Study: SARS-CoV-2 type I Interferon modulation by nonstructural proteins 1 and 2. Image Credit: NIAID

Scientists characterize type I IFN response during SARS-CoV-2 infection

In a recent study published on bioRxiv* preprint server, Canadian researchers assessed the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on interferon (IFN) response.

Although several studies have focused on the mechanism of immune evasion by SARS-CoV-2, further research is still needed to understand how the virus modulates type I interferon (IFN) release.

Study: SARS-CoV-2 type I Modulation of interferon by non-structural proteins 1 and 2. Image credit: NIAID

About the study

In the current study, researchers characterized the type I IFN response during SARS-CoV-2 infection and the immune evasion mechanisms used by the virus.

The team studied the innate immune response against the SARS-CoV-2 Wuhan strain and the SARS-CoV-2 Beta and Delta variants by infecting K18-human angiotensin-converting enzyme 2 (hACE2) mice with a lethal dose of SARS -CoV- 2. In addition, the expression of genes involved in nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), toll-like receptors (TLRs), and inducible gene-like receptors by retinoic acid (RIG) (RLR) signaling pathways were estimated. The team also monitored the expression of genes associated with IFN type 1 signaling during infection.

Additionally, the researchers infected A549-hACE2 with the Wuhan strain, followed by immunofluorescence visualization using anti-SARS-CoV-2 nucleocapsid (N) antibodies. Subsequently, IFN-beta messenger ribonucleic acid (mRNA) was quantified using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The team also explored the behavior of nonstructural protein 1 (nsp1) in the absence of nsp2 by transfecting nsp1 and nsp2-associated expression vectors into HEK293T cells.

Results

The study results showed that the number of copies of the SARS-CoV-2 envelope (E) gene or genomic RNA was almost three times higher in mice infected with SARS-CoV- 2 Beta than in those infected from Wuhan. Additionally, viral RNA loads in Delta-infected mice were higher than in Wuhan-infected mice. Similar viral loads were also observed among lung homogenates derived from groups of mice infected with Wuhan, Beta and Delta.

Cytokine mRNA and protein expression profile after infection of K18-ACE2 mice with Wuhan, Beta and Delta strains.  Lung tissue from infected or sham mice was collected three days after infection (n = 4/group).  A) SARS-CoV-2 E gene copy number was assessed by ddPCR using lung RNA and expressed as copy number per 100 copies of Rpp30 mRNA.  (B) Infectious viral titers were determined in lung homogenates and expressed as TCID50/mL.  (CD) Gene expression was assessed by RT-qPCR and cytokine concentration in lung homogenates determined using a Luminex 13-plex panel.  Cytokine gene expression and concentration levels are shown as heat maps with results expressed as fold (log2) relative to mock-infected mice.  Statistical analyzes were performed by comparing the 2(-ΔCt) values ​​for each gene in the control group and the infected groups with a nonparametric T-test and only data with a p-value less than 0.05 are presented.  (E) Absolute cytokine concentrations in lung homogenates.  The results are expressed as mean +/- SD (n=4 mice/group).  For protein quantification, statistical analyzes were performed by comparing the normalized concentration for each cytokine in the control group and the infected groups with a non-parametric T-test.  *P<0.05, **P<0.01, ***P<0.001, ****P<0,0001.

Cytokine mRNA and protein expression profile after infection of K18-ACE2 mice with Wuhan, Beta and Delta strains. Lung tissue from infected or sham mice was collected three days after infection (n = 4/group). A) SARS-CoV-2 E gene copy number was assessed by ddPCR using lung RNA and expressed as copy number per 100 copies of Rpp30 mRNA. (B) Infectious viral titers were determined in lung homogenates and expressed as TCID50/ml. (CD) Gene expression was assessed by RT-qPCR and cytokine concentration in lung homogenates determined using a Luminex 13-plex panel. Cytokine gene expression and concentration levels are shown as heat maps with results expressed as fold (log2) compared to mock-infected mice. Statistical analyzes were made by comparing 2(-∆Ct) values ​​for each gene in the control group and the groups infected with a non-parametric T test and only data with a p-value less than 0.05 are shown. (E) Absolute cytokine concentrations in lung homogenates. The results are expressed as mean +/- SD (n=4 mice/group). For protein quantification, statistical analyzes were performed by comparing the normalized concentration for each cytokine in the control group and the infected groups with a non-parametric T-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

The major cytokine genes that were upregulated by all viral variants were CXC chemokine ligand 9 (Cxcl9), Cxcl10, Cxcl11, CC chemokine ligand 2 (Ccl2), interleukin 6 (IL -6) and IFNb1. On the other hand, IL11a and IL18 were down-regulated. Additionally, the team noted that CC and CXC chemokines were sufficiently induced after viral infection. Additionally, the Wuhan strain showed high induction of gene expression leading to IFN-alpha release, while the beta and delta variants showed no such induction.

Additionally, the production of chemokines was 1000-2000 times higher than that of pro-inflammatory cytokines and type I IFNs. Overall, this showed that the innate immune response in mice against SARS-CoV -2 was driven by chemokine production.

Gene expression of the antiviral response after infection with Wuhan, Beta and Delta strains.  Heatmap representation of cytokines and genes related to inflammation (A) and genes related to type I IFN production and signaling (B).  The results are expressed in times (log2) relative to falsely infected mice.  For gene expression, statistical analyzes were performed by comparing 2(-ΔCt) values ​​for each gene from control group and infected groups with a non-parametric T-test, and only data with p-values ​​less than 0, 05 were presented.  For protein expression, statistical analyzes were performed by comparing the normalized concentration of each cytokine in the mock infected groups with groups infected with a nonparametric T-test.  *P<0.05, **P<0.01, ***P<0.001, ****P<0,0001.

Gene expression of the antiviral response after infection with Wuhan, Beta and Delta strains. Heatmap representation of cytokines and genes related to inflammation (A) and genes related to type I IFN production and signaling (B). The results are expressed in times (log2) compared to mock-infected mice. For gene expression, statistical analyzes were made by comparing 2(-∆Ct) values ​​for each gene in the control group and the groups infected with a nonparametric T-test, and only data with p-values ​​less than 0.05 were presented. For protein expression, statistical analyzes were performed by comparing the normalized concentration of each cytokine in the mock infected groups with groups infected with a nonparametric T-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

The team noted that the formation of inflammasomes and the release of pro-inflammatory cytokines involved a significant upregulation of the Mediterranean fever gene (Mefv). However, components of the inflammasome, including the apoptosis-associated speck-like protein containing a CARD (Pycard), absent in melanoma 256 2 (Aim2), proline-serine-threonine phosphatase-interacting protein 1 (Pspip1), NLR-family pyrin domain containing 3 (Nlrp3), and caspase 1 (Casp1) were not modulated in the phases initials of infection. Additionally, mitogen-activated protein kinase 14 (Mapk14) and Caspase recruitment domain-containing protein 9 (Card9) were down-regulated in response to SARS-CoV-2 infection.

Several downstream effector genes, including nuclear factor kappa-B kinase (Ikbkb) beta-subunit inhibitor, transcription factor, interleukin-1 receptor-associated kinase 1 (Irak1), and Mitogen-activated protein kinase kinase kinase 7 (Map3k7) were down-regulated during SARS-CoV-2 infection.

SARS-CoV-2 infection of A549-hACE2 showed that SARS-CoV-2 efficiently stimulated IFN-beta 1 gene transcription. However, the team found no IFN- beta 1 in the supernatant of SARS-CoV-2 infected cells. Moreover, SARS-CoV-2 infected cells actually produced a limited amount of the IFN-beta 1 protein, suggesting the impact of viral factors on mRNA translation. The team also noted that nsp1 significantly inhibited Sendai virus (SeV)-induced activation of the IFN-beta 1 promoter. On the other hand, nsp2 expression stimulated IFN-beta 1 as well as interferon responsive response element (ISRE) promoters and also amplified its response to IFN-alpha and SeV.

The team also observed that nsp1 and nsp2 had no impact on the transcription of IFN-beta 1, interferon-stimulated gene 15 (ISG15) and ISG56. In contrast, nsp1 markedly inhibited IFN-beta 1 production, while the presence of nsp2 partially reduced this inhibition. Altogether, nsp-1 decreased the IFN response by inhibiting mRNA translation.

Overall, the study results showed that SARS-CoV-2 stimulated vigorous expression of inflammatory and antiviral genes. The researchers believe that nsp2 could be a potential target for future therapeutic approaches against SARS-CoV-2 pathogenesis.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

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