Insights on interferon-independent induction of interferon-stimulated genes shaping the lung's response in early SARS-CoV-2 infection

Abstract

While mRNA vaccine efficacy against the 2019 coronavirus disease (COVID-19) outbreak remains high, research on antiviral innate immune responses in the early stages of infection is essential to develop strategies to prevent the dissemination of SARS-CoV-2. In this study, we investigated the induction of both interferon (IFN)-stimulated genes (ISGs) and IFN-independently upregulated ISGs following SARS-CoV-2 infection in Syrian golden hamsters. The viral titers were highest at 3 days post-infection (dpi). Over time, the viral titer gradually decreased while ISGs such as Mx1, Ifit2, Ifit3, Ifi44, and Rsad2 were markedly induced in the lung. The transcription of ISGs significantly increased from 2 dpi, and SARS-CoV-2-induced ISGs were maintained in the hamster lung until 7 dpi. The transcription of Ifnb and Ifng was minimally elevated, while Ifnl_2/3 was significantly induced in the lung at 5 days after SARS-CoV-2 infection. RNA sequencing results also showed that at 3 dpi, SARS-CoV-2 initiated the activation of ISGs, with lesser increases of Ifnl₂ and Ifnl₃ transcription. In addition, Ddx58 and cGAS, which encode factors for virus sensing, Stat1, Stat2, and IFN regulatory factor 7 and 9 mRNA levels were also induced at the initial stage of infection. Our data demonstrate that ISGs might be upregulated in the lung in response to SARS-CoV-2 during the early stages of infection, and the rapid induction of ISGs was not associated with the activation of IFNs. Elucidation of IFN-independent induction of ISGs could further our understanding of alternative defense mechanisms employed by the lungs against SARS-CoV-2 and provide more effective antiviral strategies for patients with severe COVID-19.

Keywords: Innate immune responses; Interferon-independent pathway; Interferon-stimulated genes; SARS-CoV-2.

Fig. 1

IFN- and ISG-related innate immune responses in the lung of CoV2+ hamsters. A. Scheme of the hamster model and the design of experiment for SARS-CoV-2 infection. Syrian golden hamsters were infected with SARS-CoV-2 (BetaCoV/Korea/SNU01/2020, 2.5 × 10⁵ TCID50/hamster) at the indicated time points (N = 3 at each indicated time point). B, C. Spike RNA levels in lung tissue (B) and BAL fluid (C) were determined at 0, 3, 7, 10, and 14 dpi. D. H&E-stained micrographs were generated from lung sections of hamsters at indicated time points of SARS-CoV-2 infection (scale bar, 100 μM). Immunohistochemical analysis of spike protein using DAB chromogen was performed in lung sections from hamsters prior to and following SARS-CoV-2 infection. Micrographs shown are representative of lung sections from three hamsters (original magnification x200). E. Differentially expressed genes (DEGs) were identified to further characterize the transcriptional changes in response to SARS-CoV-2. F. Scattered plots indicate DEGs correlated with “virus recognition” and “transcription factors of IFNs or ISGs.” Real-time PCR results were analyzed by the Mann–Whitney U test. Data are presented as mean ± SD from three independent experiments. *p < 0.05 vs. non-infected hamsters.

Fig. 2

Transcriptional alterations in the lung of CoV2+ hamsters. Syrian golden hamsters (N = 15) were infected with SARS-CoV-2 (BetaCoV/Korea/SNU01/2020, 2.5 × 10⁵ TCID50/hamster), and the lung lysate was prepared at 3 and 7 dpi (N = 3 at each indicated time point). A. Principal component analysis (PCA) was performed to reveal the similarities and dissimilarities of 3 groups (0, 3, and 7 dpi) regarding variations in DEGs. B. The GO terms in the lung of SARS-CoV-2 infection at 3 dpi. C. Volcano plot data were examined, and 490 DEGs were observed in the lungs of SARS-CoV-2-infected hamsters, especially at 3 dpi. D. The heatmap shows the top 30 significant DEGs in the lungs of hamsters following SARS-CoV-2 infection at 3 dpi. E. The GO terms in the lung of SARS-CoV-2 infection at 7 dpi. F. Volcano plot data were examined, and 2582 DEGs were observed in the lungs of SARS-CoV-2-infected hamsters, especially at 7 dpi. G. The heatmap shows the top 30 significant DEGs in the lungs of hamsters following SARS-CoV-2 infection at 7 dpi. H. The relative expression of genes associated with IFNs and IFN-specific receptors was examined in the lungs of CoV2+ hamsters, especially at 3 and 7 dpi.

Fig. 2

Transcriptional alterations in the lung of CoV2+ hamsters. Syrian golden hamsters (N = 15) were infected with SARS-CoV-2 (BetaCoV/Korea/SNU01/2020, 2.5 × 10⁵ TCID50/hamster), and the lung lysate was prepared at 3 and 7 dpi (N = 3 at each indicated time point). A. Principal component analysis (PCA) was performed to reveal the similarities and dissimilarities of 3 groups (0, 3, and 7 dpi) regarding variations in DEGs. B. The GO terms in the lung of SARS-CoV-2 infection at 3 dpi. C. Volcano plot data were examined, and 490 DEGs were observed in the lungs of SARS-CoV-2-infected hamsters, especially at 3 dpi. D. The heatmap shows the top 30 significant DEGs in the lungs of hamsters following SARS-CoV-2 infection at 3 dpi. E. The GO terms in the lung of SARS-CoV-2 infection at 7 dpi. F. Volcano plot data were examined, and 2582 DEGs were observed in the lungs of SARS-CoV-2-infected hamsters, especially at 7 dpi. G. The heatmap shows the top 30 significant DEGs in the lungs of hamsters following SARS-CoV-2 infection at 7 dpi. H. The relative expression of genes associated with IFNs and IFN-specific receptors was examined in the lungs of CoV2+ hamsters, especially at 3 and 7 dpi.

Fig. 3

Differential gene expression of IFNs and ISGs at early onset of SARS-CoV-2 infection A. Schematic of the hamster model and experimental design for SARS-CoV-2 infection. Syrian golden hamsters (N = 12) were infected with SARS-CoV-2, and the lung lysate was prepared at 0, 2, 5, and 7 dpi. B. Viral S gene RNA in lung tissue was determined at 0, 3, and 7 dpi. C, D. The mRNA levels of Ifnb1 (C) and Ifnl_2/3 (D) were determined in the lungs of SARS-CoV-2-infected hamsters at 0, 2, 5, and 7 dpi. E. The mRNA levels of Mx1, Ifit2, Ifit3, and Rsad2 were determined in the lungs of SARS-CoV-2-infected hamsters at 0, 2, 5, and 7 dpi. The real-time PCR results were analyzed by the Mann–Whitney U test and are presented as mean ± SD from three independent experiments. *p < 0.05 vs. non-infected hamsters.