Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

Abstract

Herpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry.

Importance: The alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.

FIG 1

Side-by-side comparison of EBV gH/gL and PrV gH. The EBV gH/gL (PDB code 3PHF) (A) and PrV gH (PDB code 2XQY) (B) structures (4, 6) are shown as ribbon diagrams. The four domains (D) are indicated in different colors. Previously identified structural aspects, such as the bifunctional KGD motif (red) (24), DBs (orange spheres), and syntaxin-like bundle (SLB) formed by three helices, are shown. The structural views of D-III of EBV gH/gL (C) and PrV gH (D), including the conserved DB (orange spheres) and conserved surrounding amino acids (blue sticks), are shown as ribbon diagrams. (E) Amino acid alignment of the conserved DB (orange stars) of a partial D-III (corresponding to amino acids 404 to 421 and 448 to 485 of EBV gH) is shown with the correlated secondary structures, such as alpha-helices and β-sheets (red) for PrV (top) and EBV (bottom) gH.

FIG 2

The DB and contacting amino acids approve the cell surface expression of PrV gH and EBV gH/gL. The arrangements of the conserved amino acids surrounding the conserved DB's connecting three central helices of D-III of PrV (PDB code 2XQY) (A) and EBV gH (PDB code 3PHF) (B) are shown as ribbon diagrams, and the surface expression-required amino acids are highlighted in red. The amino acids (sticks) are colored by element (C and H in gray, N in blue, O in red, and S in orange), and the hydrogen bonds are indicated as black dashes. Surface expression of wt and mutant gH of PrV (C) and EBV (D) is also shown. CELISA was performed with either the monoclonal conformation-specific antibodies against EBV gH (CL40 and CL59) and gH/gL (E1D1) or polyclonal antisera against PrV and EBV gH (HL800). Mean values and standard deviations from three independent experiments are shown.

FIG 3

The defect in cell surface expression of the DB and specific amino acid mutants strongly correlates with a loss in fusion function of PrV and EBV gH. Western blot analysis of wt and mutant gH was performed by using polyclonal antibodies against PrV gH (A) and EBV gH/gL (B) as well as anti-GAPDH as a loading control. The fusion activities of wt and mutant gH of PrV (C) and EBV (D) are also shown. For the virus-free luciferase-based fusion assay, CHO-KI cells were transfected with the fusion-required glycoproteins of either PrV (gD, gB, and gH/gL) or EBV (gp42, gB, and gH/gL). Averages with standard deviations from three independent experiments are shown.

FIG 4

The conserved tyrosine and a contacting amino acid are important for function of gH during fusion. The conserved tyrosine and contacting amino acids of PrV (PDB code 2XQY) (A) and EBV gH (PDB code 3PHF) (B) are shown as sticks and labeled by element as well as the involved secondary structures are displayed in ribbon diagrams. The surface expression-required amino acids are highlighted in red. Shown are cell surface expression and fusion activities of wt and mutant gH of PrV (C and G) and EBV (D and H). Mean values and standard deviations from three independent experiments are shown. Western blot analysis of wt and mutant gH was performed by using polyclonal antibodies against PrV gH (E) and EBV gH/gL (F) as well as anti-GAPDH.

FIG 5

Immunoprecipitation (IP) of EBV gH using monoclonal conformation-specific antibodies. Immunoprecipitation analysis for wt and selected mutant gH proteins was performed by using monoclonal conformation-specific antibodies against EBV gH (CL40 and CL59) and gH/gL (E1D1). The input is shown. Western blot analyses were done with polyclonal EBV gH/gL-specific antiserum. IP experiments were repeated at least twice.

FIG 6

The conserved DB is important for the role of gH during PrV entry. (A) Shown are images of the plaques. Scale bar represents 400 μm. (B) For analysis of the plaque size, 30 plaques were measured in three experiments, with wild-type PrV-Ka set as 100%. (C) For penetration kinetics, RK13 cells were infected on ice for 1 h and incubated for the indicated times at 37°C. Then extracellular virus was inactivated with citric acid. Averages with standard deviations from three independent experiments are shown.