Figure 7: IRG1 alters neuronal metabolism during ZIKV infection via production of itaconate.

(A) Principle component analysis following metabolomic profiling of primary cortical neurons isolated from Ripk3^−/− or Irg1^−/− mice or congenic controls at 24h post infection with ZIKV-MR766 (MOI 0.1).

(B) Expression profiles of 294 metabolites in cultures described in (A). Significant differences (>1.5 fold change, p<0.05) are noted in red (upregulated metabolites) or blue (downregulated metabolites).

(C-D) Relative ion intensities of indicated metabolites in cultures described in (A). (AUs: arbitrary units). N= 3 independent replicates.

(E) SDH activity in lysates derived from B6/N, Ripk3^−/−, or Irg1^−/− primary cortical neurons at 24h following infection with ZIKV-MR766 (MOI 0.1). Cultures were pretreated for 2h with medium containing itaconate or saline vehicle prior to infection. N= 4 independent replicates.

(F) Extracellular oxygen consumption rate (OCR) over 1h following 24h infection in neuronal cultures as described in (G). N= 4 independent replicates.

(G) B6/N, Ripk3^−/−, or Irg1^−/− primary cortical neurons were infected with ZIKV-MR766 (MOI 0.1) following 2h pretreatment with media containing malonate, lonidamine, or DMSO vehicle. Viral titers in supernatants were measured via plaque assay. N= 4 independent replicates.

(H) B6/N, Ripk3^−/−, or Irg1^−/− primary cortical neurons were infected with ZIKV-MR766 (MOI 10.0) following 2h pretreatment with media containing itaconate, citrate, or saline vehicle. Viral titers in supernatants at 24h post infection were measured via plaque assay. N= 4 independent replicates.

(I-K) Mice of the indicated genotype were incranially infected with ZIKV-MR766 (I-J) or WNV-TX (K) and viral titers were measured via plaque assay in whole brain homogenates 2 days following infection. For all animals, infection happened concurrently with intracranial administration of 4-octyl (4-O) itaconate or DMSO vehicle.

(L-M) WT B6/N mice were infected intracranially with ZIKV-MR766 (L) or WNV-TX (M) and brain viral burden was measured as in (I-K). Concurrent with infection, mice received intracranial administration of dimethylmalonate (DMM) or saline vehicle.

(N) Mice of the indicated genotype were incranially infected with ZIKV-MR766 and monitored for survival. Mice received one intracranial administration of 4-O itaconate or DMSO vehicle concurrently with infection, with a second administration on day 4 following infection. N= 10–12 mice per genotype and treatment group.

*p<0.05, **p<0.01, ***p<0.001. Error bars represent SEM. Data in (E-N) are pooled from two or three independent experiments. See also Figure S6 and Figure S7.