Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network

Abstract

During the assembly of vaccinia virus, the intracellular mature virus becomes enwrapped by a cellular cisterna to form the intracellular enveloped virus (IEV), the precursor of the extracellular enveloped virus (EEV). In this study, we have characterized the origin of this wrapping cisterna by electron microscopic immunocytochemistry using lectins, antibodies against endocytic organelles, and recombinant vaccinia viruses expressing proteins which behave as Golgi resident proteins. No labelling for endocytic marker proteins could be detected on the wrapping membrane. However, the wrapping membrane labelled significantly for a trans Golgi network (TGN) marker protein. The recycling pathway from endosomes to the TGN appears to be greatly increased following vaccinia virus infection, since significant amounts of endocytic fluid-phase tracers were found in the lumen of the TGN, Golgi complex, and the wrapping cisternae. Using immunoelectron microscopy, we localized the vaccinia virus membrane proteins W-p37, W-p42, W-p2l, and W-hemagglutinin (VV-HA) in large amounts in the wrapping cisternae, in the outer membranes of the IEV, and in the outermost membrane of the EEV. The bulk of the cellular W-p37, W-p2l, and W-p42 were in the TGN, whereas W-HA was also found in large amounts on the plasma membrane and in endosomes. Collectively, these data argue that the TGN becomes enriched in vaccinia virus membrane proteins that facilitate the wrapping event responsible for the formation of the IEV.

Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines

“…1B). The constitutively expressed MVA protein BR5 was also probed at each passage from the same lysates using the 19C2 mAb [32], and as shown in Fig. 1B, its steady state expression level was unchanged during the 10 passage evaluation.…”

“…After labeling, the cells were washed twice with PBS and either harvested immediately or chased in RPMI medium with 10% FCS (ISC-BioExpress, Kaysville, UT, USA) supplemented with excess unlabeled methionine (1 mM) and cysteine (5 mM) up to 10 h. After each time point, cells were immediately pelleted, then lysed in 1.0 mL PBS containing 1.0% Triton X-100, 1.0% sodium deoxycholate (Sigma, St. Louis, MO, USA) and 0.1% SDS in the presence of Protease Inhibitor Cocktail (Roche, Nutley, NJ, USA). Supernatants (0.5 mL) were precleared once with 50 L of protein A/G-agarose beads (Santa Cruz Biotechnology) for 1 h. Sequential incubation with 2.4 g purified mAb against CMV-pp65 (mAb 28-103 [43]) was followed by an isotope-specific mAb (19C2 [32]) for 2 h. Immune complexes were captured by incubation for 1 h with 50 L of protein A/G beads. The immune complex bound Protein A/G beads were washed 4 times with 0.1% Triton X-100 in PBS and bound proteins were eluted by boiling in 0.2% SDS, 5 mM DTT, 40 mM sodium phosphate buffer (pH 6.5) into SDS-polyacryamide gel electrophoresis (PAGE) sample buffer.…”

Virus factories: biogenesis and structural design

“…For viral genome replication, the virus first assembles cytoplasmic mini-nuclei with attached mitochondria (Tolonen et al, 2001); virus morphogenesis then starts an aggresome-like structure (Risco et al, 2002), where immature viruses assemble using an atypical membrane remodelling mechanism that has been characterized by ET (Chlanda et al, 2009). Final envelopment and viral maturation takes place in Golgi stacks (Schmelz et al, 1994).…”

Organelle dynamics and viral infections: at cross roads

“…Evidence shows that the tomato spotted wilt virus (TSWV) glycoprotein Gn localizes to Golgi membranes and induces deformation of the membranes into pseudo-circular and pleomorphic structures in tobacco plant cells [137]. Upon infection of HeLa cells, rabbit kidney cells and mouse monocytes-macrophages cell lines with VACV, viral progeny becomes enwrapped in the membrane derived from TGN cisterna to form the enveloped virus [138].…”