TY - JOUR T1 - Contributions of the four essential entry glycoproteins to HSV-1 tropism and the selection of entry routes JF - bioRxiv DO - 10.1101/2020.03.10.985325 SP - 2020.03.10.985325 AU - Adam T. Hilterbrand AU - Raecliffe Daly AU - Ekaterina E. Heldwein Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/03/11/2020.03.10.985325.abstract N2 - Herpes Simplex viruses (HSV-1 and HSV-2) encode up to 15 glycosylated and unglycosylated envelope proteins. Four of these, gB, gH, gL, and gD, are essential for entry and mediate cell-cell fusion when co-expressed in uninfected, receptor-bearing cells. However, their contributions to HSV-1 tropism and the selection of entry routes are unclear. To begin addressing this, we previously pseudotyped VSV lacking its native glycoprotein, G, with HSV-1 glycoproteins gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several aspects of HSV-1 entry: it could enter murine C10 cells, required gB, gH, gL, gD, and a cellular receptor for entry, and was sensitive to neutralization by gB and gH/gL antibodies. Here, we screened six additional HSV-1-susceptible cell lines and found that only two, C10 and CHO-HVEM cells, reproducibly supported a receptor-dependent entry by VSVΔG-BHLD. We then compared VSVΔG-BHLD and HSV-1 entry routes into these two cell lines using a combination of chemical and genetic inhibitors of cellular uptake pathways. We discovered that the VSVΔG-BHLD pseudotype not only has a narrower tropism but also uses entry pathways different from those used by HSV-1. We conclude that while the four essential HSV-1 entry glycoproteins enable entry in certain contexts, they are insufficient for entry into any HSV-1-susceptible cell nor do they specify native HSV-1 entry routes. We hypothesize that the HSV-1 envelope proteins outside the essential four (so-called “non-essential”) contribute towards the tropism and the selection of native HSV-1 entry routes. Our work draws attention to the need for systematic investigation of the HSV-1 entry mechanisms and the roles of the envelope proteins that were long considered non-essential in the selection of target cells, routes of entry, and pathogenesis.AUTHOR SUMMARY Different viruses enter cells by diverse routes, but how that choice is made is not always clear. Understanding the mechanisms behind these choices is vital for finding strategies to prevent viral infections. In enveloped viruses, viral proteins embedded in the envelope accomplish this task. While most enveloped viruses encode one or two envelope proteins, Herpes Simplex viruses (HSV) encode up to 15. Four of these are deemed essential for entry (gB, gH, gL, and gD) whereas the rest have been termed non-essential. While these four proteins are essential, their contributions to HSV-1 cellular tropism and entry pathways have not been fully elucidated. Here, we generated virions that have only the four essential HSV-1 glycoproteins on their surface. We show that the VSVΔG-BHLD pseudotype has a narrower tropism than HSV-1 and uses different entry pathways. Thus, the four essential HSV-1 entry glycoproteins alone do not define HSV-1 tropism or specify native entry routes. We hypothesize that the HSV-1 envelope proteins outside the essential four may contribute towards tropism and entry route selection. Our work emphasizes the need to investigate the roles of the so-called non-essential envelope proteins in HSV entry. This is important because HSV enters natural target cells, epithelial cells and neurons, by different, poorly defined routes. Mechanistic understanding of HSV entry is essential for understanding its pathogenesis and developing new strategies to prevent HSV entry and spread. ER -