Virginia Research Day 2021

1 Department of Microbiology and Immunology, Edward Via College of Osteopathic Medicine, University of Louisiana, Monroe, LA 71203, USA. 2 Department of Microbiology-Immunology and Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Stephen DiGiuseppe 1 , Madeline G. Rollins 2 , Helen Astar 2 , Natalia Khalatyan 2 , Jeffery N. Savas 2 , and Derek Walsh 2 Proteomic and mechanistic dissection of the poxvirus-customized ribosome

INTRODUCTION TO POXVIRUS LIFECYCLE

PHOSPHORYLATION OF BOTH SITES IN RPS2 ENHANCED POXVIRUS TRANSLATION AND SPREAD

RPS2 IS PHOSPHORYLATED DURING A POXVIRUS INFECTION BY THE VIRAL B1 KINASE

 Replicates exclusively in the cytoplasm.  Replication occurs in distinct cytoplasmic compartments called viral factories (VF) composed of viral proteins and ER- and Golgi-derived membranes.  Poxvirus does NOT encode ribosomes.  Poxvirus must hijack host ribosomes to synthesize viral protein.  Poxvirus gains control of host ribosomes by targeting: 1. Upstream regulatory signaling pathways 2. Eukaryotic initiation factors 3. Ribosomal proteins HYPOTHESIS Poxvirus induces site-specific post- translational modifications on host ribosomal proteins to promote viral protein synthesis

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Figure 1. Overview of poxvirus infection and effects on translation. After poxvirus entry into the cytoplasm, transcriptionally competent viral cores begin to produce early viral mRNAs that are translated in the cytoplasm amongst the broader pool of host transcripts. Synthesis of early proteins leads to core uncoating and entry into the post- replicative stage. This involves the degradation of a large portion of host mRNAs along with the formation of viral factories, which facilitate many processes including DNA synthesis, transcription of intermediate and late gene products, viral protein synthesis and virion assembly.

POXVIRUS CUSTOMIZES THE RIBOSOME AT THE MESSANGER RNA ENTRY & EXIT CHANNELS Fig. 5. Phosphorylation of both sites in RPS2 maximizes poxvirus protein synthesis and spread. (A) Western blot analysis of NHDFs expressing HA-RPS2 or site-specific glutamic acid (T278E; S281E), double alanine (AA), or double glutamic acid (EE) substitution mutants infected with VacV (MOI = 0.003) for 72h. (B) Quantification of relative change in viral protein levels presented as mean ± SEM (n = 8) of NHDFs expressing HA-RPS2 or site-specific glutamic acid (T278E; S281E), double alanine (AA), or double glutamic acid (EE) substitution mutants infected with VacV (MOI = 0.003) for 72h. (C) Quantification of viral titers from NHDFs expressing HA-RPS2, or double alanine (AA) or glutamic acid (EE) substitution mutants of HA-RPS2 infected with VacV (MOI = 0.003) for 72h. Data is presented as mean percent infection relative to WT infection ± SEM (n = 4). (F) Percent change in luciferase activity from poly(A)-leader reporter in NHDFs expressing site-specific single glutamic acid (T278E; S281E) or double glutamic acid (EE) mutations presented as the mean percent relative to WT ± SEM (n = 9). *p < 0.05; “NS” not statistically significant.

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PROTEOMIC ANALYSIS OF HOST RIBOSOMES DURING A POXVIRUS INFECTION

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SITE-SPECIFIC PHOSPHORYLATION OF RPS2 IS IMPORTANT FOR A POXVIRUS INFECTION Fig. 3. RPS2 is phosphorylated during a poxvirus infection. (A) MS/MS spectra of RPS2 peptides immunoprecipitated from GFP-trap complexes from VacV-infected Hap1 cells. The peptide amino acid sequence, phosphorylated residue, b-series ions (Light Blue), y-series ions (Red), charge state ( + ), and ions that support phosphorylation ( ^ ) are all indicated. (B) Structural model of RPS2 with labeled amino acid residues identified as being phosphorylated specifically during VacV infection. (C) Western blot analysis of primary human dermal fibroblasts (NHDFs) infected with VacV, Herpes Simplex virus type 1 (HSV-1), or Vesicular stomatitis virus (VSV) (MOI = 5) at 20 hpi (for VacV and HSV-1) or 6 hpi (for VSV). Arrows denote band shifted species. (D) Western blot analysis of NHDF lysates that were either mock infected or infected with VacV, then treated with recombinant shrimp alkaline phosphatase (rSAP) as indicated. Arrows point to the loss of band-shifted RPS2 in SAP-treated samples. (E) NHDFs infected with temperature sensitive (Ts) viral mutants of B1 or F10 kinase for 20h.

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Fig. 6. Poxvirus-modified RPs are positioned at the mRNA entry and exit channels of the ribosome. (A) Structural model of the 80S ribosome facing toward the mRNA entry channel highlighting proteins modified by poxvirus at specific sites (Green). (B) Rotated 90 ° structural model of the 80S ribosome facing the mRNA exit channel highlighting proteins modified by poxvirus at specific sites (Green). (C) Entire ribosome with specific proteins customized by poxvirus identified using proteomic analysis with the mRNA entry (Green) and exit (Red) channels highlighted. Note RPS2 (Light Blue) and RACK1 (Orange) are highlighted.

REFERENCES

Fig. 1. Proteomic analysis of host ribosomes during a poxvirus infection. (A) Pipeline for isolation of host ribosomes during a poxvirus infection. HAP1 cells stably expressing exogenous RACK1- eGFP fusion protein were infected with Vaccinia virus (VacV) at MOI of 5 for 20 h. GFP complexes were immunoprecipitated using GFP-Trap® (Chromotek) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). (B) Structural model of the 80S ribosome, with 40S (Green) and 60S subunits (Blue), depicting RACK1- eGFP used for immunoprecipitation. (C) Silver stain of cell lysates and GFP-Trap immunoprecipitated proteins from uninfected or VacV infected (MOI = 5) Hap1 knockout cells expressing empty vector (-) or RACK1-eGFP at 20 hpi.

Fig. 4. Site-specific phosphorylation of is important for a poxvirus infection. (A) Western blot analysis of NHDFs expressing HA-RPS2 or site-specific alanine substitution mutants (T276A; T278A; S281A) infected with VacV (MOI = 5) for 20 h. Exogenous and endogenous forms of RPS2 are indicated. Arrows point to unmodified (black) and modified (red) forms of each. (B) Western blot analysis of NHDFs expressing HA-RPS2 or site-specific alanine (T278A; S281A) or glutamic acid (T278E; S281E) substitution mutants infected with VacV (MOI = 0.003) for 72h.

1. DiGiuseppe S, Rollins MG, Astar H, Khalatyan N, Savas JN, Walsh D. Proteomic and mechanistic dissection of the poxvirus-customized ribosome. J Cell Sci. 2020 Jul 9;134(5):jcs246603. 2. Meade N, DiGiuseppe S, Walsh D. Translational control during poxvirus infection. Wiley Interdiscip Rev RNA. 2019 Mar;10(2):e1515. 3. DiGiuseppe S, Rollins MG, Bartom ET, Walsh D. ZNF598 Plays Distinct Roles in Interferon-Stimulated Gene Expression and Poxvirus Protein Synthesis. Cell Rep. 2018 May 1;23(5):1249-1258. 4. Jha S, Rollins MG, Fuchs G, Procter DJ, Hall EA, Cozzolino K, Sarnow P, Savas JN, Walsh D. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase. Nature. 2017 Jun 29;546(7660):651-655.

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