Abstract
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 NH2- and COOH-terminal specific antibodies, we show that both in vitro as well as in vivo, p21 adopts a topology where the NH2 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.
Highlights
Taken together, these data provide the first molecular evidence in support of our assembly model; they show that an essential membrane protein of the intracellular mature virus (IMV) inserts into the rough endoplasmic reticulum, but gets efficiently targeted to the intermediate compartment (IC) and membranes of the viral factory
Upon in Vitro Translation, p21 Inserts into Microsomes—As pointed out in the introduction a critical test of our assembly model is the need to find at least one membrane protein which behaved as a typical marker of the IC and that is targeted to the IC after insertion into the rough ER
This may seem a trivial point, we noticed during the mapping of the membrane proteins of the IMV that the virus lacks any typical class I or class II membrane proteins
Summary
Viruses, and Antibodies—HeLa cells (CCL2) were obtained from ATTC and grown in Dulbecco’s modified Eagle’s medium (DMEM). The cells were fixed in this solution for 10 min at room temperature, rinsed extensively with PBS and 20 mM glycine, blocked with PBS/glycine containing 5% FCS for 2 h at 4 °C, and incubated overnight at 4 °C with the antibodies (diluted in PBS/ glycine-FCS; NH2-terminal 1:100, COOH-terminal 1:300). Rough microsomes were diluted in RM buffer (50 mM Hepes-KOH, pH 7.5, 50 mM KOAC, 2 mM MgAc, 250 mM sucrose, and 1 mM DTT) to an A280 of 20 To this CaCl2 to 1 mM, 150 units/ml micrococcal nuclease (Sigma) and 2 mM PMSF were added and incubated for 10 min at 25 °C. For protease treatment of the microsomes, the samples were mixed after translation with an equal volume of protease K diluted in RM buffer at the indicated concentrations and incubated for 30 min on ice. After addition of PMSF, the samples were immunoprecipitated with the NH2- and COOH-terminal specific antibodies
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