Glycoprotein D of HSV-1 is dependent on tegument protein UL16 for packaging and contains a motif that is differentially required for syncytia formation

Abstract

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) plays a key role in multiple events during infection including virus entry, cell-to-cell spread, and virus-induced syncytia formation. Here, we provide evidence that an arginine/lysine cluster located at the transmembrane-cytoplasm interface of gD critically contributes to viral spread and cell-cell fusion. Our studies began with the discovery that packaging of gD into virions is almost completely blocked in the absence of tegument protein UL16. We subsequently identified a novel, direct, and regulated interaction between UL16 and gD, but this was not important for syncytia formation. However, a mutational analysis of the membrane-proximal basic residues of gD revealed that they are needed for the gBsyn phenotype, salubrinal-induced fusion of HSV-infected cells, and cell-to-cell spread. Finally, we found that these same gD tail basic residues are not required for cell fusion induced by a gKsyn variant.

Keywords: Cell fusion, syncytia; Cell-to-cell spread; Glycoprotein D; Herpes simplex virus; Packaging; Protein interactions; Tegument; UL16.

Fig 1.. UL16 is critical for gD packaging.

Vero cells were infected with WT, UL16-null mutant, or repaired (ΔUL16.R or ΔUL16.Rev35) viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion, and the collected cells were lysed with SDS-PAGE loading buffer. Samples were analyzed by western blotting with antibodies specific for the indicated viral proteins.

Fig 2.. UL16 interacts with the cytoplasmic tail of gD.

(A) Vero cells were infected with WT, ΔUL11, or ΔUL16 viruses. At 24 hr post infection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After 5 hr of incubation at room temperature or at 37°C, the beads were washed, boiled in sample buffer, and the proteins were analyzed by western blotting with an antibody specific for UL16. (B) Vero cells were transfected with expression plasmids for UL16-GFP or GFP-only. At 18–24 hr post transfection, cell lysates were prepared and incubated with the purified fusion proteins GST-UL11(1–50), GST-gE.CT, GST-gD.CT, or GST-only, as indicated on the top panel, and a Ponceau stain for total protein was performed. After incubation, the beads were processed as in (A) and an antibody specific for GFP was used. (C) Increasing amounts of purified His₆-UL16 produced in E. coli was incubated with GST-gD.CT, GST-gE.CT, or GST-only bound to glutathionesepharose beads in 0.5% NP-40 buffer at 37°C. After incubation, the beads were processed as in (A), a Ponceau stain for total protein was performed, and an antibody specific for the His tag was used to probe the western blot.

Fig 3.. A regulated interaction between UL16 and the gD cytoplasmic tail.

(A) Vero cells were singly transfected (top row) with plasmids that express full-length UL16-GFP, UL16 NTD-GFP, or UL16 CTD-GFP. Additionally, each of these constructs were co-expressed with gD-HA (bottom three rows). Cells were fixed, permeabilized, and stained with a monoclonal antibody against the HA tag at one day post-transfection. (B) Purified His₆-UL16(1–155) protein was incubated with the indicated GST-fusion proteins either in the presence or absence of NEM bound to glutathione-sepharose beads in 0.5% NP-40 buffer at 37°C. The beads were then washed, boiled in sample buffer, and the proteins were analyzed by western blotting.

Fig 4.. Mutants of the gD tail used in this study.

The wild-type sequence of the gD tail is underlined with basic residues indicated with pink text and the transmembrane residues indicated by purple text. The mutants are grouped into four categories based on the type of changes made. Mutants in which stop codons have been inserted are marked with asterisks. Mutants with deleted residues are marked with an underscore where the residues used to be. Mutants where residues have been altered are indicated with blue text.

Fig 5.. The UL16-gD interaction is not critical for the gBsyn phenotype.

(A) Cells were infected with gBsyn, M6/syn, ΔCT/syn, or ΔCT.R/syn at a low MOI. Images were taken with an inverted light microscope at 2–3 days post infection. (B) Vero cells were infected with the indicated viruses at an MOI of 1. At 24 hr post infection, extracellular virions in the media were pelleted through a 30% sucrose cushion and the cells were collected. Samples were analyzed by western blotting. (C) Duplicate cultures of Vero cells were infected with the indicated viruses at an MOI of 0.1. The media containing extracellular virions was collected, and the infected cells were harvested and processed separately at 6-hour time points. Infectious virus was titered by plaque assay. (D) Purified His₆-UL16 was incubated in 0.5% NP-40 buffer at 37°C for 5 hr with glutathione-sepharose beads bearing GST fusion proteins with the entire tail of gD or truncated sections of the tail. GST-UL11 and GST alone served as respective positive and negative controls. After incubation, the beads were washed, boiled in sample buffer, and the presence of bound His₆-UL16 was analyzed by western blotting.

Fig 6.. Membrane-proximal gD basic residues are important for the gBsyn phenotype and plaque size.

(A) Vero cells were infected at a low MOI with the indicated gD mutants. After infection, the cells were rinsed, overlaid with methylcellulose, and incubated at 37°C. At 4 days post infection, the cells were fixed and stained with crystal violet. The plates were imaged, and the areas of 20 representative plaques for each virus were measured using ImageJ. Plaque size is plotted as normalized to gBsyn. (B) Vero cells were infected with the indicated viruses at a low MOI. Images were taken at 2–3 days post infection with an inverted light microscope. (C) Vero cells were infected with the indicated gD mutants and analyzed as described in (A). (D) Vero cells were infected with gD.Y362A/syn or gD.Y362F/syn. Images of individual syncytia were taken 24 hr post infection.

Fig 7.. gD basic residues involvement in viral replication and cell-to-cell spread

(A) Vero cells were infected with gBsyn, M7/syn, or DED/syn viruses at an MOI of 5 for 1 hr. After infection, media and cell lysate were harvested separately in 6-hour time increments, and samples were analyzed via plaque assay. (B) A single-step growth curve with the same experimental setup as described in (A) was performed for the WT, M7, and DED viruses containing no Syn mutations. (C) Vero cells were infected with WT, M7, or DED a low MOI (0.001) for 1 hour. After infection, cells were incubated in DMEM containing 5 mg/mL pooled human IgG. At 48 hr post infection, cells were fixed, stained for VP5, and plaques were imaged with a fluorescent microscope. Scale bar is 50 μm. (D) Quantitation of the experiment performed in (C) using Olympus cellSens software. Two independent experiments were performed, 10–15 plaques per sample were measured per experiment, and a Student T-test was performed (*** P<0.001).

Fig 8.. Differential requirements for basic residues for gKsyn and salubrinal-induced fusion.

(A) Vero cells were infected with gKsyn, M7/gKsyn, or DED/gKsyn at a low MOI (0.001) for 1 hr and incubated in infection media. At 48 hr post infection, individual syncytia were imaged. (B) Quantitative data from the experiment in (A). The areas for 10–12 individual syncytia per virus were measured and plotted around the normalized mean for each virus and a Student T was performed (**** P<0.0001) (C) Vero cells were infected with WT, M7, or DED at an MOI of 3 and were incubated in 50 μM salubrinal. At 18 hr post infection, samples were harvested an analyzed via flow cytometry. This experiment was done 3 times and the % free nuclei represents a measure of total cell fusion. Statistical analysis was done by a Student T test (*** P<0.001).