Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment

The Spodoptera frugiperda cells (sfg-a c lonal isolate of Spodoptera frugiperda IPLB-Sf21-AE cells) and baculovirus expression vectors were obtained from Dr. M. Summers (Center for Advanced Invertebrate Molecular Sciences, College Station, TX). Rat PDI-cDNA (obtained from Dr. G. Banting, University of Bristol) was cloned between the Sinai and XbaI sites of the pVL1393 expression vector. This construct was used together with linearized Autographa californica nuclear polyhedrosis virus wild type-DNA to cotransfect St9 cells. The transfection was done with Lipofectin (Bethesda Research Laboratories, Gaithersburg, MD) according to manufacturer's instructions. Recombinant virus was plaque purified and infected cells were screened for PDI expression by immunofluorescence and Western blotting using a polyclonal antibody raised against purified rat PDI. For the overexpression of PDI, St9 cells were infected with the recombinant virus (9 • 106 cells, MOI = 10). As a control, St9 cells were infected with wild type virus (Luckow and Summers, 1988; Summers and Smith, 1988) . Lysates were prepared from cells harvested at 48 h after infection. To prepare the lysates, cells were washed with PBS, resuspended in 500/~1 PBS, passed through a ball bearing cell cracker (20 passes with 18 t~m clearance) and then subjected to two rounds of freezing and thawing.