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

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 degrees C, although it was delayed compared to infection at 37 degrees C. Moreover, ex vivo infection of murine epidermis demonstrated that virus entry at 7 degrees C is accomplished not only in cultured cells but also in tissue. Control experiments with cholera toxin B confirmed a block of endocytic uptake at 7 degrees C. In addition, uptake of dextran by macropinosomes and phagocytic uptake of latex beads were also inhibited at 7 degrees C. Infection of nectin-1-deficient murine keratinocytes affirmed that the entry at 7 degrees 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 degrees C, supporting both plasma membrane fusion and endocytic internalization as already observed at 37 degrees C. Overall, entry of HSV-1 at 7 degrees 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 degrees C, which led to infection of primary keratinocytes and epidermal tissue. Moreover, electron microscopy of human keratinocytes at 7 degrees 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