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 posted to the bioRxiv* preprint server, Canadian researchers assessed the impact of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on interferon (IFN) response.

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

Study: SARS-CoV-2 Type I Interferon Modulation by Nonstructural Proteins 1 and 2† Image Credit: NIAID

About the study

In the present study, researchers characterized the response of type I IFN during SARS-CoV-2 infection and the immune evasion mechanisms employed 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. Furthermore, the expression of genes involved in the nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), toll-Like receptors (TLRs), and retinoic acid-inducible gene (RIG)-like receptors (RLRs ) signaling pathways were estimated. The team also monitored the expression of genes associated with the type 1 IFN signaling during infection.

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

Results

The study results showed that the copy number of the SARS-CoV-2 envelope (E) gene or genomic RNA was almost three times higher in the SARS-CoV-2 Beta-infected mice as opposed to the Wuhan-infected mice. In addition, the viral RNA loads in the Delta-infected mice were higher than in the Wuhan-infected mice. Similar viral loads were also observed in lung homogenates derived from the Wuhan, Beta and Delta infected mouse groups.

Cytokine mRNA and protein expression profile after infection of K18-ACE2 mice with Wuhan, Beta and Delta strains.  Infected or mock lung tissues from mice were collected three days after infection (n=4/group).  A) The copy number of the SARS-CoV-2 E gene was evaluated 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 in TCID50/mL.  (CD) Gene expression was evaluated by RT-qPCR and cytokine concentration in lung homogenates determined using a 13-plex Luminex panel.  Cytokine gene expression and concentration levels are presented as heatmaps with results expressed as fold (log2) relative to mock infected mice.  Statistical analyzes were performed by comparing 2 (- Ct) values ​​for each gene in the control and infected groups by a non-parametric t-test and only data with an p value less than 0.05 are shown.  (E) Absolute cytokine concentrations in lung homogenates.  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 and infected groups using 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. Infected or mock lung tissues from mice were collected three days after infection (n=4/group). A) The copy number of the SARS-CoV-2 E gene was evaluated 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 in TCID50/ml† (CD) Gene expression was evaluated by RT-qPCR and cytokine concentration in lung homogenates determined using a 13-plex Luminex panel. Cytokine gene expression and concentration levels are presented as heatmaps with results expressed as fold (log2) versus mock-infected mice. Statistical analyzes were performed by 2 . comparable(- Ct) values ​​for each gene in the control group and infected groups with a non-parametric t-test and only data with an p value less than 0.05 are shown. (E) Absolute cytokine concentrations in lung homogenates. 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 and infected groups using a non-parametric t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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

In addition, the production of chemokines was 1,000 to 2,000 times higher compared to 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 the production of chemokines.

Antiviral response gene expression after infection with Wuhan, Beta and Delta strains.  Heatmap representation of cytokines and inflammation related genes (A) and Type I IFN production and signaling related genes (B).  Results are expressed as fold (log2) relative to mock infected mice.  For gene expression, statistical analyzes were performed by comparing 2 (- Ct) values ​​for each gene in the control and infected groups by a non-parametric t-test, and only data with p-values ​​less than 0.05 were shown.  For protein expression, statistical analyzes were performed by comparing the normalized concentration of each cytokine in the sham infected groups with infected groups using a non-parametric t-test.  *P<0.05, **P<0.01, ***P<0.001, ****P<0,0001.

Antiviral response gene expression after infection with Wuhan, Beta and Delta strains. Heatmap representation of cytokines and inflammation related genes (A) and Type I IFN production and signaling related genes (B). Results are expressed as fold (log2) versus mock-infected mice. For gene expression, statistical analyzes were performed by 2. comparable(- Ct) values ​​for each gene in the control group and infected groups with a non-parametric t-test, and only data with p-values ​​less than 0.05 were shown. For protein expression, statistical analyzes were performed by comparing the normalized concentration of each cytokine in the sham infected groups with infected groups using a non-parametric 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 implied the significant upregulation of the Mediterranean fever gene (Mefv). However, inflammasome components, including apoptosis-associated speckle-like protein containing a CARD (Pycard), are missing in melanoma 256 2 (Aim2), proline-serine-threonine phosphatase interaction protein 1 (Pspip1), NLR family pyrine domain with 3 (Nlrp3) and Caspase 1 (Casp1) were not modulated in the initial stages of infection. In addition, mitogen-activated protein kinase 14 (Mapk14) and Caspase recruitment domain-containing protein 9 (Card9) were downregulated in response to SARS-CoV-2 infection.

Several downstream effector genes, including inhibitor of nuclear factor kappa-B kinase subunit beta (Ikbkb), transcription factor, interleukin-1 receptor-associated kinase 1 (Iraq1), 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 the transcription of the IFN-beta 1 gene. However, the team found no production of IFN-beta 1 protein in the supernatant of the SARS-CoV-2 infected cells. In addition, SARS-CoV-2 infected cells effectively produced a limited amount of the IFN-beta 1 protein, suggesting the influence 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-sensitive response element (ISRE) promoters and also enhanced the response to IFN-alpha and SeV.

The team also noted that nsp1 and nsp2 did not affect the transcription of IFN-beta 1, interferon-stimulated gene 15 (ISG15) and ISG56. In contrast, nsp1 significantly inhibited the production of IFN-beta 1, while the presence of nsp2 partially reduced this inhibition. Overall, nsp-1 reduced the IFN response by inhibiting mRNA translation.

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

*Important announcement

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

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