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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

Malignancy-induced alterations to cytokine signaling in tumor cells differentially regulate their interactions with the immune system and oncolytic viruses. The abundance of inflammatory cytokines in the tumor microenvironment suggests that such signaling plays key roles in tumor development and therapy efficacy. The JAK-STAT axis transduces signals of interleukin-6 (IL-6) and interferons (IFNs), mediates antiviral responses, and is frequently altered in prostate cancer (PCa) cells. However, how activation of JAK-STAT signaling with different cytokines regulates interactions between oncolytic viruses and PCa cells is not known. Here, we employ LNCaP PCa cells, expressing (or not) JAK1, activated (or not) with IFNs (α or γ) or IL-6, and infected with RNA viruses of different oncolytic potential (EHDV-TAU, hMPV-GFP, or HIV-GFP) to address this matter. We show that in JAK1-expressing cells, IL-6 sensitized PCa cells to viral cell death in the presence or absence of productive infection, with dependence on virus employed. Contrastingly, IFNα induced a cytoprotective antiviral state. Biochemical and genetic (knockout) analyses revealed dependency of antiviral state or cytoprotection on STAT1 or STAT2 activation, respectively. In IL-6-treated cells, STAT3 expression was required for anti-proliferative signaling. Quantitative proteomics (SILAC) revealed a core repertoire of antiviral IFN-stimulated genes, induced by IL-6 or IFNs. Oncolysis in the absence of productive infection, induced by IL-6, correlated with reduction in regulators of cell cycle and metabolism. These results call for matching the viral features of the oncolytic agent, the malignancy-induced genetic-epigenetic alterations to JAK/STAT signaling and the cytokine composition of the tumor microenvironment for efficient oncolytic virotherapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38β is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38β substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38β and supports exploring p38β inhibitor development as a strategy toward creating a new class of COVID-19 therapies. SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.

The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 → Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 → Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N -glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N -glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N -glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy- d -glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

What are the mechanisms of ligand-induced allosteric transitions in proteins? A powerful method to characterize pathways and transition states of reactions is ϕ value analysis. A ϕ value is the ratio between the changes on a perturbation (e.g., mutation) in the activation and equilibrium free energies of a reaction. Here, ϕ value analysis is used to characterize the ATP-induced allosteric transitions of GroEL by using changes in ATP concentration as perturbations. GroEL consists of two stacked back-to-back heptameric rings that bind ATP with positive cooperativity within rings and negative cooperativity between rings. Evidence is presented for the existence of parallel pathways for the allosteric transition of each ring. In both allosteric transitions, there is an abrupt ATP-dependent switch from a pathway with ATP-binding sites in the transition state that are very similar to those of the initial T state (ϕ = 0) to a pathway with a ϕ value of ≈ 0.3. The ϕ value procedure outlined here should be useful in mapping the energy landscape of allosteric transitions of other proteins.

The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes. [PUBLICATION ABSTRACT]

Single cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), the causative agent of COronaVIrus Disease 2019 (COVID-19). scRNA-Seq workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We present a data processing strategy, single cell CoronaVirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to sgmRNAs or genomic RNA (gRNA). Compared to standard 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') and Chromium Next GEM Single Cell V(D)J (10X 5') sequencing, we find that 10X 5' with an extended read 1 (R1) sequencing strategy maximizes the detection of sgmRNAs by increasing the number of unambiguous reads spanning leader-sgmRNA junction sites. Using this method, we show that viral gene expression is highly correlated across cells suggesting a relatively consistent proportion of viral sgmRNA production throughout infection. Our method allows for quantification of coronavirus sgmRNA expression at single-cell resolution, and thereby supports high resolution studies of the dynamics of coronavirus RNA synthesis.

The allosteric chaperonin GroEL from E. coli assists protein folding in an ATP- dependent manner. Here, various models that describe cooperativity in ATP hydrolysis by GroEL, with respect to ATP are examined. It is demonstrated that the nested model (Yifrach, O. & Horovitz, A. (1995) Biochemistry 34, 5303-5308) remains the most appropriate, not only because it fits the kinetic data best but also because it is supported by other lines of evidence. In Chapter 2, evidence is provided for the concerted nature of the intra-ring allosteric transitions in wild-type GroEL as postulated by the nested model. It is shown that elimination of the D155-R395 salt- bridge converts the intra-ring allosteric transitions from concerted to sequential. The mechanism of this conversion appears to involve weakening of the inter-subunit R197-E386 salt-bridge that frees the apical domains and reduces the steric interference that would arise if one subunit underwent a conformational transition while its neighbors had not. In Chapter 3, it is shown that the ATPase activity of the F44W, E257A GroEL double mutant is not stimulated by non-folded proteins, in contrast with what has been observed for wild-type GroEL. Polypeptide substrate- induced stimulation of ATP hydrolysis is found to be the result (at least in part) of an increase in the off-rate of ADP which is communicated through E257 in the apical domain over a distance of ~40 Å to the nucleotide binding site. These results have provided insight into the effects of ADP on the ATPase activity of GroEL that include: (i) competitive inhibition of ATP hydrolysis by ADP bound to the cis ring and (ii) noncompetitive inhibition of ATP hydrolysis by ADP bound to trans ring.