J°urna~°l7.Genera~.Vir°l~gy~ ~ ! 9?6)~'~.77~..22.9 ~2.3~2..~.~.P.[!~te.d.!P..G~reat.Brita!P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SHORT COMMUNICATION Structural domains involved in human cytomegalovirus glycoprotein B-mediated cell-cell fusion S. Bold, 1 M. Ohlin, z W. Garten 1 and K. Radsak 1 1 Institut fur Virologie der Philipps-Universit~it, Robert-Koch-StraSe 17, D-35037 Marburg, Germany z Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, Sweden A novel fusion assay was established to determine fusion activity with cocultivated human foreskin fibroblasts of stable transfectants derived from human astrocytoma cells (U373) expressing auth- entic or mutagenized human cytomegalovirus glycoprotein B (HCHV gB; gpUL55). Compared to transfectants expressing authentic HCHV gB, those expressing gB forms with a deletion of hydrophobic domain 1 (hdl; aa714-747) or with deletions of specific segments in the cytoplasmic tail (aa 811-825 and 871-906) exhibited significantly reduced heterologous fusogenicity. HCHV gB- specific monoclonal antibodies (HAbs) as well as HAb against cellular annexin II prevented fusion of the transfectant expressing authentic gB. Compar- able surface exposure of HCHV gB or its derivatives was demonstrated in all transfectants by FACS analysis. Our observations are compatible with the notion that indigenous fusion activity of HCHV gB depends on the extracellular hdl domain and on the conformation of the cytoplasmic tail. All herpesvirus species analysed to date contain in their envelopes structural homologues of glycoprotein B (gB) of herpes simplex virus (HSV), which thus appears to be highly conserved throughout the herpesvirus family (Albrecht & Fleckenstein, 1990). In the case of HSV, it has been shown by the use of virus deletion mutants (Cai et al., 1987) that gB is essential for infectivity, i.e. virion maturation, host cell entry and cell-to-cell spread (Cai et al., 1988). The gB homologue of human cytomegalovirus (HCMV) is the abundant and immunodominant glycoprotein of the viral envelope, and has also been suggested to play a key role in productive virus infection due to its indigenous potential to mediate membrane fusion processes (Navarro et a]., 1993; Tugizov eta]., I994). The structural domains of the gB Author for correspondence: K. Radsak, Fax +49 6421 285482. molecule involved in its fusogenic potential, however, have so far not been clearly defined (Reschke eta[., I995). During our recent functional analysis of the prominent hydrophobic domains in the C-terminal portion of HCMV gB (hdI, aa 714-747; hd2, 751-771) we observed that hd2 was essential as well as sufficient for membrane anchoring (Reschke et al., 1995). Sequence alignments of hdl, on the other hand, with established fusogenic domains of envelope glycoproteins of entirely unrelated viruses revealed an intriguing similarity (Reschke et al., 1995) implying that hdI may directly participate in the fusion event. As an appropriate experimental tool for the analysis of fusogenic domains of viral glycoproteins, transient expression of the coding gene in cultured cells has been successfully used (Desnevre et aI., 1995). Compared to this approach the use of stably transformed cell lines offers the advantage of a more efficient and homogeneous expression of the protein in question. On the other hand, the establishment of stable transformants means that only cells with limited homologous fusion activity will be propagated because the procedure will select against strong fusogenicity, i.e. against syncytia which do not undergo cell division. Therefore, an alternative technique was used in the present study to reliably quantify the residual fusogenic potential of stable gB transfectants: cocultivation with permissive human foreskin fibroblasts (HFF) which exhibited reliable susceptibility for reduced fusion activities. Furthermore, gB-specific monoclonal antibodies (MAb) were used to relate fusion of gB transfectants with HFF to the product of the transfected gB gene. MAb 27-156 recognizes the C-terminal immunodominant gB epitope AD-1 (Spaete, 1990); the human MAb ITC88 binds to the N-terminal epitope AD-2 (Ohlin, 1993). For a routine fusion assay, the previously described stable astrocytoma cell derived transfectants expressing authentic or specifically mutagenized HCMV gB (Reis et al., 1993, Reschke et al., 1995) were grown in culture plates with twelve 2 cm wells; 48 h later, at 80% confluency (approximately 4 x I05 cells per well), fleshly trypsinized HFF (5 x 104 cells per well) were seeded onto the subconfluent monolayer. Incubation at 37 °C in Eagle's MEM supplemented with 10% FBS was continued until fixation with ethanol and staining with Giemsa !297 0001-3835 © 1996 SGM