Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum

Gene 9, which codes for VP7, has been cloned and sequenced (5) , and exhibits an open reading frame potentially encoding a protein 326 residues in length. Hydrophobicity analysis (17) indicates the presence of only two hydrophobic domains (hi and h2) located at the amino terminus, each preceded by an in-frame initiation codon, the second of which exhibits the stronger consensus sequence for initiation (15, 16) . Our previous studies showed that deletions at the COOH-terminal end of h2 and the adjacent 13 residues resulted in secretion of the altered VP7's (31) . More recent evidence (42) shows that h2 is cleaved after amino acid No. 50, suggesting that the distal 11 residues are significant for ER retention, rather than h2. We therefore investigated whether the region of amino acids 51-61 played a role in retention of VP7 in the ER, since they were present in the wild-type VP7 but absent in the secreted mutants A42-61, A43-61, or A47-61. VP7 coding sequences containing this 11-amino acid region or those which deleted it, were attached to those of mature mouse salivary a-amylase, a secretory protein, to assay for an effect on retention in the ER. Additional chimera were also constructed consisting of the mature amylase coding region and a larger contribution from the VP7 amino-terminal coding region of the deletions Al-14 and A47-61/dhl, which extended up to amino acid 111 and included the VP7 glycosylation site. Amylase enzymatic activity was similarly demonstrated for both wild-type amylase and the intracellularly retained chimera. We show in the present study that two regions of VP7 mediate its retention in the ER. The first lies within the region spanning amino acids 51-61 and the second in the region 62-111, the latter containing the single glycosylation site for VP7. Both regions are apparently necessary for retention, though neither is sufficient to function by itself.