Identification of picornavirus proteins that inhibit de novo nucleotide synthesis during infection

Figures

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  Fig 1. CVB3 and CVB3-2Am infection increase the levels of purine and pyrimidine metabolites in HeLa cells. 13C-glucose isotope tracing study in mock-, CVB3- and CVB3-2Am infected HeLa R19 cells (MOI 5, three replicates). Cells were infected, lysed at 2, 4, 6, 8, or 10 hours post infection (hpi) and measured by LC-MS to identify metabolites and quantify the different isotopologues. Heatmap of log2 fold changes of the total levels (i.e., the sum of all isotopologues) of the indicated metabolites between CVB3- or CVB3-2Am- and mock-infected cells. Log2 fold changes are calculated based on the mean of three replicates. Red metabolites are metabolites in the purine and pyrimidine pathway.

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  Fig 2. CVB3 infection limits de novo nucleotide synthesis, while CVB3-2Am does not. 13C-glucose tracing study in mock-, CVB3- and CVB3-2Am infected HeLa R19 cells (MOI 5, three replicates). Cells were infected, lysed at 2, 4, 6, 8, or 10 hpi and measured by LC-MS to identify metabolites and quantify the different isotopologues. A) Schematic representation of nucleotide labeling by 13C6-glucose [36]. Nucleotides can be synthesized de novo, released during degradation of nucleic acids, or recycled (i.e., called the salvage pathway), resulting in the formation of different isotopologues. PRPP, phosphoribosyl pyrophosphate; R1P, ribose-1-phosphate. Orange = phosphate group; Turquoise = pentose sugar; Purple = nucleobase. B) Isotopologue distribution and absolute peak areas of UDP. For statistical analysis, linear mixed effect models with an interaction of time and treatment and a random effect of replicate were performed. p-values between specific groups were calculated by performing a contrast analysis, in which either the total labeled fraction or the total peak area between groups was compared. *p < 0.05, **p < 0.01, ***p < 0.001. C) Isotopologue distribution of detected purine and…

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  Fig 3. Glutamine tracing studies confirm a role for 2Apro in restricting the de novo nucleotide synthesis. 13C6-glucose, 13C5-glutamine and 15N2-glutamine isotope tracing study in mock-, CVB3-2Am- and CVB3-infected HeLa R19 cells (MOI 5, three replicates per treatment and per isotope). Cells were infected, lysed at 4, 6 or 8 hpi and measured by LC-MS to identify metabolites and quantify the different isotopologues. A) Schematic representation of nucleotide labeling by 13C5-glutamine and 15N2-glutamine [38]. B) Isotopologue distribution of ADP and UDP at 6 hpi. C) Peak areas of N-carbamoyl-aspartate and orotate at 6 hpi. D) Peak areas of GMP and AMP labeled with 15N2-glutamine. E) Schematic of hypoxanthine released from RNA degradation that can be salvaged to IMP and then be converted to AMP and GMP [39]. During this latter conversion, one (labeled) nitrogen atom is added. For statistical analysis, linear mixed effect models with an interaction of time and treatment and a random effect of replicate were performed. For N-carbamoyl-aspartate, a normal distribution of the residuals could not be assumed and therefore a non-parametric linear mixed effect model with an interaction of time and…

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  Fig 4. 2Apro and L restrict de novo nucleotide synthesis, while Lpro does not. 13C6-glucose isotope tracing study in mock- and CVB3-, CVB3–2Am, CVB3–2Am + L, CVB3–2Am + Lpro infected HeLa R19 cells (MOI 5, three replicates). Cells were infected, lysed at 6 or 8 hpi and measured by LC-MS to identify metabolites and quantify the different isotopologues. Data obtained at 8 hpi are shown here. A) table of L, 2Apro and Lpro functions. B) Schematic representation of CVB3 and the CVB3 recombinant viruses that were used in this study [43]. Fig 4B was created with BioRender. C) Peak areas and isotopologue distribution of UTP and GTP at 8 hpi. D) Peak areas of N-carbamoyl-aspartate at 8 hpi. For statistical analysis, linear mixed effect models with an interaction of time and treatment and a random effect of replicate were performed. A rank transformation on the data was performed to ensure a normal distribution of the residuals. For GTP, a normal distribution of the residuals could not be assumed for the fractional data and therefore a non-parametric linear mixed effect model with an interaction of time and treatment and a random effect of replicate was performed. p-values between specific groups…

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  Fig 5. CVB3 and EMCV elevate levels of purine and pyrimidine metabolites in hiPSC-CMs. 13C6-glucose isotope tracing study in mock-, CVB3-, CVB3-2Am or EMCV infected hiPSC-CMs (MOI 5, calculated from the HeLa R19 titer, three replicates). Cells were infected, lysed at 2.5, 5, 7.5, 10 hpi and measured by LC-MS to identify metabolites and quantify the different isotopologues. A) Heatmaps showing log2 fold changes of the purine and pyrimidine metabolites between CVB3 infected- and mock-infected cells. Log2 fold changes are calculated based on the mean of three replicates. B) Isotopologue distribution of UDP and ADP. For statistical analysis, linear mixed effect models with an interaction of time and treatment and a random effect of replicate were performed. A rank transformation on the data was performed to ensure a normal distribution of the residuals. p-values between specific groups were calculated by performing a contrast analysis, in which the total labeled fraction was compared between groups. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig 6. CVB3 modulates amino acid metabolic pathways in hiPSC-CMs. 13C6-glucose isotope tracing studies in mock-, CVB3-, CVB3-2Am or EMCV-infected hiPSC-CMs (MOI 5, three replicates per experiment). Cells were infected, lysed at 2.5, 5, 7.5 or 10 hpi and measured by LC-MS to identify metabolites. A) MetaboAnalyst pathway analysis of the two independent metabolomic experiments performed on hiPSC-CMs infected with CVB3 at 10 hpi. B) representative Log2 fold changes of amino acid levels during CVB3 infection over time (from experiment 1). C) Log2 fold changes in levels of amino acids between CVB3- or CVB3-2Am- vs mock-infected hiPSC-CMs at 10 hpi. For statistical analysis, linear mixed effect models with an interaction of time and treatment and a random effect of replicate were performed. A rank transformation on the data was performed to ensure a normal distribution of the residuals. p-values between specific groups were calculated by performing a contrast analysis, in which total peak areas were compared between groups. *p < 0.05, **p < 0.01, ***p < 0.001.