Unfolded protein response

The unfolded protein response (UPR) is a cellular coping mechanism to manage and negate the effects of perturbations of the ER. Increased secretion of proteins, misfolded proteins, nutrient starvation and changes in calcium homeostasis can cause stress for the ER (Hetz and Papa, 2018; Schröder and Kaufman, 2005). Viruses hijack the cellular translation machinery during replication, thus disturb ER homeostasis and cause stress for the ER (Diehl et al., 2011; Smith, 2014). The activation of the UPR results in increased synthesis of chaperones and foldases, inhibition of mRNA translation, an enlarged ER to dilute the load of unfolded proteins and the clearance of misfolded proteins through ER-assisted protein degradation (ERAD). In cases where the UPR is unable to counteract ER stress and re-establish the normal function of the ER, apoptosis is triggered (Schröder and Kaufman, 2005; Walter and Ron, 2011).

1.3.1 Unfolded protein response pathways

Three initiation molecules of the UPR are anchored in the ER membrane: activating transcription factor 6 (ATF6), PKR-like endoplasmic reticulum kinase (PERK) and inositol requiring kinase 1 (IRE1). The binding immunoglobulin protein (BiP, also known as GRP-78) is inhibiting the activation of IRE1, PERK and ATF6 by directly binding to them. During ER stress BiP dissociates from these ER stress sensors, which results in their activation. Other reports suggest, that IRE1 and PERK can be activated by directly binding to misfolded proteins (Hillary and FitzGerald, 2018; Preissler and Ron, 2019; Wang et al., 2018).

The activation of ATF6 results in the translocation of the transcription factor from the ER to the Golgi apparatus, where two site-specific proteases, site-1 protease (S1P) and site-2

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protease (S2P), cleave of the luminal domain and the transmembrane anchor. The emerging fragment of ATF6 (ATF6f) induces multiple UPR genes related to protein folding (Figure 4) (Haze et al., 1999; Lee et al., 2002; Walter and Ron, 2011; Yamamoto et al., 2007).

The activation of PERK leads to dimerization and autophosphorylation. Activated PERK phosphorylates eukaryotic translation initiation factor 2α (eIF2α), resulting in inhibition of general protein translation. However, the selective translation of certain mRNAs is still possible, including activating transcription factor 4 (ATF4). The upregulation of ATF4 translation results in an enhanced folding capacity of the ER and protection against oxidative stress (Hetz and Papa, 2018; Smith, 2014). Furthermore, ATF4 activates the pro-apoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) and growth arrest and DNA damage-inducible protein 34 (GADD34) (Figure 4) (Novoa et al., 2003; Oyadomari and Mori, 2004; Zinszner et al., 1998).

IRE1 shows kinase and endonuclease activity. The latter cleaves 26 nt from the X-box binding protein 1 (XBP1), resulting in the removal of an intron (Calfon et al., 2002; Hetz et al., 2011; Yoshida et al., 2001). The spliced mRNA is translated into the transcription factor XBP1s, which controls genes encoding for factors controlling protein folding and ERAD (Figure 4) (Adachi et al., 2008; Lee et al., 2003).

In the case of chronical ER stress, the accumulation of misfolded/viral protein exceeds the capacity of the ER. As a result, cell death through apoptosis can occur (Sano and Reed, 2013;

Zhang and Wang, 2012). IRE1 promotes apoptosis by activation of downstream targets of Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (Sano and Reed, 2013). Both kinases can activate the pro-apoptotic protein BAX (Kim et al., 2006).

The transcriptional induction of CHOP via p38 MAPK or the PERK pathway results in modulation of gene expression favoring apoptosis, like the inhibition of Bcl-2 transcription

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and induction of BIM expression (Hu et al., 2019, p. 201; McCullough et al., 2001;

Puthalakath et al., 2007). Caspase 12 is reported to be one effector protein causing cell death after ER stress (Nakagawa et al., 2000). As part of a complex with caspase 7 and GRP78 caspase 12 is hold in its inactive preform. ER stress leads to the dissociation of GRP78 and the release of active caspase 12, which in turn was reported to activate apoptosis via direct cleavage of procaspase 3 or by the intrinsic pathway after cleavage of procaspase 9 (Hitomi et al., 2004; Rao et al., 2002a, 2002b).

Figure 4: The three pathways of UPR activation (adapted from Walter and Ron 2011). The three ER stress transducer (PERK, ATF6, IRE1) are activated upon the accumulation of unfolded proteins in the ER and induce the transcription of UPR target genes in the nucleus.

The three sensors initiate different pathways of signal transduction. ATF6 translocates to the Golgi apparatus where it is processed by site-specific proteases and emerges as an active transcription factor. PERK phosphorylates eIF2α, resulting in general translation attenuation

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except for selected mRNAs, like ATF4. Unconventional mRNA splicing of XBP1 by IRE1 results in the expression of the active transcription factor XBP1s.

1.3.2 Activation of the unfolded protein response through BVDV

Jordan et al. (2002) demonstrated the activation of the UPR after infection with the cp BVDV strain NADL. GRP-78 mRNA and protein expression were increased in cells infected with the cp BVDV strain compared to non-infected cells. PERK and eIF2α phosphorylation as well as CHOP and caspase 12 activation were detected by immunoblot analysis in cells infected with the cp BVDV strain NADL (Jordan et al., 2002). Maeda et al. (2009) infected cells with the cp BVDV strain KZ91-cp, resulting in the upregulation of GRP78 expression. A comparison of gene expression after infection of cells with the virus strains KS86-1cp and KS86-1ncp was conducted by Yamane et al. (2009). Genes typical for ER stress were induced by the cp virus, including CHOP and tribbles homolog 3 (TRB3), tryptophanyl-tRNA synthetase as well as asparagine synthetase. Furthermore, BiP protein expression in Madin-Darby bovine kidney (MDBK) cells was limited to the cp BVDV strain. Interestingly an increase of ER stress-inducible genes in primary bovine fetal muscle cells after infection with the same cp BVDV virus could not be detected. Also the expression of BiP protein after infection with KZ91-cp or treatment with the ER stress-inducer tunicamycin was not or only slightly induced, suggesting different cellular response to BVDV infections in these two cell lines (Yamane et al., 2009).

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