Downregulation of neurodevelopmental gene expression in iPSC-derived cerebral organoids upon infection by human cytomegalovirus

Abstract

Human cytomegalovirus (HCMV) is a betaherpesvirus that can cause severe birth defects including vision and hearing loss, microcephaly, and seizures. Currently, no approved treatment options exist for in utero infections. Here, we aimed to determine the impact of HCMV infection on the transcriptome of developing neurons in an organoid model system. Cell populations isolated from organoids based on a marker for infection and transcriptomes were defined. We uncovered downregulation in key cortical, neurodevelopmental, and functional gene pathways which occurred regardless of the degree of infection. To test the contributions of specific HCMV immediate early proteins known to disrupt neural differentiation, we infected NPCs using a recombinant virus harboring a destabilization domain. Despite suppressing their expression, HCMV-mediated transcriptional downregulation still occurred. Together, our studies have revealed that HCMV infection causes a profound downregulation of neurodevelopmental genes and suggest a role for other viral factors in this process.

Keywords: Biotechnology; Developmental neuroscience; Transcriptomics.

Graphical abstract

Figure 1

Three-dimensional cerebral organoid infection with HCMV (A) On Day 30 of differentiation, organoids were weighed and infected at 500 IU/ug with HCMV strain TB40/E-GFP. Bright-field and fluorescent images of one representative infected organoid taken at 4, 8, and 12 dpi are shown. (B) Images at 12 dpi of infected and uninfected organoids are displayed; one representative organoid from each pooled and sorted group is shown. (C) Representative GFP intensity plot from FACs analysis of an infected organoid. (D) Representative GFP intensity FACs from a Mock-infected organoid. (E) Percentage of living cells in the isolated populations as determined by FACs; each infected organoid group was made up of three pooled organoids. (F) Percentage of total live cells within whole infected and uninfected organoids as determined by FACs; each infected organoid group consisted of three pooled organoids. (G) qPCR analysis of viral genes (UL122, UL123, UL44, and UL99) expression within Mock compared to GFP (+) and GFP (Inter) subpopulations. (H) qPCR analysis of viral gene expression within Mock compared to the GFP (Low) subpopulation. (I) Quantification of relative viral vs. host genomes within each of the sorted infected organoid subpopulations (see STAR Methods for quantification procedure). Scale bar is 100 uM in panels A and B and error bars in E, F, G, and H represent mean ± SEM from 4 biological replicates. Also see Figures S1 and S2. Stars were assigned based on level of significance as determined by one-way ANOVA with Tukey post hoc test: ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.00001.

Figure 2

All infected organoid subpopulations cluster distinctly from mock samples with many genes being downregulated (A) PCA with components being all mapped genes across samples split by axis determined by adj p value and compared between each sample. (B) Hierarchical cluster analysis of all sequenced samples for the top 500 most variable genes according to read counts assigned. Hierarchical cluster analysis of only infected samples for the top 500 most variable genes according to read counts assigned. (C) Volcano plot of the top 250 differentially expressed genes comparing GFP (+) vs. Mock and GFP (Low) vs. Mock as determined by fold change ±3 adj p value < 0.01 as determined by DESEQ2 analysis in R. Arrows denote genes downregulated in both groups. (D) Venn diagram comparing the 2,500 most differentially expressed genes in GFP (Low) vs. Mock and GFP (+) vs. Mock. (E) Volcano plot of the differentially expressed genes found from DESEQ2 analysis of GFP (+) vs. GFP (Low) using the same significance cutoff. Also see Table S1. Differential expression analysis conducted on GFP (+) vs. Mock in R, related to Figures 2 and 4, Table S2. Differential expression analysis conducted on GFP (Low) vs. Mock in R, related to Figures 2 and 4, Table S3. Differential expression and ontology analysis conducted on GFP (+) vs. GFP (Low) in R and using G-profiler, related to Figures 2 and 4.

Figure 3

Gene set enrichment analysis (GSEA) plots comparing infected organoid populations to mock GSEA using the hallmark gene set on a list of all differentially expressed genes with fold change ± 3 and adj p value < 0.05. (A) Hallmark interferon alpha response enrichment plots and corresponding heatmaps from GFP (+) vs. Mock and GFP (Low) vs. Mock, respectively. (B) Additional enrichment plots and heatmaps (20 representative genes each) from GSEA comparing GFP (+) vs. Mock revealing significant de-enrichment in GFP (+) groups. A normalized enrichment score (NES) and normalized p value (NOM p value) for each plot are shown below; a cutoff of NOM p value < 0.05 was used to establish significance (for full GSEA analysis details see Table S4).

Figure 4

Pathway and ontology analysis conducted on GFP (+) vs. Mock and heatmaps displaying up- and downregulated genes (A) Gene Ontology analysis of the top 3,000 differentially expressed genes found from DESEQ2 analysis in R of GFP (+) vs. Mock using cutoffs of fold change ± 3 and adj p value < 0.01. Terms listed were the five most significant across categories molecular function, biological process, and cellular components (for complete list see Table S5). (B) Heatmaps showing 20 representative genes within the ontology classifications from (A); the values in the heatmap are the average raw read count number across GFP (+) or Mock samples. (C) Most significantly impacted canonical pathways as determined by Ingenuity Pathway Analysis using the same gene list as in (A, see Table S6 for complete list). (D) Heatmaps containing 20 of the most significantly upregulated genes in GFP (+) and GFP (Low) groups vs. Mock determined by adj p value and fold change. For ontology and IPA, significance is displayed as -log (adj p-value) using cutoffs stated above. Bolded genes within the heatmaps identify those used in subsequent Shield-1 experiments. Also see Figures S3, S4A–S4C, and S5 along with Tables S5 and S6.

Figure 5

Infection of NPCs with HCMV strain TB40r mGFP-IE-FKBP virus results in limited expression of viral proteins IE1 and IE2 (UL122/UL123) unless Shield-1 is administered NPCs were infected at 3 or 7 days post plate down with TB40r mGFP-IE-FKBP at an MOI of 1. One group was then supplemented with 1 μM Shield-1 daily (Shield-1 +) while the other was given vehicle (Shield-1 -). (A–D) Expression of HCMV viral genes (UL122 (IE), UL123 (IE), UL44 (E), and UL99 (L)) within NPCs infected 3- or 7- days post plate down at 24, 48, and 72 hours post infection (hpi) as determined by qPCR. A significant upregulation of viral transcripts was observed in (Shield-1 +) conditions compared to (Shield-1 -). Stars were assigned based on level of significance as determined by one-way ANOVA with Tukey post hoc test: ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.00001. (E) Immunocytochemical images at 24, 48, and 72 hpi from NPCs infected 3 days post plate down with HCMV-IE1/IE2-ddFKBP and probed for GFP (green), neurodevelopmental transcription factor SOX2 (red), UL123 (IE1) (purple), Hoescht (blue), and merge. All images taken at 40X using Zeiss LSM980 and scale bar is 20 μm. qPCR data from 3 biological replicate experiments and error bars represent mean ± SEM.

Figure 6

NPCs infected with TB40r mGFP-IE-FKBP and administered Shield-1 show positive staining for viral tegument proteins pp65 and pp71 by 24 hpi NPCs were infected at 3 dpi with TB40r mGFP-IE-FKBP at an MOI of 1. One group was then supplemented with 1 μM Shield-1 daily (Shield-1 +) while the other was given vehicle (Shield-1 –), cells were fixed at noted times post infection for staining. (A) Immunocytochemical images from the Shield-1 (−) group at 24, 48, and 72 hpi for viral tegument proteins pp65 or pp71 (purple), neurodevelopmental transcription factor HES1 (red), GFP (green), Hoechst (blue), and merge. (B) Immunohistochemical images from the Shield (+) group using the same staining targets as (A). All images taken at 40X using Zeiss LSM980 with scale bar of 20 uM.

Figure 7

Cellular gene targets are downregulated within NPCs infected with TB40r mGFP-IE-FKBP with varying dependence on Shield-1 administration qPCRs were performed for several key neurodevelopmental transcription factors (FezF2, FOXG1, DMRTA2, and EMX1) and signaling, cell-cell communication, and junctional genes (CACNA1C, CACNA1G, CAMKV, KCNF1, and GJA1) within the HCMV-IE1/IE2-ddFKBP-infected (Shield +) and (Shield –) groups plus uninfected (Mock) NPCs at 3 days post plate down. (A) No robust or consistent effect of Shield administration was observed for these targets; instead, genes were downregulated regardless of administration at all time points. (B) Downregulation of KCNF1, CACNA1C, CAMKV, CACNA1G, and GJA1was observed regardless of Shield administration; however, trends toward an effect of Shield-1 administration can be observed for CACNA1C at 48 hpi or CAMKV at 72 hpi. A significant Shield-1-dependent effect was noted for genes CACNA1G and GJA1 at both 48 and 72 hpi. Stars were assigned based on level of significance as determined by one-way ANOVA with Tukey post hoc test: ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.00001. Data from 3 biological replicate experiments and error bars represent mean ± SEM.