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Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.

PloS one (2011-10-25)
Elena Rahn, Philipp Petermann, Mei-Ju Hsu, Frazer J Rixon, Dagmar Knebel-Mörsdorf
ABSTRACT

Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methyl-β-cyclodextrin, produced by Wacker Chemie AG, Burghausen, Germany, ≥95.0% cyclodextrin basis (calculated)
Sigma-Aldrich
Cytochalasin D, Ready Made Solution, from Zygosporium mansonii, 5 mg/mL in DMSO
Sigma-Aldrich
Methyl-β-cyclodextrin, Produced by Wacker Chemie AG, Burghausen, Germany, Life Science, ≥98.0% cyclodextrin basis
Sigma-Aldrich
Cholesterol, from sheep wool, ≥92.5% (GC), powder
Sigma-Aldrich
Cholesterol, powder, BioReagent, suitable for cell culture, ≥99%
Sigma-Aldrich
Nocodazole, ≥99% (TLC), powder
Sigma-Aldrich
Methyl-β-cyclodextrin, average Mn 1310
Sigma-Aldrich
Methyl-β-cyclodextrin, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Cholesterol, Sigma Grade, ≥99%
Sigma-Aldrich
Methyl-β-cyclodextrin
Sigma-Aldrich
Cytochalasin D, from Zygosporium mansonii, ≥98% (TLC and HPLC), powder
Sigma-Aldrich
5-(N-Ethyl-N-isopropyl)amiloride
Sigma-Aldrich
Phalloidin–Tetramethylrhodamine B isothiocyanate, sequence from Amanita phalloides(synthetic: peptide sequence)
SAFC
Cholesterol, Plant-Derived, SyntheChol®, NF, Ph. Eur., JP