Structure and Assembly of Intracellular Mature Vaccinia Virus: Isolated-Particle Analysis

Abstract

In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum ( In this first study, we analyzed the IMV by on-grid staining, cryoscanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.

Cryo-electron tomography of vaccinia virus

“…Analysis of all reconstructed particles in our tomograms supports the presence of two membranes, as postulated in recent studies (18,22,23). The outer viral membrane envelops the entire virion and presents a rough appearance on the outer side.…”

“…Other subsequent studies, such as those by Roos et al (21) and Griffiths et al (22,23), applied different sample preparation and visualization techniques, including immune labeling procedures (24). These contributions led to the proposal of models speculating on how subsequent assembly intermediates could be reflected in the final virion.…”

Membrane remodelling during vaccinia virus morphogenesis

“…Initially, a single membrane was reported (Dales and Siminovitch, 1961), but the use of methods allowing better preservation of the ultrastructure led to publications claiming the existence in crescents of two closely apposed lipid bilayers (Sodeik et al, 1993). Subsequently, other studies supporting the presence in intermediate VACV particles of either one (Grimley et al, 1970;Morgan, 1976;Hollinshead et al, 1999;Heuser, 2005) or two (Griffiths et al, 2001a(Griffiths et al, , 2001bRisco et al, 2002) membranes have been published, leading to, at least, four different models to explain the evolution of membranes during VACV maturation (reviewed in Condit et al, 2006). One model involves the formation of single-membrane crescents which later enclose the DNA core which will not carry other membranes around it (Hollinshead et al, 1999).…”

The Product of the Vaccinia Virus L5R Gene Is a Fourth Membrane Protein Encoded by All Poxviruses That Is Required for Cell Entry and Cell-Cell Fusion

“…Furthermore, it has been suggested that the intracellular mature virion itself contains multiple membranes (15). The topological problems associated with the fusion of virions with multiple membranes have led to proposals of nonfusion mechanisms of entry (24).…”