A Novel Phagocytosis Uptake Mode of Herpes Simplex Virus–1 Involves Receptor Localization Within the Phagosome

Abstract

Abstract: : Purpose:To investigate the mechanism of entry of herpes simplex virus 1 (HSV–1) into human corneal fibroblasts. Methods: This study was designed to assess alternate mechanism(s) of entry of HSV–1 using primary cultures of fibroblasts from the stroma of the human cornea, to further understand HSV–1 physiopathology of the stroma. We examined electron micrographs of a purified recombinant strain of wild type HSV–1 (KOS gL86) entry into Chinese hamster ovary (CHO) cells transfected with alphaherpesvirus entry receptor, nectin–1 (or HVEM) and identified a typical phagocytosis uptake mode. We adopted an assay in which the virus as the phagocytosis effector was evaluated. Cytochalasin D and lysosomotropic agents were used to interfere with plasma membrane projections and low pH, to block HSV–1 uptake. Furthermore the role of pH in virus entry was assessed by a virus–free cell fusion luciferase assay. Heparan sulfate (HS) mediated phagocytosis engulfment in response to HSV–1 attachment was evaluated by a spinoculation technique in which the low efficiency of virus entry in HS deficient CHO–745 cells was compensated for by centrifugal force. To colocalize the receptor and phagosome we used nectin–1–EGFP, a fluorescent homologue and Texas–red tagged transferrin in cell systems infected with virus and consequently showed virus trafficking within infected cells by a green fluorescent tagged virus and a marker for early phagosome. Results: Our results show that HSV–1 entry is via a unique phagocytosis uptake mode and this route is characteristic of human corneal fibroblasts. However, within the corneal tissue there was variation of entry as related control primary trabecular meshwork cells appeared to aid attachment and fusion at the plasma membrane as previously reported. Electron micrographs showed cellular projections engulfing intact viral particles. Interestingly, we colocalized receptor and phagosome and showed virus presence within these vesicles suggesting that they are essential for tethering virion envelope to phagosomal membrane. Furthermore, low pH favored enhanced fusion of the virion envelope with the phagosomal membrane. Conclusions: We have demonstrated a unique HSV–1 entry mechanism relevant in human corneal fibroblasts. The engulfment of intact virions and release of capsids within the cell suggest a complex immune response to HSV–1 once it infects the corneal stroma, perhaps contributing to the severity of stromal keratitis.

Keywords: herpes simplex virus • cornea: stroma and keratocytes • microscopy: confocal/tunneling