Contributions of the Four Essential Entry Glycoproteins to HSV-1 Tropism and the Selection of Entry Routes

Abstract

Herpes simplex viruses (HSV-1 and HSV-2) encode up to 16 envelope proteins, four of which are essential for entry. However, whether these four proteins alone are sufficient to dictate the broad cellular tropism of HSV-1 and the selection of different cell type-dependent entry routes is unknown. To begin addressing this, we previously pseudotyped vesicular stomatitis virus (VSV), lacking its native glycoprotein G, with only the four essential entry glycoproteins of HSV-1: gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several important features of HSV-1 entry: the requirement for gB, gH, gL, gD, and a cellular receptor and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies. However, due to the use of a single cell type in that study, the tropism of the VSVΔG-BHLD pseudotype was not investigated. Here, we show that the cellular tropism of the pseudotype is severely limited compared to that of wild-type HSV-1 and that its entry pathways differ from the native HSV-1 entry pathways. To test the hypothesis that other HSV-1 envelope proteins may contribute to HSV-1 tropism, we generated a derivative pseudotype containing the HSV-1 glycoprotein C (VSVΔG-BHLD-gC) and observed a gC-dependent increase in entry efficiency in two cell types. We propose that the pseudotyping platform developed here has the potential to uncover functional contributions of HSV-1 envelope proteins to entry in a gain-of-function manner. IMPORTANCE Herpes simplex viruses (HSV-1 and HSV-2) contain up to 16 different proteins in their envelopes. Four of these, glycoproteins gB, gD, gH, and gL, are termed essential with regard to entry, whereas the rest are typically referred to as nonessential based on the entry phenotypes of the respective single genetic deletions. However, the single-gene deletion approach, which relies on robust loss-of-function phenotypes, may be confounded by functional redundancies among the many HSV-1 envelope proteins. We have developed a pseudotyping platform in which the essential four entry glycoproteins are isolated from the rest, which can be added back individually for systematic gain-of-function entry experiments. Here, we show the utility of this platform for dissecting the contributions of HSV envelope proteins, both the essential four and the remaining dozen (using gC as an example), to HSV entry.

In-vitroreconstitution of Herpes Simplex Virus 1 fusion identifies low pH as a fusion co-trigger

“…2A and D ). Since HSV-1 enters most known target cell types by pH-dependent endocytosis (10, 11, 57, 58), we tested pH ranging from pH 6.5 to 4.5 by adding predetermined amounts of HCl after incubation with a soluble receptor. Upon acidification in the presence of HVEM200t-His 8 , we observed robust fusion at pH <5.5, ∼40% ( Fig.…”

“…To test whether these four glycoproteins were sufficient for in-vitro fusion, we generated VSV virions lacking the native fusogen G and pseudotyped with HSV-1 gB, gH, gL, and gD (VSVΔG-BHLD). Previously, we showed that these VSVΔG-BHLD pseudotypes entered two types of HSV-1-susceptible cells (57, 88). Here, we found that the VSVΔG-BHLD pseudotypes fused with the target BMP LUVs.…”

“…Many enveloped viruses require cholesterol either in the host cell membrane or the viral envelope for entry (40,41,(63)(64)(65)(66)(67)(68)(69)(70). Indeed, cholesterol and other sterols, such as desmosterol, are required for efficient HSV-1 infection (57,66,68), for yet unclear reasons. In the case of HIV entry, cholesterol has been proposed to induce the formation of ordered lipid domains, specifically requiring the L o /L d interface (41) and can further regulate fusion by reducing membrane lateral mobility (63).…”

Antiviral Activities of Officinaloside C against Herpes Simplex Virus-1

“…HSV-1 entry needs four glycoproteins: -gB, gH, gL, and gD-. [ 17 ]. Therefore, it is possible that the inhibitory effect of officinaloside C on viral replication is due to the inhibition of gB expression.…”

Host Cell Neddylation Facilitates Alphaherpesvirus Entry in a Virus-Specific and Cell-Dependent Manner

“…There are few if any host cell processes known to mediate the initial internalization step of HSV entry. Endocytic uptake of HSV can occur independently of clathrin, caveolin, cholesterol, and dynamin ( 22 , 68 , 92 – 97 ). We show that neddylation is integral to internalization of HSV-1 by endocytosis in B78-nectin-1 cells and human HaCaT cells.…”