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protein structure

Structure of an infectious prion

15 September 2016 by Vincent Racaniello

prion conversionPrions are not viruses – they are infectious proteins that lack nucleic acids. Nevertheless, virologists have always been fascinated by prions - they appear in virology textbooks (where else would you put them?) and are taught in virology classes. I’ve written about prions on this blog (five articles, to be exact – look under P in the Table of Contents) and I’m fascinated by their biology and transmission. That’s why the newly solved structure of an infectious prion protein is the topic of the sixth prion article at virology blog.

Spongiform encephalopathies are neurodegenerative diseases caused by misfolding of normal cellular prion proteins. Human spongiform encephalopathies are placed into three groups: infectious, familial or genetic, and sporadic, distinguished by how the disease is acquired initially. In all cases, the pathogenic protein is the host-encoded PrPC protein with an altered conformation, called PrPsc. In the simplest case, PrPSc converts normal PrPC protein into more copies of the pathogenic form (illustrated).

The structure of the normal PrPC protein, solved some time ago, revealed that it is largely alpha-helical with little beta-strand content. The structure of PrPSc protein has been elusive, because it forms aggregates and amyloid fibrils. It has been suggested that the PrPSc protein has more beta-strand content than the normal protein, but how this property would lead to prion replication was unknown. Clearly solving the structure of prion protein was needed to fully understand the biology of this unusual pathogen.

The structure of PrPSc protein has now been solved by cryo-electron microscopy and image reconstruction (link to paper). The protein was purified from transgenic mice programmed to produce a form of  PrPSc protein that is not anchored to the cell membrane, and which is also underglycosylated. The protein causes disease in mice but is more homogeneous and forms fibrillar plaques, allowing gentler purification methods.

prion structureThe structure of this form of the PrPSc protein reveals that it consists of two intertwined fibrils (red in the image) which most likely consist of a series of repeated beta-strands, or rungs, called a beta-solenoid. The structure provides clues about how a pathogenic prion protein converts a normal PrPC into PrPSc . The upper and lower rungs of beta-solenoids are likely the initiation points for hydrogen-bonding with new PrPC molecules – in many proteins with beta-solenoids, they are blocked to prevent propagation of beta-sheets. Once added to the fibrils, the ends would serve to recruit additional proteins, and the chain lengthens.

The authors note that the molecular interactions that control prion templating, including hydrogen-bonding, charge and hydrophobic interactions, aromatic stacking, and steric constraints, also play roles in DNA replication.

The structure of PrPSc protein provides a mechanism for prion replication by incorporation of additional molecules into a growing beta-solenoid. I wonder if incorporation into fibrils is the sole driving force for converting PrPCprotein into PrPSc, or if PrPC is conformationally altered before it ever encounters a growing fibril.

 

Filed Under: Information Tagged With: beta-solenoid, beta-strand, cryo-electron microscopy, cryo-EM, prion, protein structure, scrapie, transmissible spongiform encephalopathy, TSE, viral, virology, virus, viruses

TWiV 394: Cards in a hand

19 June 2016 by Vincent Racaniello

Vincent and Alan speak with Erica Ollmann Saphire about her career and her work on understanding the functions of proteins of Ebolaviruses, Marburg virus, and other hemorrhagic fever viruses, at ASM Microbe 2016 in Boston, MA.

You can find TWiV #394 at microbe.tv/twiv, or listen or watch the video below.

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Filed Under: This Week in Virology Tagged With: cryoEM, ebolavirus, filovirus, marburg virus, protein, protein structure, structural biology, viral, virology, virus, viruses, X-ray structure

Viruses in the extreme

28 May 2015 by Vincent Racaniello

RudivirusMany microbes live in extreme environments, encountering conditions that are very hot, very cold, highly acidic, or very salty. The viruses that infect such microbes must also be able to retain infectivity in extreme conditions. How do they do it?

Clues come from the observations that the genomes of viruses that infect Archaea in extreme geothermal environments encode proteins that have never been seen before. The idea is that such unusual proteins must endow these viruses with the ability to maintain infectivity under extreme conditions.

The hosts of Rudiviruses (rudi=small rod in Latin), the Archea Sulfobolus islandicus, live at high temperatures (80° C) and low pH (3.0). These non-enveloped viruses consist of double-stranded DNA wrapped in a helical manner with thousands of copies of a 134 amino acid protein (illustrated; image credit). The three-dimensional structure of Sulfobolus islandicus rod-shaped virus 2 (SIRV2) reveals a new type of organization of virus particles, and provides clues about how it retains infectivity in extreme environments.

Resolution of the SIRV2 structure reveals that it consists of dimers of a single protein which forms helices that are tightly wrapped around the DNA genome. The result is a coiled DNA protected by a coat of protein that stabilizes and protects the genome. Without DNA, over half of the capsid protein is unstructured. Only in the presence of DNA does the viral protein form an alpha helix that wraps around the nucleic acid.

The DNA genome of SIRV2 is in the A-form, in contrast to B-form DNA which is found in most other organisms. The two types of DNA differ in their geometry and dimensions. It was previously thought that A-DNA occurs only when the nucleic acid is dehydrated.

These two usual properties of SIRV2 are also found in gram positive bacteria which form desiccation and heat resistant spores when starved of nutrients. Sporulation is accompanied by a change in the bacterial genome from B-DNA to A-DNA, which is caused by the binding of small acid-soluble proteins. Like the SIRV2 capsid protein, small acid-soluble proteins of spore-forming bacteria are unstructured in solution, and become alpha helices when bound to DNA. These observations suggest that binding of the SIRV2 capsid protein changes the viral DNA to the A-form, conferring stability in extreme environments.

Filed Under: Basic virology, Information Tagged With: A-DNA, alpha helix, archaea, B-DNA, extremophile, helical symmetry, hyperthermophilic, protein structure, rod-shaped virus, Sulfobolus islandicus, viral, virology, virus

How ZMapp antibodies bind to Ebola virus

25 November 2014 by Vincent Racaniello

antibodies bound to Ebola virus GPZMapp, a mixture of three antibodies against Ebola virus, became a household name after it was used to treat two Americans who were infected while working in Liberia. The structure of these antibodies bound to the Ebola virus glycoprotein suggest how they inhibit infection and ways to improve ZMapp.

The three monoclonal antibodies that comprise ZMapp (called c13C6, c2G4, and c4G7) were produced by immunizing mice with a recombinant vesicular stomatitis virus in which the glycoprotein was replaced with that from Ebola virus. Antibodies that bound the viral glycoprotein and protected mice from infection were identified, and three were made to resemble human antibodies and produced in tobacco plants. Ecco Zmapp!

Embedded in the membrane of the filamentous Ebola virus particle are many copies of the Filovirusglycoprotein, seen as club-shaped spikes in the image to the right (image credit: ViralZone). The viral glycoprotein is essential for entry of the virus into cells. The antibodies in ZMapp are directed against the viral glycoprotein.

To determine how the antibodies bind the virus particle, they were individually mixed with purified Ebola virus glycoprotein, and the structures were determined by electron microscopy and image reconstruction. The results, shown in the illustration, indicate precisely where each antibody binds to the Ebola virus glycoprotein. The individual antibodies colored red (c2G4), yellow (c4G7), and purple (c13C6) are bound to a single Ebola virus glycoprotein in white, with the viral membrane below (Image credit).

The structures reveal that c13C6 (purple) binds at the tip of the viral glycoprotein, perpendicular to the plane of the viral membrane. The other two antibodies (red, yellow) bind at the base of the viral glycoprotein. Their binding sites overlap but are not identical (the Ebola virus glycoprotein is a trimer, and in the image, the yellow and red antibodies are shown binding to different subunits for clarity). Two other antibodies that block Ebola virus infection also bind at the base of the glycoprotein.

Antibody c13C6, which binds to the tip of the viral glycoprotein, does not neutralize viral infectivity. Nevertheless, it can protect animals from Ebola virus infection. This observation suggests that the c13C6 antibody may work in concert with complement, a collection of serum proteins, to block virus infection. It is not known why c13C6 antibody is non-neutralizing, but one possibility is that it binds to a part of the viral glycoprotein that is removed by an endosomal protease, cathepsin, before receptor binding in late endosomes.

Antibodies c2G4 and c4G7, which bind to the membrane-proximal part of the viral glycoprotein, neutralize viral infectivity. How they do so is not known, but one possibility is that they prevent structural changes of the viral protein that are essential for the fusion with the endosomal membrane, a process that delivers the viral nucleic acid into the cell cytoplasm.

These studies reveal two general areas of the Ebola virus glycoprotein that are important targets for antibodies that protect animals from Ebola virus infection. Those directed at the base of the glycoprotein neutralize infectivity while those that bind the tip do not. This information can now be used to isolate additional antibodies that bind either site. These can be used in animal protection studies to design mixtures that are even more potent than ZMapp.

Filed Under: Basic virology, Information Tagged With: antibody, ebola virus, ebolavirus, electron microscopy, monoclonal antibody, neutralize, protein structure, viral, virology, virus, ZMapp

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by Vincent Racaniello

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