Chapter 2: Characterizing unfolded protein response modulators in Huh7 cells and
2.1 Introduction
Flaviviruses are a class of positive-sense, single stranded RNA viruses that include dengue, Zika, and yellow fever. While the yellow fever vaccine has been one of the most reliable and successful vaccines developed, no other widespread vaccination options exist for flaviviruses, which is surprising given that half the world’s population is estimated to be at risk of dengue infection1. Additionally, no therapeutics exist for the treatment of flavivirus infection, though several efforts have been made to characterize and develop direct-acting antiviral compounds2,3.
To begin investigating dependencies of flaviviruses on host machinery, we focused on the endoplasmic reticulum, an organelle basally used as a hub for host protein folding4. Specifically,
Figure 2.1 Small molecules can selectively regulate UPR branches. The above structures show the small molecules designed to activate or inhibit (green/red symbols respectively) the three branches of the unfolded protein response. The molecules for regulation of IRE1 and ATF6 will be discussed further.
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we took advantage of the observation that DENV infection selectively modulates the unfolded protein response, a stress response pathway intended to return the cell to a homeostatic state under conditions of increased protein folding load or stress from the buildup of unfolded proteins or if prolonged ER stress persists, induce apoptosis.5.
The UPR consists of three branches; PERK, IRE1/XBP1s (referred to as IRE1 from hereon) and ATF6. Each of these three branches is ultimately responsible for upregulating distinct but overlapping functional outputs in order to increase the folding capacity of the ER and/or decrease cellular translation rates in order to allow the cell to play catch-up on a current protein folding load6.
We began with the selection of four molecules that activate or inhibit the IRE1 and ATF6 branches of the UPR (Figure 2.1). All of these molecules are recently discovered, and provide a novel method of selectively regulating each of these branches7–10.
The UPR branches are named after the transmembrane sensors that localize inside of the ER membrane and are able to direct communication between sensing stress in the ER lumen and relay the response to the cytosol or other organelles6. On the ER luminal side, under non-stressed conditions, these transmembrane sensors are bound by binding immunoglobulin protein (BiP, also referred to as GRP78). BiP is the sole ER luminal Hsp70 chaperone that, upon sensing an accumulation of unfolded proteins, will dissociate from the UPR sensor in an attempt to correct the misfolding. This dissociation is what thought to initiate the cascade of UPR activation, though alternative mechanisms have been proposed11–14.
BiP dissociation from PERK induces dimerization and autophosphorylation, which leads to full activation of its kinase domain. This domain then phosphorylates eukaryotic initiation factor 2α, which results in translational attenuation via the ribosome15–17. Counterintuitively, this leads to the selective translation of specific transcripts containing upstream open reading frames (uORFs), one of which is activating transcription factor 4 (ATF4)17,18. ATF4 protein trafficks to the nucleus and upregulates specific targets, two of which are CHOP and GADD3419. CHOP is another
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transcription factor which begins a further cascade of upregulating components of apoptosis; thus, prolonged PERK activation leads to this mechanism of controlled cell death. GADD34/PPP1R15A is the regulator which closes the negative feedback loop, serving as a specific regulatory subunint of the protein phosphatase 1 complex that dephosphorylates eIF2α and allows translation to resume normally20. The phosphorylation of eIF2α marks the intersection of the UPR with the integrated stress response (ISR), which is a broader cellular response activated under a range of physiological changes or stimuli21. Due to the interplay between these two pathways, small molecules have been developed to study the ISR at large, some of which bind PERK directly22. Sephin-1, an ISR ‘activator’, works via inhibition of the GADD34 phosphatase subunit, thus prolonging eIF2α phosphorylation10. ISRIB, short for integrated stress response inhibitor, works by enhancing activity or eIF2B, another component of the initiation complex. The activity enhancement is able to counteract the phosphorylation of eIF2α and prevent the translation attenuation9.
In a similar fashion, BiP dissociation from IRE1 also causes dimerization and autophosphorylation of this sensor12,23. However, in this instance an endonuclease domain within IRE1 is activated after phosphorylation. The endonuclease domain degrades selective mRNA substrates via regulated IRE1-dependent decay (RIDD), but also cleaves a 26 nucleotide sequence from the mRNA of the X-box binding protein 1 (XBP1)24–26. This non-canonical splicing event causes a frameshift, and results in translation of an alternate protein product labeled as XBP1s. XBP1s is a transcription factor that upregulates some chaperones and other folding factors, but also upregulates genes involved in lipid biosynthesis and ER-associated degradation to increase overall folding capacity and help rid the organelle of misfolded proteins27. Inhibitors of both the kinase and endonuclease domains of IRE1 have been discovered and characterized28–30. The last sensor is ATF6, whose mechanism of activation is less defined than either PERK or IRE1. ATF6 is localized in the ER membrane as an oligomer, with intramolecular disulfide bonds linking monomers together. After BiP dissociation, these oligomers are reduced to a specifically
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linked dimer, and these dimers of ATF6 are trafficked to the Golgi apparatus31,32. Here, ATF6 is cleaved by S1P and S2P proteases to release a cytosolic active transcription factor, which traffics to the nucleus and again upregulates chaperones and other folding factors which help restore protein homeostasis33. The two compounds designed to preferentially modulate this pathway are the activator 147 and the inhibitor Ceapin-A77,8. 147 was discovered in a high-throughput screen that looked at ATF6 activation at both the transcript and protein levels, and selected molecules which initially enhanced activation of a luciferase under the ERSE (BiP) promoter (indicative of ATF6 cleavage), while counterscreening against those molecules which also activated XBP1.
Several other molecules were looked at as potential candidates after both screens, including compound 263, which will be discussed in chapter 4 in this dissertation. A follow-up study showed that this compound does not bind ATF6 directly, but rather targets protein disulfide isomerases, which are hypothesized to control the oligomeric state of ATF6 in the membrane via oxidation and reduction of disulfide bonds34. Ceapin-A7 prevents trafficking of ATF6 from the ER membrane to the Golgi apparatus, thus inhibiting its ability to form a functional transcription factor35. It does this by tethering the cytosolic domain of the sensor to a second transmembrane protein, ABCD3, found within the membrane of the peroxisome36.
This chapter details efforts to characterize the selectivity of the ATF6 and IRE1 modulators in Huh7 cells, later used as a model system for flavivirus infection. After characterization, these modulators were tested in viral infections to monitor the effects of modulation of ATF6 and IRE1 modulation on viral replication.