• A scaffold with a twist: Cryo-EM reveals

    From ScienceDaily@1:317/3 to All on Thu Mar 31 22:30:46 2022
    A scaffold with a twist: Cryo-EM reveals the building blocks of poxvirus


    Date:
    March 31, 2022
    Source:
    Okinawa Institute of Science and Technology (OIST) Graduate
    University
    Summary:
    Researchers have revealed how poxviruses build their scaffold -
    a temporary protein coat that forms and disappears as the virus
    matures.



    FULL STORY ========================================================================== Researchers at the Okinawa Institute of Science and Technology Graduate University (OIST) have revealed how poxviruses build their scaffold --
    a temporary protein coat that forms and disappears as the virus matures.


    ========================================================================== Reporting today in Nature Communications, the scientists revealed
    the structure of a protein called D13, in near-atomic resolution, and
    showed how it assembles with other copies of D13 to form scaffold-like structures.

    "D13 is a key target for research, because if you know how the scaffold
    is assembled, you can design new drugs that prevent it from forming,"
    said Professor Jaekyung Hyun, a former staff scientist in the OIST
    Molecular Cryo- Electron Microscopy Unit, and now Assistant Professor at
    Pusan National University in South Korea. "If the scaffold can't form,
    then replication of the virus stops." D13 is a trimer protein, as it
    is formed from three identical protein chains.

    Once synthesized, it acts as a scaffold building block for the Vaccinia
    virus - - a harmless strain developed in the laboratory as a vaccine
    against smallpox.

    Researchers now use the Vaccinia virus as a model for all poxviruses.

    "Smallpox is the most famous and lethal disease caused by a poxvirus,
    with 1 in 3 infected people dying," said Professor Wolf, who leads
    the Molecular Cryo- Electron Microscopy Unit. "But while smallpox has
    been eradicated in the wild, there are fears that it could be used
    as a bioweapon. Also, numerous other poxviruses still infect humans
    and livestock, so further research into how these viruses replicate is essential." The scaffold seen in immature poxviruses is of particular
    interest to scientists, as the structure differs from the protein coats typically seen in viruses.



    ========================================================================== While most viruses have regular and symmetrical structures, poxvirus
    scaffolds have roughly spherical honeycomb lattices, which vary in shape between each viral particle.

    To determine how these blocks assemble into these spherical honeycomb
    lattices, the research team used cryo-electron microscopy (cryo-EM) --
    a technique in which samples are frozen in liquid nitrogen and probed by electrons -- to generate 3D images of both single D13 protein trimers and
    two connected D13 protein trimers, at the highest resolution seen so far.

    They found that the two proteins joined together with a slight twist
    between their trimer axes, creating a curve that is key for forming a
    spherical shape.

    However, when the team used computer modelling to extend the interaction
    to multiple D13 proteins, they didn't fit together properly.

    "This told us that there must be at least one other way for the two
    proteins to interact, that we hadn't yet seen," said Prof. Hyun.

    The researchers also found that when they compared the single D13 protein
    to the two D13 proteins joined together, a small helix structure at the
    end of the protein chains had shifted. Previously, the helix structure was buried deep into the pocket where the two proteins interact, suggesting
    that its repositioning was critical for the two proteins to connect.



    ==========================================================================
    To explore the role of the helix structure further, the research team
    made modified D13 proteins and then used cryo-electron tomography to look
    at how they self-assembled when placed in solution. When a purification
    tag was added to the helix, the proteins formed spherical structures
    similar to the immature poxvirus scaffold. And when the helix was
    completely removed, the researchers were surprised to see the formation
    of cylindrical tubes.

    Using cryo-EM, the research team were able to capture high-res images of
    these cylinders and zoom in on the honeycomb structure. They identified a second way for the dimers to interact and found that when they modelled alternating patterns of interaction, the D13 proteins fit together to
    form the hexagonal honeycomb pattern.

    Both modes of how the proteins interacted required the small helix
    structure to shift, with the proteins then held together by the attraction between positively and negatively charged amino acids.

    Overall, the researchers proposed that displacement of the helix was
    essential for forming poxvirus scaffold, and likely acted as a trigger
    for the assembly to begin.

    "When the poxvirus replicates inside cells, the scaffold forms in
    association with a lipid membrane," explained Prof. Wolf. "The helix
    structure is hydrophobic, which means that it would shift towards to
    the water-free environment of the lipid membrane, freeing up the pocket
    where the D13 proteins interact." The discovery of the helix's role in assembly could be a promising new avenue of research for antiviral drug discovery, emphasized Prof. Hyun.

    "If we can design a drug that binds really strongly into the pocket
    where the helix usually sits, it would interfere with formation of the scaffold," he said. "This is one of my next goals."

    ========================================================================== Story Source: Materials provided by Okinawa_Institute_of_Science_and_Technology_(OIST)
    Graduate_University. Original written by Dani Ellenby. Note: Content
    may be edited for style and length.


    ========================================================================== Journal Reference:
    1. Jaekyung Hyun, Hideyuki Matsunami, Tae Gyun Kim, Matthias
    Wolf. Assembly
    mechanism of the pleomorphic immature poxvirus scaffold. Nature
    Communications, 2022; 13 (1) DOI: 10.1038/s41467-022-29305-5 ==========================================================================

    Link to news story: https://www.sciencedaily.com/releases/2022/03/220331101527.htm

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