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 the assembly of vaccinia virus is the formation of a novel cisternal domain of the intermediate compartment (IC) between the endoplasmic reticulum and the Golgi complex (Sodeik, B., Doms, R. W., Ericsson, M., Hiller, 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 vaccinia, the intracellular mature virus (IMV). In this study we describe the characterization of an abundant membrane protein of the IMV, the gene product of A17L, a 21-kDa protein that has recently been shown to be essential for the formation of the viral membranes (Rodriguez, D., Esteban, M., and Rodriguez, J. R. (1995) J. Virol. 69, 4640-4648). Upon translation in vitro, p21 associated with rough microsomal membranes in a co-translational manner. Using NH 2 - and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH 2 and COOH termini are cytoplasmically orientated. Immunocytochemical experiments demonstrated that p21 is a component of the inner of the two cisternal membranes of the immature virus as well as of membranes of the IC, identified using antibodies against Rab1. Taken together, these data provide the first molecular evidence in support of our assembly model; they show that an essential membrane protein of the IMV inserts into the rough endoplasmic reticulum, but gets efficiently targeted to the IC and membranes of the viral factory.