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

To determine whether VP7 sequences distal to the second hydrophobic domain (h2) extending up to amino acid 63 could target the secretory protein salivary amylase to the ER, the VP7-amylase chimeric products expressed by transfected cells were examined in cell lysates and the media in the presence and absence of endo-H. Wild-type VP7, as shown previously (31) , was immunoprecipitable from intracellular lysates in an endo-H sensitive form (Fig. 4, lanes//and 12) but not from the media of these cells (lanes 23 and 24) . On the other hand, when wild-type amylase (Am), or the VP7amylase chimera VP763/Am, Al-1463/Am or A51-6163/dhl/ Am, were transfected into COS7 cells, products were immunoprecipitable in each case from both intracellular lysates ( Fig. 4 , lanes 1-8) and the media (Fig. 4, lanes 13-20) after a 4-h labeling period, and these products did not change in size after endo-H treatment (Fig. 4) or when transfected cells were grown in the presence of tunicamycin (data not shown).