First and foremost, I am grateful to Lars for allowing me to be one of the founding members of his lab in 2017

First and foremost, I am grateful to Lars for allowing me to be one of the founders of his lab in 2017. Their work opened doors for me to continue asking questions and finding answers, and I am grateful for their time and talent.

Exploiting viral dependencies: Identifying and targeting host endoplasmic reticulum

Role of the ER in protein homeostasis and in viral infections

For larger aggregates, autophagy of part of the ER (colloquially called ERphagy) is used to remove the aggregate from the ER17. For this reason, targeting the proteostasis network for therapeutic purposes has been a long-standing goal.

Figure 1.1. The proteostasis network. Protein folding in the ER consists of a wide variety of functions to ensure a protein takes its properly folded state

Identifying host-virus interactions with the protein folding network

SARS-CoV-2 nsp3.1 interacts with ATF6 and inhibits the ATF6 branch of the unfolded protein response (UPR). The C-terminal part of the nsp3.1 fragment for SARS-CoV and SARS-CoV-2 consists of a SARS-unique domain (SUD).

Figure 1.2. Methods for identifying virus-host interactions. (A) RNAi (top) or plasmid (bottom) libraries can be used to determine the effects on virus levels when genes are knocked down or overexpressed, respectively

Perturbing host-virus interactions within the protein folding network to block

Summary and conclusions

This review summarizes some of the ways in which virus-host interactions have been identified, with particular emphasis on those interactions of viral proteins with the host endoplasmic reticulum machinery. We then examine ways in which these interactions or dependencies can be targeted using host-directed therapies to inhibit viral replication.

Scope of dissertation

Several interactors are conserved between SARS-CoV-1 and SARS-CoV-2 nsp2, and between SARS-CoV-1, SARS-CoV-2 and hCoV-OC43 nsp4 homologues. We discovered that the nsp3 N-terminal fragment (nsp3.1) interacted with the UPR sensor ATF6, and follow-up studies showed that nsp3.1 of SARS-CoV-2 is able to modulate the ATF6 stress response.

Genome-wide mutagenesis of dengue virus reveals the plasticity of the NS1 protein and enables the generation of infectious tagged reporter viruses. Modulation of the unfolded protein response pathway as an antiviral approach in airway epithelial cells.

Characterizing unfolded protein response modulators in Huh7 cells and

Introduction

We began by selecting four molecules that activate or inhibit the IRE1 and ATF6 branches of the UPR (Figure 2.1). The branches of the UPR are named after transmembrane sensors that are localized within the ER membrane and can direct communication between sensing stress in the ER lumen and transmitting the response to the cytosol or other organelles6.

Results

After infection, the inoculum was removed, the cells were washed and medium containing DMSO or 32 µM 4 µ8c was added. After infection, the inoculum was removed, the cells were washed and medium containing 6 µM Ceapin-A5 or 6 µM Ceapin-A7 was added.

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

Discussion

As discussed above, the increased expression of Erdj4 may be due to the gene's lack of selectivity in Huh7 cells, rather than the lack of selectivity of the molecule. The global proteomics experiment also suggested that Ceapin-A7 might also slightly suppress IRE1 gene activation (Figure A2.1).

Materials and Methods

After infection, the inoculum was removed, the cells were washed with DPBS, and media containing chemical compounds or DMSO were added for the remainder of the experiment. The cells were overlaid with a 1:1 mixture of 2x nutrient overlay (2x Earle's Balanced Salt solution, 2x Ye-Lah Medium, 4% FBS, 0.4% sodium bicarbonate, 0.1 mg/ml gentamycin, 0.5 mg/ ml amphotericin B) and 2.4% methylcellulose in water. The cells were left on ice for at least 10 minutes and the lysate was cleared for 10 minutes at 17,000 x g.

Structure and molecular mechanism of ER stress signaling by the unfolded protein response signal activator IRE1. The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response. Unconventional splicing of XBP1 mRNA occurs in the cytoplasm during the mammalian unfolded protein response.

ER proteostasis regulator 147 acts as a broad-spectrum inhibitor of dengue and Zika

Introduction

Genetic screens have identified many proteostasis factors as essential for DENV replication, including several components of the oligosaccharyl transferase (OST) complex, as well as the ER membrane protein complex27–30. Since flavivirus replication and polyprotein translation occur at the ER membrane, infection leads to expansion of the ER and remodeling of the ER proteostasis environment 31,37,38. The first two branches mainly control the upregulation of chaperones and other proteostasis factors to expand the protein folding capacity of the ER.

Results

Whole-cell proteomics data comparing DENV-infected cells with uninfected Huh7 cells are shown in Figure A3.1C-D. TMP-dependent upregulation of ATF6-regulated targets BiP, PDIA4 and GRP94 in Huh7 cells (Figure A3.3D-F). Chemical genetic activation of ATF6 did not lead to a measurable reduction in viral protein levels (Figure 3.3F, Figure A3.3G).

Figure 3.2. Treatment with small molecule 147 reduces viral RNA, protein, and titer levels

Discussion

To validate that the observed antiviral effects were independent of the viability reduction, we repeated the experiments at 1 µM 147, the approximate IC50 observed in Figure A3.2B. None of the PDI inhibitor compounds significantly reduced DENV viral titers, suggesting that 147 elicits its antiviral activity through an alternative mechanism of action. These results highlight broad utility of the proteostasis regulator compound to reduce flavivirus infection by targeting conserved host cell processes commonly exploited by the virus.

Materials and Methods

The inoculum was removed, cells were washed, and medium containing compounds or DMSO was added until the end of the experiment. Huh7 cells were seeded in 96-well plates and treated with 100-fold dilutions of compounds in culture medium. 16 h after treatment, medium was removed and cells were infected as described above (no virus was added for -DENV samples).

Dengue virus selectively annexes endoplasmic reticulum-associated translation machinery as a strategy to co-opt host cell protein synthesis. An Inducible Heat Shock Protein 70 Small Molecule Inhibitor Demonstrates Anti-Dengue Virus Activity, Validating Hsp70 as a Host Antiviral Target. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53.

ER proteostasis regulator 263 acts as a broad-spectrum inhibitor of dengue and Zika

Introduction

Pharmacological modulation of the proteostasis network, for example with inhibitors of Hsp70 and the oligosaccharyl transfer complex (OST), can prevent flavivirus replication in both cellular and mouse models24-26. 263 was originally discovered in the same high-throughput screen that identified 147 as an activator of the ATF6 branch of UPR35. However, target enrichment studies using a competition-based chemoproteomic approach suggest that the targets of the molecules may only partially overlap.

Results

To validate the competition experiment, we confirmed the observation of PDIs as the main targets of 147 (Figure 4.4d). Inhibition of the oxidation step with resveratrol (a broad-spectrum P450 inhibitor) or competition of the nucleophilic addition step with beta-mercaptoethanol) resulted in recovery of viral titers. No changes in cell viability were observed upon co-treatment of 263 with any of the above compounds (Figure A4.9).

Figure 4.1. 263 inhibits DENV2 propagation in Huh7 cells. a) Structures of compounds 147 (left) and 263 (right), the two proteostasis regulators of interest

Discussion

An additional study showed that an aldehyde dehydrogenase was one of the enzymes found in yeast responsible for the metabolism of these compounds43. This may indicate that another member of the ALDH family is responsible for the antiviral activity, or otherwise for the metabolism of the compounds in liver cells, where dehydrogenase enzymes are highly expressed44. Considering this fact, the mechanism by which 263 appeared to compete for binding to 147-20 targets raises the question of how these two molecules bind some of the same proteins (or at least ALDH1A1).

Materials and methods

Cleared lysate concentrations were normalized using Bio-Rad Protein Assay Dye, and normalized gel samples were separated by SDS-PAGE gel at 175V. For detection, samples were prepared and run on SDS-PAGE gels as described above. Collected samples were acidified using a SpeedVac and resuspended in 95% water/5% acetonitrile/0.1% formic acid and pH adjusted to 2 if necessary.

A global interactome map of the Dengue virus NS1 identifies virus restriction and host factors. Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections. Review of the role of alcohol dehydrogenase and aldehyde dehydrogenase and their variants in the development of alcohol-related pathology.

Comparative multiplexed interactomics of SARS-CoV-2 and homologous

Introduction

A previous AP-MS study of SARS-CoV-1 nsp2 identified multiple host interactors including prohibitin 1/2 (PHB1/2)17. Nsp2 is dispensable for infection in SARS-CoV-1 20 and has marked amino acid sequence differences between coronavirus strains (Figure 5.1B, Figure A5.1A). Furthermore, early sequence analysis of SARS-CoV-2 identified regions of positive selection pressure in nsp221.

Results

Using the strictest cutoff, we identified 6 and 11 high-confidence interactors for SARS-CoV-2 and SARS-CoV-1 nsp2, respectively (Figure 5.2C-D). In contrast, cluster 2 and 4 contained unique interactors enriched for OC43 and SARS-CoV-1 nsp4, respectively. We identified several common interactors between SARS-CoV-1 and SARS-CoV-2 that were absent in OC43.

Figure 5.1. Design and validation of expression of CoV nsp2 and nsp4 constructs for affinity purification

Discussion

SARS-CoV-2 nsp3.1 was able to inhibit activation by small molecules, suggesting its role in suppressing the UPR (Figure 6.5 A-B). Therefore, we were particularly interested in finding ATF6 as a high-fidelity interactor of SARS-CoV and SARS-CoV-2 nsp3.1 fragments. Sequences for generating SARS-CoV nsp3 fragments were selected based on Pan et al, 2008, PLoS One21.

Materials and Methods

Severe acute respiratory syndrome coronavirus nonstructural protein 2 interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling. Non-canonical function of IRE1α Determines mitochondria-associated endoplasmic reticulum composition to control calcium transport and bioenergetics. RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation.

Comparative host interactomes of the SARS-CoV-2 nonstructural protein 3 and

Introduction

However, the remaining parts of nsp3 have been missing from SARS-CoV-2 interaction studies so far, likely because their complex topology and large size make protein expression difficult. We expressed each fragment from each of the five viruses listed above (hCoV-229E, hCoV-OC43, MERS-CoV, SARS-CoV, SARS-CoV-2). Previously, we demonstrated the use of AP-MS and TMT technologies in comparing the interactions of the nsp2 and nsp4 coronavirus proteins from three strains: SARS-CoV-2, SARS-CoV, and hCoV-OC4322.

Results

Venn diagram showing the number of unique and shared host interactors among all five CoV nsp3.1 homologues. Venn diagram showing the number of unique and shared host interactors among all five CoV nsp3.2 homologues. Venn diagram showing the number of unique and shared host interactors among all five CoV nsp3.3 homologues.

Figure 6.1. Design and expression of CoV nsp3 truncations for affinity-purification mass spectrometry (AP-MS)

Discussion

Orf6 of SARS-CoV and SARS-CoV-2 inhibits STAT1 signaling by blocking nuclear import of phosphorylated STAT1. Nuclear localization of SARS-CoV and SARS-CoV-2 orf3b has been demonstrated, as well as for N proteins. Our results indicate that the N-terminal regions of SARS-CoV-2 nsp3 directly interact with ATF6 to suppress its activation.

Materials and Methods

After three PBS washes, cells were blocked in PBS with 1% BSA and 0.1% saponin (blocking buffer) for 1 h at room temperature. After blocking, cells were incubated with anti-PDIA4 primary antibody (Protein Tech, 14712-1-AP) in blocking buffer (1:1000 dilution) overnight at 4°C. Cells were then stained with M2 FLAG primary antibody (SigmaAldrich, F1804) and AlexFluor 594-conjugated anti-mouse goat antibody (ThermoFisher, A-11005) using the same conditions.

Conclusions and future directions

Summary of Work

Ongoing and future experiments

Conclusion

Supplemental figures and tables for chapter 2