THE ROLE OF A 21-KDA VIRAL MEMBRANE-PROTEIN IN THE ASSEMBLY OF VACCINIA VIRUS FROM THE INTERMEDIATE COMPARTMENT

We have recently provided morphological evidence that a key event in t he assembly of vaccinia virus is the formation of a novel cisternal do main of the intermediate compartment (IC) between the endoplasmic reti culum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hi ller, G., Machamer, C. E., van't Hof, W., van Meer, G., Moss, B., and Griffiths, G. (1993) J. Cell Biol. 121, 521-541). This tightly apposed cisternal domain incompletely surrounds the spherical immature virus that matures into the first of the two distinct infectious forms of va ccinia, the intracellular mature virus (IMV). In this study we describ e the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown t o be essential for the formation of the viral mem branes (Rodriguez, D ., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640-4648). Upon translation in vitro, p21 associated with rough microsomal membra nes in a co-translational manner. Using NH2- and COOH-terminal specifi c antibodies, we show that both in vitro as well as in vivo, p21 adopt s a topology where the NH, and COOH termini are cytoplasmically orient ated. Immuno cytochemical experiments demonstrated that p21 is a compo nent of the inner of the two cisternal membranes of the immature virus as well as of membranes of the IC, identified using antibodies agains t Rab1. Taken together, these data provide the first molecular evidenc e in support of our assembly model; they show that an essential membra ne protein of the IMV inserts into the rough endoplasmic reticulum, bu t gets efficiently targeted to the IC and membranes of the viral facto ry.