Targeting of rotaviral glycoproteins to the endoplasmic reticulum

Abstract

This dissertation utilizes the simian rotavirus SA11 as a model system to study the mechanism of retention of glycoproteins in the endoplasmic reticulum (ER). Rotavirus encodes two glycoproteins, VP7 and NS28, which are localized to the ER. The oligomerization of both VP7 and NS28 in infected and transfected cells was examined. Both were found to form homoligomers of dimers, tetramers and higher order species. In addition, a heteroligomer, necessary for viral maturation, was detected consisting of VP7, NS28, and the viral fusion protein, VP4. Previous work in this laboratory demonstrated that the first NH{dollar}\sb2{dollar}-terminal 61 amino acids of VP7 are sufficient and necessary for ER retention. Current studies utilized site-specific mutagenesis to define the signal more closely. It was observed that Ile{dollar}\sb9{dollar}, Thr{dollar}\sb{lcub}10{rcub}{dollar}, and Gly{dollar}\sb{lcub}11{rcub}{dollar} within this sequence were necessary for retention. It was demonstrated by in vitro transcription/translation that signal cleavage of the second hydrophobic domain, (Hs), did not occur in a secreted VP7 mutant. This rules out a sustained involvement of H2 in retention. Though a minimal sequence was defined which allowed secretion when deleted, it was necessary to define the minimal context which would allow it to function in retention. Thus, varying lengths of the NH{dollar}\sb2{dollar}-terminus of VP7 were placed on the NH{dollar}\sb2{dollar}-terminus of mature mouse salivary {dollar}\alpha{dollar}-amylase, a secreted protein. It was shown that Ile{dollar}\sb9{dollar}, Thr{dollar}\sb{lcub}10{rcub}{dollar}, and Gly{dollar}\sb{lcub}11{rcub}{dollar} were sufficient to retain the enzymatically active chimeric protein in the ER only when there was 31 or more residues of VP7 present. Introduction of deletions in the context of the chimera revealed that larger deletions are required for the secretion of the VP7/amylase chimera than are required for the secretion of VP7. Thus, it is clear that the VP7 retention signal functions within a folded structure and depends very much on the adjacent structure. It was also observed that VP7 residues presented in certain chimera were able to abolish amylase enzymatic activity, presumably because the complex was denatured. These inappropriately folded molecules are degraded within the ER.