Activation and modulation of Ire1-Xbp1 pathway in SARS-CoV-2 infected Vero cells (B.1.1.7 Alpha, B.1.617.2 Delta, B.1.1.529 Omicron variants)

Abstract

BACKGROUND: The emergence of new variants of the SARS-CoV-2 virus at the end of 2020, which became a source of increased risk for global public health, prompted the study of their molecular biological characteristics and pathogenic effects. It is known that one of the reasons for the viruses’ pathogenic action is their interaction with the defense mechanisms of the cell. Ire1 (inositol-requiring enzyme 1)-mediated splicing of Xbp1 mRNA (X-box binding protein 1) is a protective mechanism that is activated in response to the accumulation of misfolded proteins in the cell, a situation that occurs due to uncontrolled synthesis of viral proteins during infection. Studying the interaction of different variants of the SARS-CoV-2 virus with this protective mechanism will help to shed light on various aspects of the pathogenesis of a new coronavirus infection.

AIM: Study of Ire1-Xbp1 defense mechanism activation and modulation in SARS-CoV-2 infected Vero cells.

MATERIALS AND METHODS: We studied the activation of the Ire1 enzyme in Vero cells infected with various variants of the SARS-CoV-2 virus using immunoblotting and antibodies to various forms of this protein in the cell. In addition, we studied the activation of Xbp1 mRNA splicing under conditions of infection with various variants of the SARS-CoV-2 virus in a PCR reaction with specific primers.

RESULTS: Reproduction of B.1.1.529 (Omicron) strain in Vero cells is slower than B.1.1.7 (Alpha) and B.1.617.2 (Delta) strains of SARS-CoV-2. The whole reproduction cycle is 48 hours.

Ire1-dependent defense mechanism is activated after 12 hours of SARS-CoV-2 infection with either of three variants. However, despite the activation of the Ire1 endonuclease domain, there is no Xbp1 mRNA splicing in SARS-CoV-2 infected cells. Inhibition of Xbp1 mRNA splicing occurs slower in Vero cells infected with the B.1.1.529 — Omicron variant.

CONCLUSIONS: The paper describes the reproduction of various variants of the SARS-CoV-2 virus in Vero cell culture and the activation of the Ire1-Xbp1 defense mechanism during infection. The Ire1 endonuclease is phosphorylated, however, mRNA splicing of the Xbp1 transcription factor is impaired in SARS-CoV-2 infected cells. A decrease in the rate of inhibition of this protective mechanism in Vero cells infected with the Omicron (B.1.1.529) variant of the SARS-CoV-2 virus may be the reason for its lower pathogenicity described in various studies.

Keywords

SARS-CoV-2 / endoplasmic reticulum stress / Ire1 / Xbp1

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