RETENTION BY THE ENDOPLASMIC-RETICULUM OF ROTAVIRUS VP7 IS CONTROLLEDBY 3 ADJACENT AMINO-TERMINAL RESIDUES

The rotavirus outer capsid glycoprotein, VP7, is an endoplasmic reticu lum (ER) membrane-associated glycoprotein in both infected and transfe cted cells. It was previously demonstrated in this laboratory and by o thers that both the cleaved signal sequence (H2) and the first NH2-ter minal 61 amino acids of VP7 are sufficient and necessary for ER retent ion of this molecule. Using site-specific mutagenesis and transfection techniques, we show that residues Ile-9, Thr-10, and Gly-ll were spec ifically necessary for ER retention. These results further define the ER retention sequence of VP7 and demonstrate that conservative changes , apparently innocuous in only three adjacent amino acids, can lead to major solubility and compartmentalization changes. It was found that placement of the first 31 mature NH2-terminal residues of VP7, in addi tion to the cleaved ER translocation signal sequence, was sufficient t o retain the enzymatically active chimeric alpha-amylase in the ER; th is enzyme is normally secreted. Deletions of the residues Ile-9, Thr-1 0, and Gly-ll within the amylase chimera containing 31 VP7 amino acids resulted in secretion of enzymatically active protein. It was also ob served that the residues of VP7 presented in certain chimeras were abl e to abolish cy-amylase enzymatic activity. These chimeras are presuma bly misfolded since it was demonstrated by pulse-chase experiments tha t these molecules are degraded in the ER. We surmise that a favorable conformation is necessary for retention since ER retention and activit y of the chimeras depend on the primary sequence context.