Chapter 2: Characterizing unfolded protein response modulators in Huh7 cells and
2.2 Results
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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.
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important checkpoint. At this point, the structures and functions of 147, the IRE1 inhibitor 4µ8c, and Ceapin-A7 were known. Studies on the preferential IRE1 activator 474/IXA4 (referred to hereon as 474) studies had not yet been released, and thus data from this molecule is excluded from these studies.
Initial 4µ8c experiments tested the molecule over a various range of concentrations, and the ability of the drug to inhibit XBP1 splicing was dose-dependent29. To determine the optimal concentration for use in Huh7 cells, cells were seeded into 12-well dishes and treated with 0.5 µM final concentration thapsigargin (Tg), a universal UPR activator, or DMSO as a control37. Wells treated with thapsigargin were then treated with increasing concentrations of 4µ8c in DMSO. After 6 hours, samples were harvested for RT-PCR and qPCR to measure effects on XBP1 splicing.
cDNA was synthesized from the collected RNA for each sample and a PCR product of the XBP1 gene was amplified using primers that spanned the splice site (Table A2.1, splice site is nucleotides 494-519 of the coding sequence), thus creating two products with 26 nucleotide difference. These products were separated on a 3% agarose gel, which provided the resolution to visualize two bands; one slightly above 200 bp, and one slightly below (Figure 2.2). These bands correspond to the unspliced and spliced products respectively. Under basal conditions (no thapsigargin or other ER stress), the majority of XBP1 mRNA remains unspliced. After thapsigargin addition, these conditions are reversed and a significant portion becomes
Figure 2.2. XBP1 splicing inhibition by 4µ8c is dose-dependent. Samples were treated with the indicated compounds (thapsigargin, where indicated, was dosed at 0.5 µM) for 6 hours. RNA was isolated, and the complimentary cDNA was synthesized and separated on an agarose gel. Upper band represents unspliced mRNA, lower band represents excision of 26bp after UPR activation. Saturated pixels not shown.
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spliced. As the dose of 4µ8c increases, the splicing ratio decreases; by 32 µM, no splicing is seen. Thus, 32 µM was chosen as the concentration for later experiments; experiments down to 16 µM would likely have also given valid results, as the majority of splicing is inhibited at this concentration.
A second XBP1 splicing assay was performed to determine how long the effects of 4µ8c last in Huh7 cells. If a compound is quickly metabolized or otherwise disappears from the intracellular space, the effects on its target should not be expected to last a long time. To test this in Huh7 cells, samples were treated with a pulse of 0.5 µM thapsigargin, with or without 4µ8c. After the initial treatment, sequences of wells were treated with a second pulse of thapsigargin, pairing a 4µ8c treated and untreated well. If 4µ8c levels in the cell were sufficiently reduced, this second pulse of thapsigargin would result in an increase in splicing due to the decreased availability of the endonuclease inhibitor. However, even when the second pulse came 24 hours after the first, little splicing was seen in the presence of 4µ8c (Figure 2.3). Attempts to extend the timecourse were unsuccessful, likely due to the high toxicity of thapsigargin.
A similar experiment was performed with Ceapin-A7, the ATF6 inhibitor. Because an mRNA splicing event does not occur as with XBP1, a further downstream event needs to be measured.
In this case, quantification of HspA5, a marker selective for ATF6, was used27. Again, samples were treated with thapsigargin with or without Ceapin-A7. A second pulse of thapsigargin was added at the indicated hour post treatment, and transcription was measured 6 hours post-
Figure 2.3. XBP1 splicing inhibition by 4µ8c lasts at least 24 hours. Cells were treated with thapsigargin and 4µ8c (where indicated) simultaneously. A second dose of thapsigargin was given at the indicated hour post treatment (hpt). Levels of XBP1 splicing were measured 6 hours after the second treatment by RT-PCR. Saturated pixels not shown.
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treatment. Similarly to the results with 4µ8c, Ceapin-A7 was able to inhibit ATF6 activation up to 24 hours post treatment (Figure 2.4). The BiP upregulation at the 6 hours post treatment timepoint even in the presence of Ceapin-A7 was unexpected, and given the continued effect at 12 and 24 hours post treatment suggests Ceapin-A7 was aberrantly omitted from the sample.
After ensuring that the molecules are effective during the length of times required for infection experiments, a more general exploration of the selectivity of these molecules for each UPR branch was carried out. Although designed for inhibition or activation of a single branch, it is conceivable that this selectivity may vary slightly from cell line to cell line. To characterize the effects on reporter gene levels of each compound, a combination of qPCR and proteomics was used. Reporter genes were chosen based on studies by Shoulders et al., and Grandjean et al., characterizing genes upregulated by a single stress pathway (so as to avoid genes upregulated by multiple branches)27,38. To measure changes at the transcript/mRNA level, Huh7 cells were treated with the indicated compounds and harvested 6 hours post-treatment for qPCR or RT-PCR
Figure 2.4. ATF6 inhibition by Ceapin-A7 lasts 24 hours. Cells were treated with thapsigargin and Ceapin-A7 (where indicated) simultaneously. A second dose of thapsigargin was given at the indicated hour post treatment (hpt). Levels of HspA5 as an ATF6 reporter gene were measured 6 hours after the second treatment by qPCR.
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to measure XBP1 splicing. To measure changes at the translational/protein level, which is downstream of transcription, a timepoint of 20 hours post-treatment was used.
When examining transcript levels after treatment with each molecule, a discrepancy was observed between expected and experimental results. First, XBP1 splicing was measured using the same assay as outlined in Figures 2.2 and 2.3. Thapsigargin was used as a universal UPR activator to measure upregulation of all three branches. Figure 2.5 shows the results of examining XBP1 splicing in the presence of single compounds or combination treatments; RP22 was used as an inactive analog of 147, and Ceapin-A5 as an inactive analog of Ceapin-A7. As expected, addition of thapsigargin induced a large amount of XBP1 splicing, which was reversed by 4µ8c but not Ceapin-A7. However, 147 also appeared to induce a significant amount of splicing, indicating it may possess IRE1 activating potential as well as ATF6 activating potential in the Huh7 cell line. This splicing was not reversed by Ceapin-A7 but it was inhibited by the IRE1 inhibitor 4µ8c.
For qPCR experiments to measure reporter gene levels of each branch, CHOP was used as a PERK marker (it should be noted this gene may also respond to ATF6 activation), HspA5 as an ATF6 marker, and Erdj4 as an IRE1 marker. RiboP was used as a housekeeping gene and all transcripts were normalized to the level in each sample.
Figure 2.5. Selectivity of small molecules for XBP1s. Cells were treated with the indicated compounds and harvested 6 hours post-treatment. RT-PCR was performed using primers spanning the XBP1 splice site and samples were run on a 3% agarose gel at 80V for approximately 1 hour.
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Figure 2.6. Selectivity of small molecules by qPCR. Cells were treated with the indicated compounds for 6 hours. RNA was harvested, cDNA was synthesized from 500ng cellular RNA, and qPCR primers for the genes listed above were used for amplification. Primers listed in Table A2.1.
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When measuring CHOP levels, which were the most drastically changed in each of the treatments compared to background, thapsigargin and 147 both resulted in a greater than a 10 fold increase in expression (with Tg inducing closer to 20 fold). This was expected with thapsigargin, the global activator, but not with 147, which should be selective for ATF6 and thus levels should not be as high. 4µ8c was able to reduce levels of expression in the Tg treated samples, but not the 147 treated samples. Ceapin-A7, on the other hand, did reduce CHOP expression in the presence of 147 (Figure 2.6).
As expected, HspA5 levels were raised in both the Tg and 147 treated sample. 4µ8c was able to attenuate this increase in both cases, suggesting it is capable of reversing ATF6 activation.
Ceapin-A7 was also able to reduce HspA5 levels, to a greater extent than 4µ8c. However, Ceapin-A7 was also able to reduce expression levels of Erdj4, a marker intended to be selective for IRE1.
2.2.2 Effects of IRE1 and ATF6 inhibitors on DENV infection
Given that DENV upregulates the IRE1 and ATF6 branches of the UPR, our initial hypothesis was that inhibition of these branches using small molecules would lead to deleterious effects on virus replication. Intriguingly, in the initial study knockout of either ATF6 or XBP1 did not affect replication of the virus. Knockout of IRE1, however, did lead to a decrease in replication; this implicates a function of IRE1 other than XBP1 splicing being important in the DENV life cycle5. In this study, an endonuclease inhibitor was used to specifically inhibit the RNA splicing/degradation function of IRE1, while in theory leaving the kinase domain active. Huh7 cells were seeded and infected with DENV2 strain BID-V533 at MOI 3 for 3 hours. Inoculum was removed, cells were washed, and media containing DMSO or 32 µM 4µ8c was added. Cells were harvested between 12 and 60 hours post-infection at 12 hour intervals. Lysate was used to compare viral protein levels between samples, and media was collected to evaluate infectious viral titer. At 12 hours, neither viral protein nor viral titers were observed. This is likely too early in the replication cycle
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(approximately 24 hours) for output to be monitored, or a more sensitive assay would need to be used to obtain measurements. At all timepoints, no effect on viral protein levels was seen, suggesting the molecule does not affect production of the viral polyprotein. At most timepoints, no effect on viral titer was seen either. However, a small reduction in titer, approximately 50%
was seen at the 24 hour timepoint (Figure 2.7). The selectivity for a decrease in titer but not Figure 2.7. Effects of 4µ8c on the viral life cycle. Samples were infected with DENV2 BID- V533 for 3 hours. After infection, inoculum was removed, cells were washed, and media containing DMSO or 32 µM 4µ8c was added. Cells were harvested at the indicated intervals post-infection; samples were also taken at 12 hours, but no signal was observed at the titer or protein level.
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protein suggested a mechanism by which assembly and secretion of progeny virions was affected; however, with a small effect, this needed to be confirmed.
To confirm the effect seen with 4µ8c, another inhibitor of the IRE1 endonuclease domain was used, Kira628. This time, the 24 hour timepoint was used as a representative sample, rather than completing the entire timecourse, since this is where effects were initially seen with 4µ8c. This time, no effects were seen with either molecule (Figure 2.8), leading to the conclusion that the endonuclease domain of IRE1 and/or subsequent activation of downstream UPR reporter genes is not necessary for flavivirus infection. Notably, the significant change with 4µ8c observed in initial experiments was not seen again, even with a pre-treatment step added.
Experiments were repeated with the ATF6 inhibitor Ceapin-A7. Given that ATF6 knockout did not affect viral replication in Peña and Harris, it was hypothesized that the addition of this molecule would not have a significant effect on viral titers. This proved to be true, as both protein and titer levels were unaffected by Ceapin-A7 up to 60 hours post infection (Figure 2.9).
Figure 2.8. Viral titers after treatment with IRE1 inhibitors. Cells were pre-treated with inhibitors for 16 hours, prior to infection with DENV2 BID-V533 for 3 hours. Inoculum was removed, cells were washed, and media and treatments were replaced. Media was collected for focus forming assay 24 hours post infection. No significant differences in titers seen.
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2.2.3 Effects of IRE1 and ATF6 activators on DENV infection
Although the inhibitors were initially of interest, given the availability of complementary activators of the IRE1 and ATF6 branches we were also intrigued to characterize the effects of these Figure 2.9. Effects of Ceapin-A7 on the viral life cycle. Samples were infected with DENV2 BID-V533 for 3 hours. After infection, inoculum was removed, cells were washed, and media containing 6 µM Ceapin-A5 or 6 µM Ceapin-A7 was added. Cells were harvested at the indicated intervals post-infection; samples were also taken at 12 hours, but no signal was observed at the titer or protein level.
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molecules on viral infection. Previously, selective activation of each UPR branch was not possible;
an ‘activator’ of the PERK branch was available, but this worked by inhibition of the GADD34 phosphatase rather than direct activation of PERK itself10.
Given that DENV upregulates the IRE1 and ATF6 UPR branches, we hypothesized that treatment with pharmacologic activators may enhance viral infection, leading to increased protein and titer levels. To test this hypothesis, we pre-treated Huh7 cells with 10 µM 474 or 147 before infection with DENV2 strain BID-V533 at an MOI of 3 for 3 hours. 474 showed no effect on viral titers 24 hours post-infection (Figure 2.10); initial experiments by a rotation student (data not shown) indicated that it may increase titers, as was hypothesized, but this data was not reproducible in later experiments shown here.
When the treatments were repeated with 147, a remarkable decrease in titer was observed at 24 hours, and up to 36 hours post-infection (Figure 2.11). This decrease neared 2 orders of magnitude, or a 99% reduction in virus levels. The change at the protein level was still significant, but on the order of a ~75% decrease instead of maintaining two orders of magnitude. After this Figure 2.10. Effect of IRE1 activator 474 on DENV infection. Samples were pre-treated with 10 µM 474 or DMSO and infected with DENV2 BID-V533 for 3 hours. After infection, inoculum was removed, cells were washed, and media containing DMSO or 10 µM 474 was added.
Media samples were collected for focus forming assay 24 hours post infection.
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observation, 147 became the topic of focus for this portion of the dissertation project, and the effects of this molecule are much further discussed in chapter 3.
Figure 2.11. Effect of ATF6 activator 147 on DENV infection. Samples were pre-treated with 10 µM 147 or DMSO and infected with DENV2 BID-V533 for 3 hours. After infection, inoculum was removed, cells were washed, and media containing DMSO or 10 µM 147 was added.
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