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

The evidence that h2 is cleaved led us to more specifically assess the role in ER retention, of the amino acid domain 51-61, since the absence of the coding region for these amino acids was the only difference between the mutants which were secreted and wt VP7 or AI-14, which were not secreted. The retention function of this region of VP7 was tested by constructing chimera of portions of VP7 with mouse salivary amylase, a normally secreted glycoprotein. Genes for VP7/amylase, Al-14/amylase and A51-61/dhl/amylase, each consisted of VP7 sequences up to the codon for the 63rd amino acid of the open reading frame attached to the gene for amylase lacking its normal cleavable signal sequence (Fig. 1) . The chimeric genes, VP763/Am, Al-1463/Am and A51-6163/dhl/Am, which each did not include the glycosylation site of VP7, were cloned into the transcription vector pGEM3 and translation products were examined in the absence or presence of microsomes to assess signal cleavage in the chimeric molecules. In the absence of membranes a product corresponding to the size expected, for wt amylase, or slightly greater for the chimera VP763/Am, Al-1463/Am or A51-6163/dhl/Am, ~54 kD decreased in size upon the addition of membranes, likely indicating signal cleavage (data not shown).