Hydrophobic alpha-helices 1 and 2 of herpes simplex virus gH interact with lipids, and their mimetic peptides enhance virus infection and fusion

Abstract

Entry of herpes simplex virus into cells occurs by fusion and requires four glycoproteins. gD serves as the receptor binding glycoprotein. Of the remaining glycoproteins, gH carries structural and functional elements typical of class 1 fusion glycoproteins, in particular alpha-helix 1 (alpha-H1), with properties of a candidate fusion peptide, and two heptad repeats. Here, we characterized alpha-H2 and compared it to alpha-H1. alpha-H2 (amino acids 513 to 531) is of lower hydrophobicity than alpha-H1. Its deletion or mutation decreased virus infection and cell fusion. Its replacement with heterologous fusion peptides did not rescue infection and cell fusion beyond the levels exhibited by the alpha-H2-deleted gH. This contrasts with alpha-H1, which cannot be deleted and can be functionally replaced with heterologous fusion peptides (T. Gianni et al., J. Virol. 79:2931-2940, 2005). Synthetic peptides mimicking alpha-H1 and alpha-H2 induced fusion of nude lipid vesicles. Importantly, they increased infection of herpes simplex virus, pseudorabies virus, bovine herpesvirus 1, and vesicular stomatitis virus. The alpha-H1 mimetic peptide was more effective than the alpha-H2 peptide. Consistent with the findings that gH carries membrane-interacting segments, a soluble form of gH, but not of gD or gB, partitioned with lipid vesicles. Current findings highlight that alpha-H2 is an important albeit nonessential region for virus entry and fusion. alpha-H1 and alpha-H2 share the ability to target the membrane lipids; they contribute to virus entry and fusion, possibly by destabilizing the membranes. However, alpha-H2 differs from alpha-H1 in that it is of lower hydrophobicity and cannot be replaced with heterologous fusion peptides.

FIG. 1.

(A) Linear map of HSV gH showing the amino acid positions of α-H1 (i.e., candidate fusion peptide; FP), α-H2, HR-1 and HR-2, signal sequence (SS), and the transmembrane (TM) domain. C2 and C4, cysteines 2 and 4. (B) Amino acid sequence of α-H2 and adjacent sequences in wt-gH, in gH_WGW, and in α-H2-deleted gH (gH_Δ513-531). Heterologous sequences show the amino acid sequences of the fusion peptides of HIV gp41, VSV G, and the putative fusion peptide of VZV gH or the amino acid sequences obtained by cloning the DNA fragments in the antisense orientation. The hererologous sequences were cloned in gH_Δ513-531, in the sense or antisense orientation, thus generating the plasmids gH-αH2_VSVG, gH-αH2_AS-G, gH-αH2_gp41, gH-αH2_AS-gp41, gH-αH2_VZV, and gH-αH2_AS-VZV.

FIG. 2.

Cell surface expression of wt-gH, gH_Δ513-531, gH_WGW, gH-αH2_VSVG, gH-αH2_AS-G, gH-αH2_gp41, gH-αH2_AS-gp41, gH-αH2_VZV, and gH-αH2_AS-VZV. (A to I) Paraformaldehyde-fixed COS cells transfected with wild-type or mutant gH plasmids and a gL-encoding plasmid and reacted with MAb 53S. The positive reactivity denotes gH-gL heterodimer formation and trafficking to the plasma membrane. (J) CELISA quantification of cell surface expression of gH mutants in COS cells cotransfected with a gL plasmid. Each assay was performed in triplicate. The bars represent the mean percentage relative to wt-gH ± standard deviation.

FIG. 3.

(A) Cell-cell fusion and (B) infectivity complementation of mutant forms of gH. (A) Cell-cell fusion of COS cells transfected with the gH mutant plasmids plus plasmids encoding gL, gB, and gD. Fusion was quantified by means of a T7 promoter-driven reporter luciferase gene, as luciferase units (L.U.), according to reference , and is expressed as a percentage of fusion induced by wt-gH. Each assay was performed in triplicate. The bars represent the mean ± standard deviation. (B) COS cells were transfected with mutant or wt-gH plasmids and superinfected with the gH deletion mutant virus SCgHZ 4 h later. Virus was harvested 24 h after transfection and titrated in F6 cells. The bars represent the mean of triplicates ± standard deviation.

FIG. 4.

(A) Sequence and coordinates of synthetic peptides to HSV-1 gH α-H1, α-H2, HR-1, HR-2, and gD. (B and C) Fusion of large unilamellar vesicles induced by mimetic peptides to α-H1 and α-H2, and, as a reference, by herpes simplex virions. Large unilamellar vesicles were mixed with pyrene-loaded vesicles in the presence of herpes simplex virions or the indicated peptides (30 μM) in a 3:1 lipid/peptide ratio. Extent of fusion was measured as decrease of the excimer fluorescence emission (I_E) and increase in monomer fluorescence emission (I_M) and is expressed as percent I_E/I_M ratio. Fusion induced by synthetic peptides was expressed as a percentage relative to the fusion induced by herpes simplex virions. (B) Induction of lipid vesicle fusion by single peptides (30 μM). (C) Induction of lipid vesicle fusion by a mixture of peptides mimicking α-H1 and α-H2 (each at 15 μM). All assays were performed in triplicates. The bars represent the mean ± standard deviation.

FIG. 5.

Enhancement of HSV-1, PrV, BoHV-1, and VSV infection by synthetic peptides or a mixture thereof. (A to D) Effect of the peptides on infection with HSV-1(R8102) (A), PrV (B), or BoHV-1 (C) recombinants carrying the LacZ reporter gene and VSV (D). J-nectin1, Madin-Darby bovine kidney, or Vero cells were exposed to the indicated peptides from time zero (start of virus adsorption) until harvest. Infection was quantified as β-Gal activity (R8102, PrV, and BoHV) or as the number of plaques (VSV). The abscissa shows the μM peptide concentration. In panel D, the scale is logarithmic. (E to H) Effect of simultaneous exposure to mixtures of mimetic peptides, each one present at a 200 μM concentration, on HSV-1 (E), PrV (F), BoHV-1 (G), and VSV (H) infection. In panel H, the scale is logarithmic. The bars represent the mean of triplicates ± standard deviation.

FIG. 6.

Enhancement of cell-cell fusion by synthetic peptides or mixtures thereof. Cell-cell fusion in BHK cells transfected with plasmids encoding wt-gH, gL, gB, gD, and LacZ and exposed to the indicated peptides. (A and E) Fusion was quantified by means of β-Gal activity. (A to D) Peptides at 450 μM each. (B to D) Representative examples of polykaryocytes formed in the absence of added peptides (B) or in the presence of the indicated mimetic peptides (C and D). (E) Additive effect on cell-cell fusion of mixtures of mimetic peptides, each one present at a 200 μM concentration. The bars represent the mean of triplicates ± standard deviation.

FIG. 7.

Sedimentation analysis of soluble forms of gH_792t.gL (A and B), gB_730Hist (C and D), gD_285t (E and F), and IgG (G and H), preincubated (A, C, E, and G) or not (B, D, F, and H) with liposomes, through discontinuous preformed sucrose gradients. Gradient fractions 1 to 4 contained 5, 15, 25, and 50% sucrose (wt/vol), respectively. After centrifugation, the fractions were concentrated by Amicon Y10 filters, and the proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enhanced chemiluminescence. The liposome content in each fraction was determined by means of phosphorous determination. A typical example is represented in panel I. Vertical bars represent standard deviation.