CHARACTERIZATION OF CD4-GP120 ACTIVATION INTERMEDIATES DURING HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 SYNCYTIUM FORMATION

The mechanism by which cells expressing HIV envelope glycoproteins pro gress from binding CD4(+) cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical an d biochemical tools (temperature shifts, soluble CD4, protease inhibit ors, and a battery of anti-CD4 monoclonal antibodies) to isolate discr ete steps during syncytia formation. Previously (Fu et al., J Virol 19 93;67:3818), we found that preincubation of cells stably expressing HI V-1 gp160 (TF228.1.16) with CD4(+) SupT1 cells at 16 degrees C, a temp erature that is nonpermissive for syncytia formation, resulted in an i ncreased rate of syncytia formation when the cocultures were shifted t o the syncytia-permissive temperature of 37 degrees C. We have since f ound that syncytia formation is further enhanced by shifting the cocul tures from 16 to 4 degrees C prior to incubation at 37 degrees C. Toge ther, these data suggest that two discrete states, which we term the f irst and second activation intermediates (FAI and SAI), are involved i n syncytia formation. We have found that acquisition of the FAI (by pr eincubation at 16 degrees C) is sensitive to some serine protease inhi bitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoc lonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions o f domain 1 on CD4. Expression of the FAI (formation of syncytia by shi fting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb th at bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4 , shedding of gp120, and MAb directed toward CDR-1/2. In contrast, exp ression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose tha t syncytia formation, mediated by HIV-1 envelope glycoproteins, procee ds by a multistep cascade.