Dissecting the structure of the pox virion, one layer at a time

Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization

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One of the great challenges in the study of the biology of poxviruses is to understand the morphogenesis steps that culminate with the production of the infectious virus particle. To accomplish this task one needs to understand the structure of the virion.

Poxviruses replicate in the cell cytoplasm using a DNA genome and must therefore package in the virion the complete apparatus for synthesis of early viral RNA. The result is a uniquely complex virion structure. The virion of vaccinia virus, the prototypic member of the family Poxviridae, contains approximately 70 proteins, however the localization of a substantial fraction of the proteins within the virion is unknown.

The vaccinia virion can be thought of as a layered structure in which an inner nucleocapsid is surrounded by a core wall, which in turn is flanked by lateral bodies, all of which is encased in a proteolipid membrane. Nearly one third of the virion proteins are located in the virion membrane and are responsible for binding and entry of the particle into cells. The lateral bodies comprise a payload of proteins some of which may thwart the innate antiviral response early in infection. The core wall is a proteinaceous structure that protects the nucleocapsid and provides a novel cytoplasmic compartment for early viral transcription after loss of the membrane and lateral bodies during entry. It is assumed that the nucleocapsid contains the genome and transcription enzymes. The protein composition of the virion membrane is well defined, however the localization of proteins within the core and nucleocapsid is unknown. In this study we used controlled degradation of whole mount preparations of purified virus on electron microscope grids to successively peel away the layers of the virion. We then localized specific virion proteins to the resulting subdomains using immunogold labeling.

Removing the lipid layer of virus on the grid was an easy task, and we were able to identify lateral body proteins and pore-like structures on the surface of the core. However, to break open the core and release the nucleocapsid presented a major challenge. Surprisingly, when we applied our standard controlled degradation technique to a vaccinia mutant in a major core wall protein, the core burst open and the nucleocapsid was released onto the grid. Using this approach we were able to localize one of the core wall proteins to the internal layer of the wall. A bigger surprise occurred when we were able to detect the virion DNA-binding protein L4 only after DNase treatment, suggesting that L4 is wrapped in DNA in a nucleosome-like structure in the nucleocapsid.

Combining our results with published data, we proposed that during virus morphogenesis there is a volume contraction of the particle, during which water is excluded from the internal milieu. In this constricted form, the virion will be impermeable to water and inactive for transcription. During the entry process, the virion will lose its membrane, hydration of the internal milieu will occur, and the transcription machinery will be activated. From a broader perspective, this phenomenon could be a general mechanism for viruses that contain a transcription apparatus associated with the particle and that require the removal of the membrane or capsid protein to be activated.

Figure VirolBlog

Figure Legend
A model for vaccinia virus structure. A) Last steps of morphogenesis starting with immature virion (IV), followed by the incorporation of a DNA molecule to form the immature virions with nucleoid (IVN), proteolysis of specific proteins and exclusion of water to form the mature virion (MV). B) Virus in the constricted form. Fine detail of a sagittal section (see inset, lower left) of the MV showing the different virion sub-domains as specified on the left side of the figure. C) Membrane-less virion (equivalent to the virion core) after hydration. Same as B but after infection the virus loses the membrane and the nucleocapsid expands.

Introducing the Authors
Moussatche&Condit
Nissin Moussatche and Richard Condit (University of Florida) are friends and collaborators for 37 years.

About the Research
Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization
Virology
, Volume 475, January 2015, Pages 204–218
Nissin Moussatche, Richard Condit

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