CLEAVAGE OF RHESUS ROTAVIRUS-VP4 AFTER ARGININE-247 IS ESSENTIAL FOR ROTAVIRUS-LIKE PARTICLE-INDUCED FUSION FROM WITHOUT

We recently described our finding that recombinant baculovirus-produce d virus-like particles (VLPs) can induce cell-cell fusion similar to t hat induced by intact rotavirus in our assay for viral entry into tiss ue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:3555 -4563, 1997). The conditions required for syncytium formation are simi lar to those for viral penetration of the plasma membrane during the c ourse of viral infection. This VLP-mediated fusion activity was depend ent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred o nly with cells that are permissive for rotavirus infection. Here we be gin to dissect the role of VP4 in rotavirus entry by examining the imp ortance of the precise trypsin cleavage of VP4 and the activation of V P4 function related to viral entry. We present evidence that the elimi nation of the three trypsin-susceptible arginine residues of VP4 by sp ecific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium forma tion, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recom binant VLPs in our syncytium assay will aid in understanding the confo rmational changes that occur in VP4 involved in rotavirus penetration into host cells.