Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1

Abstract

Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection which was not blocked by a nectin1 antibody and hence was not a consequence of residual nectin1 expression. Strikingly at later times, the population of cells originally resistant to HSV1 infection had also become infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a ΔUL34 virus defective in capsid export to the cytoplasm. Moreover, newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. Neutralizing human serum also failed to block virus transmission in nectin1 KO cells, which was dependent on the receptor binding protein glycoprotein D and the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission.

Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function

“…and fixed after 5 h. Cells were then either processed directly for immunofluorescence to stain only cell surface proteins or were permeabilized and stained to detect intracellular as well as cell surface protein. As previously shown in uninfected cells [27], nectin1 was localized to the cell surface of nTERT keratinocytes and the nectin1 antibody detected the protein at the cell surface, confirming that it recognized the extracellular domain of the protein (, mock). However, in HSV1-infected cells the characteristic cell surface pattern of nectin1 had disappeared by 5 h and nectin1 was not detectable within the permeabilized and stained cells, suggesting that nectin1 had been depleted during infection (, HSV1-GFP22).…”

“…Cell surface staining of HSV1-GFP22-infected nTERT cells with an antibody for the transferrin receptor revealed that this receptor was present on the cell surface of both mock and infected cells at 5 h, and although the morphology of the cells had slightly changed in response to HSV1 infection, there was little or no downregulation of transferrin receptor (). In addition, staining of Wt-infected nTERT cells fixed at 4 and 8 h for the highly related protein nectin2, which we have previously shown does not function as an entry receptor for HSV [27], indicated that as for transferrin and in contrast to nectin1, nectin2 remained at the cell surface of HSV1-infected cells (). This suggests that HSV1 infection specifically targets nectin1 for downregulation from the cell surface.…”

“…As a preliminary investigation of endogenous nectin1 in HSV1-infected nTERT keratinocyte cells, we wished to measure the overall level of nectin1 after virus infection. To date we have not found an antibody that detects endogenous nectin1 by Western blotting, even in nTERT cells that express a high level of the protein, but nectin1 is detectable in these cells by immunofluorescence and flow cytometry [ 27 ]. We have previously shown that HSV1 enters and replicates rapidly in nTERT keratinocyte cells [ 22 ] and hence, to initially determine the effect of virus infection on nectin1 localization, nTERT cells were infected with HSV1 expressing GFP-tagged VP22 [ 30 ] at high m.o.i.…”

“…Of note, HSV1 enters and replicates rapidly in these human keratinocytes [22]. Although HSV1 has been shown to use a number of different receptors [8, 23–26], we have recently demonstrated that nectin1 is the major receptor for HSV1 entry into nTERT keratinocyte cells [22, 27]. Importantly, unlike other cell types that have been used in the field, nTERT cells express relatively high levels of nectin1, and endogenous nectin1 is readily detectable at the cell surface by flow cytometry or immunofluorescence [27].…”

“…Although HSV1 has been shown to use a number of different receptors [8,[23][24][25][26], we have recently demonstrated that nectin1 is the major receptor for HSV1 entry into nTERT keratinocyte cells [22,27]. Importantly, unlike other cell types that have been used in the field, nTERT cells express relatively high levels of nectin1, and endogenous nectin1 is readily detectable at the cell surface by flow cytometry or immunofluorescence [27]. The nTERT keratinocyte system therefore represents a valuable tool for investigating the downstream effect of HSV1 infection on its endogenous receptor in a physiologically relevant cell type.…”