THE ROTAVIRUS NONSTRUCTURAL GLYCOPROTEIN NSP4 POSSESSES MEMBRANE DESTABILIZATION ACTIVITY

During a unique morphogenetic process, rotaviruses obtain a transient membrane envelope when newly synthesized subviral particles bud into t he endoplasmic reticulum (ER), Pis rotavirus particles mature, they lo se their transient membrane and a layer of the glycoprotein VP7 forms the virion outer capsid shell, The nonstructural glycoprotein NSP4 fun ctions as an intracellular receptor in the ER membrane (K. S. Au, W. K . Chan, J. W. Burns, and M. K. Estes, J. Virol. 63:4553-4562, 1989), a nd it has been hypothesized that NSP4 is involved in the removal of th e envelope during viral morphogenesis (M. K. Estes and J. Cohen, Micro biol. Rev. 53:410-449; 1989; B. L. Petrie, M. K. Estes, and D. Y. Grah am, J. Virol. 46:270-274, 1983), The purpose bf the present study was to determine if NSP4 has a direct membrane destabilization activity (M DA) by using liposome leakage assays and electron microscopic visualiz ation of liposome, microsome, and viral envelope disruption, The fluor escent marker (calcein) incorporated into liposomes was released when the liposomes were incubated with purified NSP4, A region correspondin g to amino acid residues 114 to 135 of NSP4 also released calcein from liposomes, NSP4(114-135) peptide-specific antibody completely blocked the MDA of the purified NSP4 protein, These results suggest that this region contains at least part of the functional domain of NSP4, Lipos omes composed of phosphatidylcholine and microsomes (to simulate ER me mbranes) were broken when observed by electron microscopy after incuba tion with NSP4 or the NSP4(114-135) peptide, In contrast, the envelope of Sendai virus, which is derived from cytoplasmic membranes, and ery throcytes were not disrupted by NSP4 and the NSP4(114-135) peptide, Th ese results provide direct evidence that NSP4 possesses MDA and sugges t that it can cause ER membrane damage, Therefore, NSP4 might play an important role in the removal of the transient envelope from budding p articles during viral morphogenesis, A model for the MDA of NSP4 in vi ral morphogenesis is proposed.