Mammalian reovirus is a diverse family of virus that has segmented double-stranded RNA genome contained in two protein shells, which distinguishes them from many other viral families. Despite causing disease in wide range of mammalian hosts, reovirus is not typically considered as a human pathogen due to the absence of obvious symptoms or known clinical impact. Because of this, it becomes a perfect 'model organism' for studying cellular processes that can be safely handled in the lab, including translation, which is the main subject of this paper. It is the last step in converting DNA into proteins that are the actual workers of our cells.
In our cells, translation is made possible by the presence of a hat (5'cap) and a tail (poly-A tail) appended to mRNAs (the temporary carrier of genetic information), which helps them to exit the nucleus, protect them from enzymes that can degrade them, and help initiate translation (circularize the mRNA and reload translation machinery). Then mRNA will be converted into protein in the cytoplasm. In comparison, reovirus RNA genome gets translated within viral factories, which are little houses with no walls (often in membrane-less compartments called liquid-liquid phase separation) containing necessary tools for baby viruses to assemble and mature. Reovirus µNS protein forms the scaffold of these houses and interact with the core, where viral mRNAs are transcribed with the hat but without the tail. Surprisingly, these viral mRNAs can be readily translated in infected cells without the assistance and protection of the poly-A tail! This paper seek to find out the host cell factor that reoviruses has 'kidnapped' to produce viral proteins for themselves.
Since µNS protein is the important building block of viral factories where viral translation takes place, the 'manipulated' cellular protein probably interact with µNS protein. Based on this logic, the researchers used a technique called proximity-dependent Biotin identification, which is basically a spray-paint process. µNS protein is tagged with the spray---biotin protein ligase, which will enable the biotinylation of nearby proteins---paint. The 50 painted proteins labeled with biotin are identified and underwent a protein-protein interaction cluster analysis. There's one cluster that matches well with the expectations: proteins associated with SG (stress granule) and translation initiation. Followed by a long process of screening that identified proteins that would lead to failure in viral replication if deleted and if temporarily suppressed through CRISPR and siRNA whole genome screening respectively, a single common candidate that pop out in all filtered results is identified: ATXN2L.
Then the hypothesis that ATXN2L is the essential mediator of viral translation without poly A tail is tested stepwise. If ATXN2L indeed facilitates the translation of viral protein, it should be indispensable for viral replication. The researchers hence used CRISPR/Cas9 gene editing technique, which basically messes up the nucleotides at the specific desired site (gene ATXN2L) and render it nonfunctional, and it resulted in greatly impaired viral replication compared to the control group with undisrupted ATXN2L gene (WT) and with ATXN2L gene re-introduced (KO+). The green fluorescence is the virus-specific the antibody staining, indicating the infectivity.
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Images:
https://www.creative-biolabs.com/proximity-dependent-biotin-identification-bioid-service.html
https://www.nature.com/articles/s41580-021-00417-y
https://www.preprints.org/manuscript/202506.0175/v1