Simultaneous Detection of RIG-1, MDA5, and IFIT-1 Expression Is a Convenient Tool for Evaluation of the Interferon-Mediated Response

Abstract

In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.

Evaluation of the antiviral effect of exogenous human IFN-lambda mRNA against influenza virus in vitro

“…To assess the antiviral activity, we used the MDCK (canine kidney) cell culture permissive against in uenza viruses. We have previously shown that human hIFN-λ1 is able to bind to its receptor and cause antiviral effect [10].…”

“…Real-time PCR assays were performed using the CFX96 Real-Time PCR System (Bio-Rad, USA). Multiplex qPCR [10] was performed in 25 µL nal reaction volumes containing 12.5 µL BioMaster HS-qPCR mix (2x) (BioLabMix, Russia) and 2 µL cDNA.…”

Exosomes Released by Influenza-Virus-Infected Cells Carry Factors Capable of Suppressing Immune Defense Genes in Naïve Cells

“…The fact that the key "marker" of viral infection MxA [64], as well as SOCS1 (important in regulation of cytokine signaling), did not change their expression in response to E or EV treatment, further supports an absence of influenza virions in the isolated exosome samples. There is a close relationship between the cytosolic RNA sensors RIG1, MDA5 and IFIT1, which forms a unique system of cellular protection from foreign RNAs [66]. Annexin A1 (AnxA1) is able to enhance the expression of RIG1 and type I IFN, but its activity The innate immune response requires IFN induction and activation of interferonstimulated genes (ISGs) [64].…”

“…There is a close relationship between the cytosolic RNA sensors RIG1, MDA5 and IFIT1, which forms a unique system of cellular protection from foreign RNAs [66]. Annexin A1 (AnxA1) is able to enhance the expression of RIG1 and type I IFN, but its activity is associated with apoptosis during influenza infection.…”