Characterization of fusion from without induced by herpes simplex virus

Abstract

The process of fusion from without (FFWO) induced by herpes simplex virus (HSV) was analyzed by using various inhibitors and compared to fusion from within (FFWI). The fate of certain elements of the cytoskeleton after FFWO was also investigated. Our experiments demonstrate FFWO as a very suitable system for study of early virus-cell interactions. Zn++ ions proved inhibitory for penetration whilst pretreatment of cells with Ca++ ions before infection enhanced FFWO activity. Dissociation of penetration from the fusion process itself was possible by use of Zn++ ions, low pH-treatment and antiserum on the one hand and N-ethylmaleimide and cytochalasin D on the other. Penetration itself needs only 6 min or less to proceed. FFWO is independent of inhibitors of glycosylation (tunicamycin) and intracellular vesicular traffic (monensin), protein-synthesis (cycloheximide) and energy-delivery (2.4 dinitrophenol and Na-azide). Analyzed strains of HSV-1 and -2 producing FFWI could be subgrouped into three categories: Strain ANG with high, strain HFEM and Lux with low and strains IES, Len, MP, US with no FFWO activity. The results of these experiments indicate that the property of FFWO is not purely a consequence of the number of PFU but depends on certain inherent properties of the virus particles. Addition of heparin as well as treatment of cells with heparitinase effectively prevented FFWO, indicating identical virus receptors for entrance of virus into cells and FFWO. During our studies several calf sera were found to inhibit FFWO-activity. Inhibition of FFWO by a glycoconjugate (ferritin coupled with oleic acid) indicates specific stereochemical hindrance of FFWO by this compound. Shortly after FFWO the actin filaments rearrange to form long fibres and surface fibronectin is being lost from the cell membrane.

Differentiation of herpes simplex virus-induced fusion from without and fusion from within by cyclosporin A and compound 48/80

“…FFWO is produced by the virus particles themselves 30 to 45 min after infection and cannot be inhibited by inhibitors of transcription or translation (Falke et al, 1985). Furthermore, it was possible to show that strains of HSV-I and -2 producing FFWI could be divided into two groups: strains producing FFWI and FFWO, and strains exhibiting FFWI activity only (Walev et al, 1991). Recently, McKenzie et al (1987) observed inhibition of FFWI produced by strain MP (HSV-I) after addition of cyclosporin A (CyA) without any influence on penetration of this HSV strain.…”

“…Recently we have shown that penetration can be dissociated from FFWO by some substances: antibody and low pH treatment as well as addition of Zn 2÷ ions inhibit penetration, whereas treatment with Nethylmaleimide (NEM) and cytochalasin B blocks the fusion process itself about 10 rain later (Walev et al, 1991). NEM inhibits FFWO, possibly by binding to a cellular fusion protein; cytochalasin B causes disruption of the microfilaments involved in FFWO (Walev et al, 1991). Because we were looking for cellular constituents or functions participating in FFWO or FFWI, we used CyA and Cpd 48/80 as potential inhibitors of fusion processes.…”

“…Moreover we have to differentiate between FFWI and FFWO generated by HSV (Falke et al, 1985). Recently we have shown that penetration can be dissociated from FFWO by some substances: antibody and low pH treatment as well as addition of Zn 2÷ ions inhibit penetration, whereas treatment with Nethylmaleimide (NEM) and cytochalasin B blocks the fusion process itself about 10 rain later (Walev et al, 1991). NEM inhibits FFWO, possibly by binding to a cellular fusion protein; cytochalasin B causes disruption of the microfilaments involved in FFWO (Walev et al, 1991).…”

“…We used the HSV-1 strains ANG (Falke et al, 1985), HFEM (Walev et al, 1991), IES (Falke & Kahl, 1971), MP (Heeg et al, 1986), Len (Heeg et al, 1986), 17 TK 1301 (Sanders et al, 1982), L4-1 and L3-2S (Falke et al, 1985). Strains KOS-804 (syn) and KOS-78R (Little & Schaffer, 1981) were provided by P. Schaffer, Boston, Mass., U.S.A., strains 17 syn and 17 TK 1301 were provided by Professor J. H. SubakSharpe (Glasgow, U.K.) and HSV-2 strains Lux syn and US-A12 by K. E. Schneweis (Bonn, Germany).…”

“…Vero cells (2 x 104 to 4 x 104 were seeded into 96-well Nunclon plates and kept overnight before the FFWO tests. Medium 199 was used with 5 ~ selected, inhibitor-free foetal calf serum (Walev et al, 1991). FFWO and FFWI assay. In order to produce FFWO, subconfluent Vero cell monolayers in 96-well plates were infected with 100 ~tl of virus at a concentration of 5 x 107 to 10 × 107 p.f.u, per ml.…”