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. 2005 Jun;79(11):6655-63.
doi: 10.1128/JVI.79.11.6655-6663.2005.

Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus type 1

Richard S B Milne  1 , Anthony V Nicola, J Charles Whitbeck, Roselyn J Eisenberg, Gary H Cohen
Affiliations

Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus type 1

Richard S B Milne et al. J Virol. 2005 Jun.

Abstract

Two herpes simplex virus type 1 (HSV-1) entry pathways have been described: direct fusion between the virion envelope and the plasma membrane, as seen on Vero cells, and low-pH-dependent endocytosis, as seen on CHO nectin-1 and HeLa cells. In this paper, we studied HSV entry into C10 murine melanoma cells and identified a third entry pathway for this virus. During entry into C10 cells, virion envelope glycoproteins rapidly became protected from the membrane-impermeable chemical cross-linker BS3 and from proteinase K. Protection was gD receptor dependent, and the time taken to detect protected protein was proportional to the rate of virus entry. Ultrastructural examination revealed that virions attached to the surface of C10 cells were localized to membrane invaginations, whereas those on the surface of receptor-negative B78 cells were peripherally attached. Virus entry into C10 cells was energy dependent, and intracellular enveloped virions were seen within membrane-bound vesicles consistent with endocytic entry. Entry was not inhibited by bafilomycin A1 or ammonium chloride, showing that passage of the virion through a low-pH environment was not required for infection. Resistance to similar reagents should therefore not be taken as proof of HSV entry by a nonendosomal pathway. These data define a novel gD receptor-dependent acid-independent endocytic entry pathway for HSV.

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Figures

FIG. 1.
FIG. 1.
gD receptor-dependent protection of glycoprotein B from BS3 cross-linking during virus entry. A. Virus was attached to C10 cells at 4°C. Entry was then initiated by incubation at 37°C for the indicated times and then stopped by placing the cells on ice. Cells were then cross-linked with BS3 or mock treated and then lysed. gB was detected by immunoprecipitation with MAb BD60 and then Western blotting with polyclonal antibody R69. B. The cross-linking assay was carried out on C10 cells or receptor-negative B78 cells. After virus attachment, samples were held at 4°C (lanes 1, 2, 5, and 6) or placed at 37°C for 10 min (lanes 3, 4, 7, and 8) and then chilled on ice. Samples were then cross-linked with BS3 (lanes 1, 3, 5, and 7) or mock treated (lanes 2, 4, 6, and 8). C. The assay was carried out using virus that was pretreated with the indicated gD-specific MAb. MAb-treated virus was also titrated on Vero cells to measure the degree of neutralization. Titers (log10 PFU/ml) are shown below the panel. Open arrows indicate high-molecular-weight gB-containing complexes formed by cross-linking. Solid arrows point to unit-length gB. nv, no virus.
FIG. 2.
FIG. 2.
Protection of gB from cross-linking occurs with kinetics comparable to the rate of virus entry. A. Rate of virus entry into C10 cells at 37°C, 30°C, and 22°C. A standard acid inactivation entry assay was carried out (see Materials and Methods). B. After attachment of virus to C10 cells at 4°C, entry was initiated by incubation at the indicated temperatures for the indicated times. Cross-linking was then carried out as before. For clarity, only the unit-length gB band is shown. nx, no cross-linking; nv, no virus.
FIG. 3.
FIG. 3.
Rapid, gD receptor-dependent protection of viral glycoproteins from proteinase K digestion. A. Virus was attached to B78 and C10 cells for 45 min at 4°C. Entry was then initiated by incubation at 37°C for the indicated times. Cells were treated with proteinase K, and gB was detected by immunoprecipitation and Western blotting. B. C10 cells were infected with HSV gD-GFP virus as for panel A. Samples were proteinase K treated and then lysed. gD-GFP was immunoprecipitated with MAb DL6 and detected by Western blotting with polyclonal antibody R7. nv, no virus. C. Virus was attached to B78 (lanes 1, 2, 5, and 6) or C10 (lanes 3, 4, 7, and 8) cells for 45 min at 4°C. Cells were then incubated at 37°C for 10 min (lanes 2, 4, 6, and 8) or held at 4°C (lanes 1, 3, 5, and 7), treated with proteinase K or mock treated for 1 h at 4°C, and lysed in the presence of PMSF, and glycoproteins were detected by immunoprecipitation and Western blotting.
FIG. 4.
FIG. 4.
Protection of viral gB from proteinase K correlates with endocytic entry. After virus attachment for 45 min at 4°C, cells were either held at 4°C (lanes 1 and 3) or incubated at 37°C for 10 min (lanes 2 and 4) and then chilled on ice. Cells were then digested (lanes 3 and 4) or mock treated (lanes 1 and 2) with proteinase K and lysed in the presence of PMSF. gB was immunoprecipitated from the lysates and detected by Western blotting.
FIG. 5.
FIG. 5.
Energy dependence of HSV entry into C10 cells. C10 cells were treated with energy depletion medium, and then the effect on virus entry was determined using a GFP reporter virus. The number of infected, GFP-expressing cells in the mock-treated sample was set to 100%.
FIG. 6.
FIG. 6.
EM analysis of HSV localization on C10 and B78 cells. The localization of HSV on C10 cells (A to F) and B78 cells (G and H) was examined by EM after virus attachment at 4°C (A to C, G, and H) or after initiation of entry by incubation at 37°C for 4 (D and F) or 10 min (E). Original magnification: ×25,000 (A); ×100,000 (B, C, and F); ×50,000 (D, E, G, and H). Bars, 100 nm.
FIG. 7.
FIG. 7.
Entry of HSV into C10 cells is not affected by bafilomycin A1 or ammonium chloride. C10 cells were treated with the indicated concentrations of bafilomycin A1 (A) or ammonium chloride (B) for 1 h. Virus was added in the presence of fresh inhibitor. After 90 min the cells were refed with medium containing the inhibitor. After a further 4 h, the inhibitor was washed away and a semisolid overlay was added. Plaques were counted at 24 h (for VSV) or 48 h (for HSV) postinfection.
FIG. 8.
FIG. 8.
Model of entry pathways. The figure depicts four pathways by which HSV can enter cells. Pathways A, C, and E result in viral gene expression, and pathway B does not. However, all four pathways are rapid and specific. A. Vero cells: receptor-dependent fusion between virus envelope and plasma membrane. B. CHO K1 cells: gD receptor-independent endocytic internalization of virions leading to degradation of virions. C. CHO nectin-1 cells: involvement of gD receptor in virus internalization is unknown. Acidification is required for successful egress of virions from endosome. D. B78 cells: no rapid internalization. Virions remain on the cell surface and are peripherally attached, without any cell surface alterations at point of contact. (Note that, by EM, virions remain on the surface of B78 cells after 10 min of incubation at 37°C and that no protection of viral glycoproteins from cross-linker is seen on these cells for as long as 30 min at 37°C. However, the ultimate fate of these cell surface virions is unknown.) E. C10 cells: gD receptor-dependent internalization preceded by enwrapment of virions by plasma membrane invaginations. Release of virions from endocytic vesicle does not require endosome acidification.
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