Virginia Research Day 2021

Faculty Research Biomedical

05 Proteomic and Mechanistic Dissection of the Poxvirus-Customized Ribosome

Stephen DiGiuseppe; Madeline G Rollins; Helen Astar; Natalia Khalatyan; Jeffrey N Savas; Derek Walsh Corresponding author: sdigiuseppe@ulm.vcom.edu

Via College of Osteopathic Medicine-Louisiana Campus Northwestern University

Poxviruses are double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells. During infection, poxviruses must co-opt host ribosomes to synthesize viral protein. Ribosomes are protein synthesis machines comprised of 80 ribosomal proteins (RPs). Despite this, ribosomes are often viewed lacking intrinsic regulatory capacity. Evidence has emerged that ribosomes can functionally diversify via changes to RP composition or via post-translational modifications (PTMs) of RPs. Ribosome modifications have been linked to changes in embryonic development, cellular stress responses, viral infections, congenital diseases, and even some cancers. Changes to the ribosome can be utilized by the cell to control translation via preferential selection of subsets of mRNAs. Recently, it was uncovered that poxviruses phosphorylated unique sites on receptor for activated C kinase 1 (RACK1) to enhance

viral protein synthesis. To identify other RPs being modified during a poxvirus infection, I co-developed an approach for large-scale proteomic analysis of ribosomes isolated from cells infected different viruses. We identified additional phosphorylated residues within RPS2 and RPS28 that arise during poxvirus infection, but not other viruses tested. The modified sites lie within unstructured loop domains that position around the mRNA entry and exit channel, respectively, and site-substitution mutants revealed that each modified residue contributed differently to poxvirus replication. Our findings reveal the broader extent to which poxviruses customize host ribosomes and provide new insights into how ribosomes can functionally diversify.

Figure. Poxvirus-modified ribosome . Structural model of the 80S ribosome facing the mRNA entry and exit channels with RACK1 (Blue), RPS2 (Purple), and RPS28 (Red) and specific phosphorylated residues during a poxvirus infection (Orange; Yellow; Green respectively)..

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