Inhibition of Herpes Simplex Virus Type 1 Penetration by Cytochalasins B and D

SUMMARY Internalization of herpes simplex virus type 1 (HSV) KOS strain by HEp-2 cells was reversibly inhibited by pretreatment of cells with cytochalasins B and D. Internalization of virus following preincubation at 4 °C and temperature shift to 37 °C was normally preceded by a 5 to 8 min lag period and was complete within 20 to 30 min. A similar lag period followed HSV addition at 37 °C. Cytochalasin D was fivefold more active on HSV entry than cytochalasin B, with 50~ inhibition at 2 ~tM and 10 ~tM respectively. Inhibition was completely reversible, such that all cell-bound infectious virus was recovered upon removal of cytochalasin. In conjunction with previous reports, the activity of cytochalasin on HSV entry suggests that a change in cytoskeletal structure following virus attachment triggers a microfilament activity important for internalization of HSV by HEp-2 cells. The involvement of the cytoskeleton in herpes simplex virus type 1 (HSV-1) attachment and penetration was suggested by fluorescence photobleaching recovery measurements (Rosenthal et al., 1984). An initial limitation of cell surface protein mobility, characteristic of anchorage modulation of cell surface protein mobility was observed. This is mediated by multivalent ligand crosslinking of cell surface proteins interacting with the cytoskeleton (Gall & Edelman, 1981). The anchorage modulation was released concurrent with initiation of virus penetration with a return to control levels following completion of virus internalization. These results suggest that cytoskeletal function is involved in HSV penetration into the target cell. The microfilament component of the cytoskeleton is important for contractile processes involved in motility, capping, phagocytosis and cell surface protein movement (Tanenbaum, 1978) but not in uptake by clathrin-coated vesicles. The role of the microfilaments in HSV entry into the target cell was investigated using the selective inhibitors cytochalasin B (CB) and D (CD). The cytochalasins bind to actin, preventing its proper polymerization into microfilaments (MacLean-Fletcher & Pollard, 1980). CB has been shown to partially inhibit the penetration of other enveloped viruses such as vesicular stomatitis and Sindbis viruses (Coombs et al., 1981) but not influenza (Patterson et al., 1979) or Sendal viruses. However, Sendal virus-induced cellcell fusion is inhibited by CB (Asano & Okada, 1977; Miyake et al., 1978). Recently, Lycke et al. (1984) have shown that CB and CD inhibit the axoplasmic transport of HSV in neurons, indicating the role of microfilaments in transporting virion capsids within the cytoplasm. In this study we show that cytochalasins also inhibit the internalization of HSV into the cell. Stocks of HSV-1 (KOS strain, originally provided by P. Schaffer, Dana Farber Cancer Institute, Boston, Mass., U.S.A.) were prepared by low multiplicity infection of HEp-2 cells (0.01 p.f.u./cell) and infectivity was evaluated by plaque assay on Vero cells using a 1.5~ methyl cellulose overlay (Dreesman & Benyesh-Melnick, 1967). Cells were maintained in Eagle's MEM (Auto-Pow; Flow Laboratories) supplemented with foetal calf serum (10~) and antibiotics (penicillin G, 25 U/ml; L-glutamine, 29-2 ~tg/ml; gentamicin 50 ~tg/ml). Maintenance