CONICET | Buscador de Institutos y Recursos Humanos

The single-shelled particle binding domain(s) on NS28 was examined by testing the ability of different truncated forms of NS28 to bind single-shelled particles (ssp). Deletion of amino acids (aa) 161 to 175 of NS28 abolished ssp binding activity. Deletion of the last three aa (173-175) of NS28 diminished, but did not abolish, the ligand binding activity in our assay conditions. An internal deletion of NS28 (aa 110 to 155) also significantly diminished ssp binding activity in standard binding assays. As an alternative approach to study the ssp binding domain on NS28, we mapped the epitope of binding of monoclonal antibody BA/55, which was found to block ssp binding to NS28. Immunoprecipitation experiments done with truncated mutants of NS28 located the epitope of BA/55 to aa 149-160 of NS28, immediately adjacent to or partially overlapping the putative ssp binding domain. Experiments using synthetic peptides mimicking the carboxy end of NS28, found these peptides were not able to compete for ssp binding. Together, these results suggest that the ssp binding site in NS28 (aa 161-172) is highly dependent on the conformational integrity of the cytoplasmic C-terminus of NS28. NS28 truncation mutants also were assayed for interactions with rotavirus VP4 expressed in baculovirus. Amino acids 112 to 148 of NS28 were found to be critical for NS28-VP4 binding. Unexpectedly, aa 149 to 175 not only were nonessential for interaction with VP4, but mutants lacking those aa showed improved binding activity. We hypothesize that the VP4 binding domain may be buried in the NS28 cytoplasmic domain, and that the binding of ssp and VP4 may be an interdependent process that functions in conjunction with triggering of the budding of the whole complex into the endoplasmic reticulum. These results demonstrate the pleiotropic properties of NS28 in the unique rotavirus morphogenetic process.

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