IFN alpha inducible protein 27 (IFI27) acts as a positive regulator of PACT-dependent PKR activation after RNA virus infections

TL;DR

IFI27 enhances PKR activity during RNA virus infections, which could lead to new antiviral treatments.

Contribution

The first identification of IFI27 as a positive regulator of PACT-dependent PKR activation.

Findings

IFI27 interacts with PACT and PKR, enhancing PKR activity and reducing protein translation.

IFI27 increases PKR function in cells infected with SARS-CoV-2 and VSV.

IFI27 promotes stress granule formation, correlating with PKR activation.

Abstract

Protein kinase R (PKR) expression is induced by interferons. This protein is activated by double-stranded (ds) RNAs or RNAs containing duplex regions, produced after different stimuli, such as after viral infections, leading to the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), and subsequently inhibiting cellular and viral protein translation. This function may lead to different effects such as to impairing the replication of RNA viruses by inhibiting viral protein translation, and to modulating the innate immune responses after viral infections by affecting the translation of effector proteins. In this work, we identify, for the first time, an interaction of IFN alpha inducible protein 27 (IFI27) with PKR-activating protein (PACT or PRKRA) and with PKR, showing that the interaction of IFI27 with PACT is likely mediated by dsRNAs or RNAs containing duplex regions, and that the interaction of IFI27 with PKR is PACT-dependent. Interestingly, using IFI27 knocked-down, knocked-out and overexpressing tumour-derived, established cells, we show that these interactions trigger a potentiation of the activity of PKR and, therefore, a decrease in protein translation. Moreover, we find that IFI27 increases PKR function in cells infected with different RNA viruses such as Severe Acute Respiratory virus 2 (SARS-CoV-2), and Vesicular Stomatitis virus (VSV), and in cells transfected with the dsRNA analog poly(I:C), suggesting a broad effect of IFI27 on PKR activation. Moreover, we show that IFI27 expression increases the formation of stress granules (SGs) at the cell cytoplasm, correlating with the increased PKR activation mediated by IFI27, as it has been shown that the translational arrest induced by activated PKR leads to the formation of SGs. Mechanistically, we describe that this ability of IFI27 to activate PKR is dependent on its interaction with PACT. Further understanding of the regulation of PKR activity will allow us to develop new antiviral drugs to modulate this signalling axis, which is crucial in RNA virus infections.

Protein kinase R (PKR) is a protein whose expression is induced after innate immune responses are triggered. Its activation involves the binding to double-stranded (ds)RNAs or RNAs containing duplex regions, produced after different stimuli, such as viral infections. This PKR activation leads to eIF2α phosphorylation, and subsequent inhibition of protein translation, that may lead to impairing viral replication, and to modulating host innate immune responses. In this manuscript, we identify a completely novel interaction of IFI27 with PACT and with PKR. The interaction of IFI27 with PACT is likely mediated by RNAs, and the interaction of IFI27 with PKR depends on PACT. Importantly, these interactions increase PKR activity and, therefore, decrease protein translation after the infection with different viruses. IFI27 increases PKR function dependent on its interaction with PACT, in cells infected with Severe Acute Respiratory virus 2 (SARS-CoV-2), and Vesicular Stomatitis virus (VSV), and in cells transfected with poly(I:C), suggesting that the effect of IFI27 on PKR activity is broad. Further understanding of the regulation of PKR activity will allow us to develop new molecules to modulate PKR activity.

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