Characterization of Vesicular Stomatitis Virus Pseudotypes Bearing Essential Entry Glycoproteins gB, gD, gH, and gL of Herpes Simplex Virus 1

Abstract

Herpes simplex viruses (HSVs) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the herpes simplex virus 1 (HSV-1) entry mechanism is complicated by the presence of more than a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated vesicular stomatitis virus (VSV) virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSV⌬G-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytium formation. The HSV pseudotyping system established in this study presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms. IMPORTANCEHerpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells-a prerequisite for a successful infection-is a complex process that involves multiple viral and host proteins and occurs by different routes. Detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development, yet the presence of more than a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. In this study, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered in this work opens doors for future systematic exploration of the herpesvirus entry mechanisms.T o enter living cells to replicate, viruses must overcome the barrier of the cellular membrane. Enveloped viruses accomplish this task by facilitating the merger of their envelope with a target cell membrane, during which capsids are delivered into the cytosol and infection ensues. Entry is initiated by binding of a virus to an appropriate receptor on the surface of the host cell and is catalyzed by a virus-encoded membrane fusogen. In most enveloped viruses, the receptor binding and the fusogenic functions are executed by a single protein (1).Herpesviruses are double-stranded DNA, enveloped viruses with intricate envelopes that contain...

Contributions of the four essential entry glycoproteins to HSV-1 tropism and the selection of entry routes

“…As expected, HSV-1 entered all receptor-bearing cells but not receptor-negative B78H1 or CHO-K1 cells (Fig 2A). VSVΔG-BHLD pseudotype also readily infected C10 cells but not parent B78H1 cells (Fig 2B), consistent with previous observations (27). VSVΔG-BHLD pseudotype also infected CHO-HVEM and CHO-nectin-1 cells in a receptor-dependent manner albeit with much lower efficiency (Fig 2B).…”

“…Previously, we showed that the VSVΔG-BHLD pseudotype efficiently entered C10 cells and that its entry recapitulated several important features of HSV-1 entry into susceptible cells: the requirement for gB, gH, gL, gD, and a gD receptor, and sensitivity to anti-gB and anti-gH/gL neutralizing antibodies (27). Here, we expanded this study to six additional HSV-1-susceptible cell lines and found that out of seven, only C10 and CHO-HVEM cells supported appreciable VSVΔG-BHLD entry.…”

“…To begin addressing this question, we previously generated VSV lacking its native glycoprotein G and pseudotyped with HSV-1 gB, gH, gL, and gD through trans -complementation, which we refer to as the VSVΔG-BHLD pseudotype (27). The VSVΔG-BHLD pseudotype efficiently entered C10 cells (B78 murine melanoma cells expressing nectin-1), and its entry recapitulated several important features of HSV-1 entry into susceptible cells, namely, the requirement for gB, gH, gL, gD, and a gD receptor, as well as sensitivity to anti-gB and anti-gH/gL neutralizing antibodies (27). However, this study left unclear whether the VSVΔG-BHLD pseudotype could enter any HSV-1 susceptible cell types or specify native entry routes.…”

Peptides Derived from Glycoproteins H and B of Herpes Simplex Virus Type 1 and Herpes Simplex Virus Type 2 Are Capable of Blocking Herpetic Infection in vitro

“…We believe that these peptides may be interacting with glycoprotein D, which is the least conserved protein between both viruses (85%) and is responsible for the activation of the heterodimer gH-gL [42,51]. …”

“…For herpes simplex viruses, the viral entry process takes place with the aid of 4 viral glycoproteins: gD, gB, gH, and gL, all of which have been proven to be essential for these viruses to carry out membrane fusion [42]. …”