Role of the Intermediate Compartment between the Rough ER and Golgi in the Biogenesis of Vaccinia Virus
Beate Sodeik and Gareth Griffiths, EMBL, Heidelberg, Germany Vaccinia virus is the best studied example of the pox virus family, a group of complex, DNAcontaining viruses that replicate in specialized areas of cytoplasm, referred to as factories, within infected cells. During its biogenesis the virus is sequentially enveloped by four distinct membrane layers. A surprising dogma has been accepted in the field that the first of these membranes results from de novo synthesis of membrane, presumably due to enzymes encoded by the virus (Dales and Pogo, 1981). This 'fact' has actually been cited as the only exception to the general mechanism of membrane biogenesis, namely, synthesis on pre-existing membrane templates that occurs mostly, if not exclusively, in the rough ER in eukaryotic cells (Palade, 1983).
We have studied the acquisition of membranes by Vaccinia in tissue culture cells by a combination of immunofluorescence and immunoelectron microscopy, using thawed cryosections. For this antibodies were available against several Vaccinia membrane-or membrane-associated antigens as well as against defined cellular compartments. Additionally, the viral DNA could be identified using either Hoechst dye at the light microscopy level or anti-DNA antibodies for EM studies. An important tool in these studies was the use of rifampicin, a drug which selectively and reversibly blocks the assembly of the immature (one-membrane enclosed) virions.
Our results (see Figs.
) suggest that the first two of the viral inner membranes are acquired from a pre-existing cellular compartment, the so-called intermediate compartment between the rough endoplasmic reticulum and the Golgi complex that can be specifically marked with antibodies against the GTP-binding protein Rab 2 (Chavrier et al., 1990) and a 53 Kd protein (Schweizer et al., 1989). This intermediate compartment is an extensive structure with extremely complex organization. A 62 Kd protein (the target of rifampicin) was predominantly associated with the first budding membrane of immature virions (using an antibody provided by Drs. B. Moss and B. Doms). In the presence of rifampicin the membranes of the intermediate compartment were also labelled with an antibody against a 14 Kd membrane protein (provided by Dr. M. Esteban). Noteworthy however, was the fact that, under normal conditions, the immature virions were not labelled with this marker whereas on the twomembraned, intracellular naked virions (INV) the outer membrane was strongly labelled. Our tentative model to explain these findings is that the virus buds into the lumen of the intermediate compartment, acquiring the first, 62 Kd-containing membrane in the process. Subsequently, the virus buds out again into the cytosol therebyobtaining its second, 14 Kd-containing membrane. Our data show that the INV subsequently become engulfed by a cisterna that is enriched in two viral membrane proteins, the 85 Kd haemagglutinin (antibody provided by Dr. H. Shida) and a 37 Kd protein (antibody provided by Dr. G. Hiller). In the presence of brefeldin A the latter process does not occur and only INV are produced.