BREAKING! COVID-19 News: Weil Cornell Scientists Discover That CIART Is A Key Regulator of SARS-CoV-2 Infection!

COVID-19 News: A new study led by scientists from Weill Cornell Medicine, New York-USA had identified CIART, the circadian-associated repressor of transcription as the key regulator of SARS-CoV-2 infection.


 
The study also involved researchers from New York University-USA and Albert Einstein College of Medicine, Bronx, New York, USA
 
Using organoid models, the study team systematically examined changes in transcript profiles caused by SARS-CoV-2 infection at different multiplicities of infection for lung airway organoids, lung alveolar organoids and cardiomyocytes, and identified several genes that are generally implicated in controlling SARS-CoV-2 infection, including CIART, the circadian-associated repressor of transcription.
 
Interestingly, it was found that lung airway organoids, lung alveolar organoids and cardiomyocytes derived from isogenic CIART−/− human pluripotent stem cells were significantly resistant to SARS-CoV-2 infection, independently of viral entry.
 
Subsequent single-cell RNA-sequencing analysis further validated the decreased levels of SARS-CoV-2 infection in ciliated-like cells of lung airway organoids.
 
Importantly, CUT&RUN, ATAC-seq and RNA-sequencing analyses revealed that CIART controls SARS-CoV-2 infection at least in part through the regulation of NR4A1, a gene also identified from the multi-organoid analysis.
 
Transcriptional profiling and pharmacological inhibition led to the discovery that the Retinoid X Receptor pathway regulates SARS-CoV-2 infection downstream of CIART and NR4A1.
 
This breakthrough study findings provides potential therapeutic targets for protection against COVID-19 across organ systems.
 
The study findings were published in the peer reviewed journal: Nature Cell Biology.
https://www.nature.com/articles/s41556-023-01095-y
 
A broad spectrum of human cells can be infected by SARS-CoV-2, encompassing cells in lung alveoli, lung airways, the small intestine and colon, the brain, choroid plexus, heart, liver, pancreas, kidneys, blood vessels, and tonsil organoids. While progress has been made in understanding cellular tropism and host reactions, some crucial aspects of infection regulation are still unclear. Specifically, it has yet to be determined if there are common factors that can be targeted to inhibit or counteract SARS-CoV-2 infection across different tissue types, which would be highly beneficial in addressing the multi-systemic effects of the disease. To explore this question, we conducted a comprehensive examination of the transcriptional alterations resulting from SARS-CoV-2 infection in various unique hPSC-derived cell types and organoids.
 
The study team discovered that the nuclear transcription factor CIART plays a crucial role in the regulation of SARS-CoV-2 infection. CIART, also referred to as CHRONO, C1orf51, and GM129, was initially recognized for its function in controlling a circadian-clock feedback loop. While CIART has not been linked to any human illnesses, the influence of host cell circadian rhythm on viral replication is an emerging area of interest.
 
A recent investigation utilizing the Calu-3 lung cancer cell line indicated that the suppression of Bmal1 hinders SARS-CoV-2 entry.
https://pubmed.ncbi.nlm.nih.gov/34545347/
 
The study team performed a comprehensive analysis of the biological function and downstream mechanism of CIART regulation in relation to SARS-CoV-2 infection.
 
Lung organoids comprise various cell types; hence, single-cell RNA sequencing (scRNA-seq) was employed to investigate SARS-CoV-2 infection in specific lineages of hPSC-AWOs. Both WT and CIART-null AWOs were exposed to SARS-CoV-2 (multiplicity of infection = 0.1) and dissociated into individual cells for scRNA-seq analysis at 24 hours post-infection.
 
In line with earlier findings, the proportion of viral reads in infected CIART-deficient AWOs was considerably reduced compared to WT AWOs. The study team identified five distinct clusters, including basal cells (cluster 0), ciliated-like cells (clusters 1 and 2), proliferating basal cells (cluster 3), and neuroendocrine cells (cluster 4).
 
The analysis of signature genes' correlation further confirmed the identity of the hPSC-derived ciliated-like cell group, demonstrating a high resemblance to mature human ciliated cells.
 
The study team then assessed the proportion of unique viral molecular identifier (UMI) counts in every cluster of WT and CIART-null AWOs after SARS-CoV-2 infection. The majority of viral UMIs were observed in the ciliated-like cell cluster 1. The percentage of viral UMIs in each cluster of CIART-deficient AWOs was notably reduced compared to their corresponding WT AWO groups.
 
The study team further examined individual viral transcripts in ciliated-like cells and found that all viral transcripts were significantly decreased in CIART−/− ciliated-like cells.
 
Collectively, these data further confirm that loss of CIART significantly impairs SARS-CoV-2 infection.
 
The study team also performed cleavage under targets and release using nuclease (CUT&RUN) chromatin profiling, assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA-seq to identify downstream targets and signaling pathways regulated by CIART in hPSC-CMs.
 
The researchers identified 6,379 peaks using CUT&RUN, most of which were close to the transcription start site. More than 50% of the identified peaks were located in proximal promoter regions. The ATAC-seq peaks derived from WT and CIART−/− hPSC-CMs clustered separately with 12,529 sites gained and 564 sites lost in CIART−/− hPSC-CMs compared with WT hPSC-CMs.
 
 From the RNA-seq data, clustering and principal component analyses (PCA), the study team confirmed that WT and CIART−/− hPSC-CMs are distinguishable at the transcript level.
 
When the study team combined the CUT&RUN, ATAC-seq and RNA-seq assays, 671 peaks (associated with 560 genes) that are bound by CIART in WT hPSC-CMs and are significantly changed between WT and CIART−/− hPSC-CMs at both transcriptional and chromatin accessibility levels were identified.
 
Significantly, one of these genes, NR4A1, was also identified along with CIART as an upregulated hit gene following SARS-CoV-2 infection in the initial hPSC-derived multi-organoid platform.


 a, Schematic of the experimental design. b, Levels of subgenomic viral transcripts, determined by qRT-PCR, in hPSC-derived AWOs, ALOs and CMs at 48 h.p.i. with SARS-CoV-2 at different m.o.i. (m.o.i. = 0.01, 0.10 and 1.00). The dashed red line indicates the detection limit. c, Three-dimensional analysis of transcriptional changes in hPSC-derived AWOs, ALOs and CMs infected at 48 h.p.i. (m.o.i. = 0.01, 0.10 and 1.00). The genes that were significantly changed (log2(fold change) > 0.75, base mean > 10 and adjusted P < 0.05) in each condition are highlighted in purple. d, Heatmap of the protein-coding genes that were increased for at least seven of nine conditions in hPSC-derived AWOs, ALOs and CMs at 48 h.p.i. (m.o.i. = 0.01, 0.10 and 1.00). c,d, Data are presented as an integration of all biological replicates. e,f, Representative confocal images (e) and the calculated percentage of SARS-N+ cells in the cTnT+ subpopulation (f) of hPSC-CMs infected with lentivirus carrying Cas9 and sgRNAs targeting hit genes. P values were calculated using an unpaired two-tailed Student’s t-test. The red text and red bar highlight that knockout of CIART showed the greatest resistance to SARS-CoV-2 infection. b,e,f, Data are the mean ± s.d. b,c–f, n = 3 independent biological replicates.
 
The peaks associated with NR4A1 in the CUT&RUN assay) were correlated in the ATAC-seq data and significantly changed in CIART−/− hPSC-CMs compared with WT hPSC-CMs. Interestingly, some ATAC-seq peaks were enhanced in the mutant cells, suggesting an altered chromatin structure with loss of CIART binding.
 
Finally, qRT-PCR assays confirmed that the transcript levels of NR4A1 were significantly decreased in both mock- and SARS-CoV-2-infected CIART−/− hPSC-CMs, consistent with the RNA-seq data.
 
In order to determine whether NR4A1 impacts SARS-CoV-2 infection, hPSC-derived CMs, AWOs and ALOs were infected with lentivirus expressing Cas9 and one of two different sgRNAs targeting NR4A1. CMs, AWOs and ALOs expressing sgRNA targeting NR4A1 (sgNR4A1) or scrambled sgRNA control were infected with SARS-CoV-2 (m.o.i. = 0.1). At 24 h.p.i., both sub-genomic viral RNAs and the percentage of SARS-N+ cells in cTnT+ CMs, FOXJ1+ cells in AWOs and SP-B+SP-C+ cells in ALOs were significantly decreased in cells expressing sgNR4A1 compared with those expressing scramble sgRNA.
 
Ingenuity pathway analysis highlighted Retinoid X receptor (RXR) signaling pathways in CIART−/− hPSC-AWOs, CIART−/− hPSC-ALOs and CIART−/− hPSC-CMs.
 
Heatmaps showed the downregulation of RXR pathway-associated genes in CIART−/− hPSC-AWOs, hPSC-ALOs and hPSC-CMs.
 
Furthermore, RXR pathway-associated genes were also downregulated following SARS-CoV-2 infection in CIART−/− hPSC-AWOs, hPSC-ALOs and hPSC-CMs.
 
Past studies reported that NR4A1 could heterodimerize with RXR and increase the potential of RXR to modulate gene expression.
https://pubmed.ncbi.nlm.nih.gov/7705655/
 
The downregulation of RXR pathway-associated genes in hPSC-AWOs, hPSC-ALOs and hPSC-CMs expressing sgNR4A1 was confirmed using qRT-PCR assays.
 
Lastly, hPSC-AWOs, hPSC-ALOs and hPSC-CMs were treated with RXR inhibitors, followed by SARS-CoV-2 infection. Treatment with the RXR inhibitors HX531 and PA452 blocked SARS-CoV-2 infection in hPSC-CMs, hPSC-AWOs and hPSC-ALOs at 24 h.p.i., as determined using qRT-PCR and immunostaining assays.
 
Together, these data confirm that inhibition of the RXR pathway suppresses SARS-CoV-2 infection.
 
The study also found that several fatty acids, including palmitic acid, stearic acid, 11-eicosenoic acid, arachidic acid and myristic acid, were decreased in CIART−/− and WT + HX531 hPSC-AWOs compared with the control WT without treatment.
 
Past studies reported that inhibition of fatty-acid synthesis could suppress SARS-CoV-2 infection.
https://pubmed.ncbi.nlm.nih.gov/34731648/
 
https://pubmed.ncbi.nlm.nih.gov/34580494/
 
Collectively, these data suggest that loss of CIART decreases fatty-acid synthesis, which results in diminished SARS-CoV-2 infection.
 
The study team told COVID-19 News reported at TMN, “We found that CIART regulates SARS-CoV-2 infection through an entry-independent mechanism. Loss of CIART blocks SARS-CoV-2 infection by downregulating the RXR pathway, at least in part through NR4A1, leading to decreased fatty-acid synthesis, thereby impairing viral infection.”
 
Interestingly, this study highlights the potential role of the host-cell circadian rhythm on viral infection. Interestingly, studies have been performed to develop RXR modulators as drug candidates, and in that sense, this study could aid in the advancement of strategies for the development of antiviral drugs.
https://pubmed.ncbi.nlm.nih.gov/31298602/
 
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