic agent, ammonium chloride, strongly inhibited HSV-1 entry suggesting a role for endosomal acidification. Ultrastructural analyses in turn revealed free capsids in the cytoplasm as well as virus particles in vesicles after infection at 7°C supporting both plasma membrane fusion and endocytic internalization as already observed at 37°C. Overall, entry of HSV-1 at 7°C suggests that the virus can efficiently adopt nectin-1-dependent unconventional vesicle uptake mechanisms in keratinocytes strengthening the role of endocytic internalization for successful infection. IMPORTANCE The human pathogen herpes simplex virus 1 (HSV-1) relies on multiple internalization pathways to initiate infection. Our focus is on the entry in human keratinocytes, the major in vivo target during primary and recurrent infection. While antivirals reduce the severity of clinical cases, there is no cure or vaccine against HSV. To develop strategies that interfere with virus penetration, we need to understand the various parameters and conditions that determine virus entry. Here, we addressed the impact of virus internalization via vesicles by blocking endocytic processes at low temperature. Intriguingly, we detected entry of HSV-1 even at 7°C which led to infection of primary keratinocytes and epidermal tissue. Moreover, electron microscopy of human keratinocytes at 7°C support that internalization is based on fusion of the viral envelope with the plasma membrane as well as vesicle membranes. These results provide novel insights into conditions that still allow endocytic internalization of HSV-1.

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Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent

PLoS ONE ◽

10.1371/journal.pone.0025464 ◽

2011 ◽

Vol 6 (10) ◽

pp. e25464 ◽

Cited By ~ 51

Author(s):

Elena Rahn ◽

Philipp Petermann ◽

Mei-Ju Hsu ◽

Frazer J. Rixon ◽

Dagmar Knebel-Mörsdorf

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Human Keratinocytes ◽

Simplex Virus

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Assembly of VP26 in herpes simplex virus-1 capsid implicated by 3-D structure of recombinant capsid

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140579 ◽

1995 ◽

Vol 53 ◽

pp. 840-841

Author(s):

Z. Hong Zhou ◽

Jing He ◽

Joanita Jakana ◽

J. D. Tatman ◽

Frazer J. Rixon ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

3D Structure ◽

Structure Study ◽

Herpes Simplex Virus 1 ◽

Shell Proteins ◽

Life Threatening ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus-1 (HSV-1) is a ubiquitous virus which is implicated in diseases ranging from self-curing cold sores to life-threatening infections. The 2500 Å diameter herpes virion is composed of a glycoprotein spike containing, lipid envelope, enclosing a protein layer (the tegument) in which is embedded the capsid (which contains the dsDNA genome). The B-, and A- and C-capsids, representing different morphogenetic stages in HSV-1 infected cells, are composed of 7, and 5 structural proteins respectively. The three capsid types are organized in similar T=16 icosahedral shells with 12 pentons, 150 hexons, and 320 connecting triplexes. Our previous 3D structure study at 26 Å revealed domain features of all these structural components and suggested probable locations for the outer shell proteins, VP5, VP26, VP19c and VP23. VP5 makes up most of both pentons and hexons. VP26 appeared to bind to the VP5 subunit in hexon but not to that in penton.

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Application of EM to differentiate herpes virus from pox virus in genital specimens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169043 ◽

1994 ◽

Vol 52 ◽

pp. 262-263

Author(s):

K. Rekrut ◽

K. Schleuter

Keyword(s):

Tissue Culture ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Fresh Tissue ◽

Culture Cells ◽

Viral Transport ◽

Carbon Coated ◽

Immuno Assay ◽

Simplex Virus ◽

Enzyme Immuno Assay

Confirmation of herpes simplex virus (HSV) from genital lesions of obstetrical (OB) patients may affect both the management of the delivery and of the neonate.(l,2) During 1992 and 1993, 4,450 genital specimens from OB patients were submitted in viral transport media for herpes culture. The specimens were inoculated into MRC-5, Vero, and A-549 tissue culture tubes, incubated, and examined daily for 7 days for cytopathic effect (CPE). The original specimens were frozen at −70° C until final reports were issued. Culture tubes with CPE were tested by the Dupont Herpchek enzyme immuno assay (EIA) to confirm the presence of herpes simplex virus (HSV). (3,4) 170 OB patient specimens were positive by culture and confirmed by EIA.There were also 63 cultures exhibiting CPE ressembling HSV which were negative by EIA testing, which failed to pass in fresh tissue culture cells or progress to more enhanced CPE in culture. These original specimens were screened by electron microscopy after direct ultracentrifugation employing the Beckman airfuge with the EM 90 rotor on to formvar carbon-coated 300 mesh copper grids and negatively stained with 2% PTA.

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