Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus

Abstract

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors.

Importance: Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

FIG 1

Genomic structures of recombinant HSVs. The schematics show representations of the genomes of the recombinant viruses used in this study. U_L, unique long segment; U_S, unique short segment; CMVp, human cytomegalovirus immediate early (IE) promoter; scEGFR, anti-EGFR scFv-fused gD; scEpCAM, anti-EpCAM scFv-fused gD; NT, D285N/A549T mutations in gB that increase the rate of virus entry (38); Bh, R858H mutation in gB; Kt, A40T mutation in gK. Closed boxes show terminal and internal inverted repeats.

FIG 2

Plaque morphologies of syncytial, EGFR-retargeted HSVs. (A) Vero cells infected for 24 h with the viruses indicated above the panels were stained with wheat germ agglutinin-Alexa Fluor 594 (red) and Hoechst 33342 (blue) for detection of cell membranes and nuclei, respectively. Dotted lines indicate the margins of plaques (white, nonsyncytial plaques; yellow, syncytial plaques). (B) Vero cells infected for 24 h with the viruses indicated above the panels were incubated with the anti-gD MAb DL6 and stained with a Cy3-conjugated secondary antibody. Red, Cy3 signals; green, EGFP signals. Rescued, a rescued virus derived by replacement of the gB:R858H and gK:A40T mutations of KGNE-BhKt BAC with the respective wild-type residues.

FIG 3

Lateral spread of syncytial, EGFR-retargeted HSVs on human carcinoma cell lines. (A) Surface expression of EGFR as analyzed by flow cytometry. Closed histograms represent staining using an isotype-matched negative-control IgG as the primary antibody. Open histograms represent staining using the anti-EGFR MAb 528 as the primary antibody. (B, C, and E) The cell lines listed above the panels were infected for 2 h with the viruses indicated to the left and then overlaid with methylcellulose-containing medium. EGFP signals were recorded at 3 days postinfection. Photographs of representative plaques are shown. Bars, 500 μm (B), 500 μm (C), and 1 mm (E). (D and F) Mean areas of plaques (n = 15) in panels C and E, normalized to the respective means of KGNE plaque areas. Error bars represent standard deviations. White bars, KGNE; horizontally striped bars, KGNE-Bh; vertically striped bars, KGNE-Kt; black bars, KGNE-BhKt. *, P < 0.05 by the Steel-Dwass test (D) or the Welch t test (F); n.s., not significant.

FIG 4

Cell killing activity of syncytial, EGFR-retargeted HSVs. (A and B) The cell lines indicated above the panels were infected at MOIs ranging from 0.003 to 0.3 for 96 h, and percent cell viability relative to that of uninfected cells was measured by MTT assay. Means for 6 replicates are shown, and error bars represent standard deviations. White bars, KGNE; horizontally striped bars, KGNE-Bh; vertically striped bars, KGNE-Kt; black bars, KGNE-BhKt.

FIG 5

Specificity of entry by syncytial, EGFR-retargeted HSVs. The cell lines indicated to the left of the photographs were infected for 12 h (A) or 8 h (B) with the viruses indicated above the panels at an MOI of 3, and EGFP fluorescence was visualized. Bars, 125 μm.

FIG 6

Specificity of lateral spread by KGNE-BhKt. (A) CT26-EGFR cells were infected with the viruses indicated above the panels (MOI of 10). Extracellular viruses were inactivated by an acidic wash, and equal numbers of infected (donor) cells were added to monolayers of the uninfected (acceptor) cells indicated to the left. The mixed cultures were overlaid with methylcellulose-containing medium, and EGFP signals were recorded at 2 days postinfection. Bars, 500 μm. Arrows show single green cells or small foci. (B) Mean areas of plaques or foci (n = 15) in the wells examined for panel A. Error bars represent standard deviations. White bars, CT26 (mock-transduced) cells; black bars, CT26-EGFR cells. *, P < 0.05 by the Welch t test; n.s., not significant.

FIG 7

Lateral spread of syncytial, EpCAM-retargeted HSV on human cancer cells. (A) The cell lines listed above the panels were infected for 2 h with the viruses indicated to the left and then overlaid with methylcellulose-containing medium. EGFP signals were recorded at 3 days postinfection. Photographs of representative plaques are shown. Bars, 500 μm. (B) Mean areas of the plaques (n = 15) from panel A normalized to the respective means of KGNE plaque areas. Error bars represent standard deviations. White bars, KGNEp; black bars, KGNEp-BhKt. *, P < 0.05 by the Welch t test. (C) Inhibition of KGNEp-BhKt entry by pretreatment of AsPC-1 cells with 100 μg/ml anti-EpCAM MAb MY24 or an isotype-matched negative-control antibody, as indicated below the columns. Pretreated cells were incubated with KGNEp-BhKt at an MOI of 0.01 for 2 h, extracellular viruses were inactivated, and cells expressing EGFP were counted at 12 h postinfection. Means for 3 replicates are shown, and error bars represent standard deviations. Ab (−), no antibody; NC, isotype-matched negative-control antibody (MG1-45). *, P < 0.05 by the Welch t test; n.s., not significant.