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

VP7jAm, Al-1463/Am or A51-6163/dhl/Am expression vectors were constructed by restricting the plasmids pJC9, Al-14, each previously described (31) , or A51-61/dhl, with Xho I and initially isolating the 1,100-bp fragment (reference to fragment sizes are approximate) encoding wild-type or a deletion of VP7. The 100-200-bp fragment encoding the 5' end of each of the three variations of VP7 was purified after Nco I digestion, and ligated to the large Xho I/Apa I fragment of pJC/Am (pJC/Am-sig). pJC/Am-sig was thus pJC/Am lacking the wild-type signal sequence coding region. Precise deletion of the 5' signal sequence coding region of amylase was achieved by Apa I restriction of pJC/Am, blunting of the end which formed the junction between VP7 and amylase with T4 polymerase, followed by Xho I digestion which removed the small Xho I/Apa I fragment encoding the amylase signal but which retained the exact amino terminus of mature amylase (39) . The VP7-amylase coding sequences were each ligated at their junctions after treatment with Klenow enzyme, which blunted the overhang left by Nco I digestion of VP7.