A Coupled Mathematical Model of the Intracellular Replication of Dengue Virus and the Host Cell Immune Response to Infection

Dengue virus (DV) is a positive-strand RNA virus of the Flavivirus genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle in silico and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets

Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts

Dengue virus (DENV) represents a major disease burden in tropical and subtropical regions of the world, and has shown an increase in the number of cases in recent years. DENV is transmitted to humans through the bite of an infected mosquito, typically Aedes aegypti, after which it begins the infection and replication lifecycle within human cells. To perform the molecular functions required for invasion, replication, and spread of the virus, proteins encoded by DENV must interact with and alter the behavior of protein networks in both of these hosts. In this work, we used a computational method based on protein structures to predict interactions between DENV and its human and insect hosts. We predict numerous interactions, with many involved in known cell death, stress, and immune system pathways. Further investigation of these predicted protein-protein interactions should provide targets to combat the clinical manifestations of this disease in humans as well as points of intervention focused within the mosquito vector

TMEM203 is a putative co-receptor of innate immune adaptor protein STING

Acute inflammation is the innate immune defence against environmental disturbances. Macrophages are one of the central immune cells that react to infections and maintain tissue homeostasis, and they exhibit their functions via numerous inflammatory signalling regulators. In addition to previously identified immune mediators, novel proteins involved in inflammation continue to emerge. A previous cDNA functional screening in murine macrophages has identified a novel protein named transmembrane protein 203 (Tmem203) displaying pro-inflammatory characteristics. Tmem203-promoted inflammatory activities were found to be TLR independent but dependent on STING, a cytosolic innate immune adaptor for DNA detection. STING responds to upstream DNA sensors and microbial cyclic dinucleotides, and instigates type I interferon response via TBK1-IRF3 axis. The work in this thesis investigated the function of TMEM203 in STING-dependent type I interferon responses. TMEM203 has been found to colocalise, interact and migrate with STING. Further studies revealed a critical role for TMEM203 in STING-dependent type I interferon response in both human and mouse primary macrophages. We showed that TMEM203-STING association was highly dependent on STING’s N-terminal transmembrane domains. Finally, TMEM203 showed a distinct regulation of STING-interferon signalling between stimulation by natural and synthetic STING ligands, and this difference was also reflected in TMEM203-STING interaction. Thus, this novel mechanism of TMEM203-dependent STING regulation has brought new insights to better understand critical regulators of pathogen infections and interferon-associated autoimmune diseases. Additionally, a brief research was conducted to explore STING regulation in flavivirus infected primary macrophages. Flaviviruses Dengue virus and Zika virus infect humans to cause global pandemics. Dengue virus is known to specifically and potently interrupt STING-interferon pathway. The emerging flavivirus Zika virus is genetically-closely related to Dengue virus and thus it has been hypothesised to adopt similar strategies in STING antagonism. We have investigated Dengue and Zika virus-induced type I interferon stimulated ISG response in the M-CSF differentiated primary macrophage model, and tested the role of STING in such conditions. Contradictory to previous report, our experiments showed a potent and persistent ISG induction in virus-infected macrophages. Prior virus infections were unable to intercept ISG induction cause by STING ligands, whereas the downregulation of STING dampens virus-induced ISG response. Therefore, this primary macrophage model highlights alternative regulatory mechanisms via STING in response to Dengue and Zika virus

Host Cell Responses to Zika Virus Infection

The re-emergence of Zika virus (ZIKV) in 2015 as a significant human pathogen causing neurological diseases in infants as well as adults is a serious global health concern. A clear understanding of the mechanisms involved in ZIKV replication in infected host cells as well as the host responses to virus infection are keys to the development of therapeutic strategies against ZIKV. Studies conducted in this dissertation demonstrate that ZIKV infection induces the activation of mTOR signaling cascade that promotes viral protein accumulation and infectious progeny production. While both mTORC1 and mTORC2 are essential for ZIKV replication, the observation that depletion of Raptor, the unique component of mTORC1, imposes a robust negative effect on ZIKV protein expression and progeny production also suggests a mTOR- independent role played by Raptor. Additionally, the activation of autophagy at early times of infection indicates an antiviral role for autophagy in ZIKV infection. The observation that pharmacological inhibition of autophagy led to increased accumulation of ZIKV proteins further strengthens this contention. Since infection-induced oxidative stress contributes to ZIKV pathogenesis, studies reported in this dissertation also show that ZIKV infection alters the redox homeostasis in infected cells and triggers Nrf2- mediated antioxidant response. Depletion of Nrf2 downregulates the cellular pool of GSH and NADPH leading to enhanced ZIKV replication thereby underscores a role for cellular antioxidants in the suppression of ZIKV replication. The dependency of ZIKV replication on host cell glycolysis is highlighted by significant reduction in viral protein expression and virus yield due to pharmacologic inhibition. When glycolysis is inhibited, gluconeogenesis was found to facilitate ZIKV replication by compensating carbon input via oxidative mitochondrial metabolism. Further experimentation comparing the metabolic profiles of mock- and ZIKV-infected cells may provide important information in understanding the role of cellular metabolism in virus replication. Advisors: Asit K. Pattnaik and Rodrigo Franco Cru

Advances in Developing Therapies to Combat Zika Virus: Current Knowledge and Future Perspectives

Zika virus (ZIKV) remained largely quiescent for nearly six decades after its first appearance in 1947. ZIKV reappeared after 2007, resulting in a declaration of an international “public health emergency” in 2016 by the World Health Organization (WHO). Until this time, ZIKV was considered to induce only mild illness, but it has now been established as the cause of severe clinical manifestations, including fetal anomalies, neurological problems, and autoimmune disorders. Infection during pregnancy can cause congenital brain abnormalities, including microcephaly and neurological degeneration, and in other cases, Guillain-Barré syndrome, making infections with ZIKV a substantial public health concern. Genomic and molecular investigations are underway to investigate ZIKV pathology and its recent enhanced pathogenicity, as well as to design safe and potent vaccines, drugs, and therapeutics. This review describes progress in the design and development of various anti-ZIKV therapeutics, including drugs targeting virus entry into cells and the helicase protein, nucleosides, inhibitors of NS3 protein, small molecules, methyltransferase inhibitors, interferons, repurposed drugs, drugs designed with the aid of computers, neutralizing antibodies, convalescent serum, antibodies that limit antibody-dependent enhancement, and herbal medicines. Additionally, covalent inhibitors of viral protein expression and anti-Toll-like receptor molecules are discussed. To counter ZIKV-associated disease, we need to make rapid progress in developing novel therapies that work effectually to inhibit ZIKV

Chemokine Induction by Dengue Virus Infection: Mechanisms and the Role of Viral Proteins: a Dissertation

The focus of this thesis is the role of dengue virus in the induction of chemokines. Dengue virus (DENV) occurs as four distinct serotypes, called DENV 1,2,3,and 4. Symptomatic DENV infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. Many factors including T cell cross reactivity, viral burden, antibody dependent enhancement and induction of chemokines and cytokines have been reported in DHF and may play a role in the pathogenesis of DENV infection. Cytokines have been shown to modulate endothelial cell permeability [1-3]. Recent studies have shown that DENV-infected endothelial cells secrete the chemokine, interleukin (IL)-8 in vitro [4]. In addition, the permeability of an endothelial cell monolayer was found to be increased by interleukin-8 (IL-8) in vitro[5]. This thesis examines the effects of DEN2V infection on the induction of chemokines, and specifically, which DEN2V viral protein(s) are involved in the induction of IL-8. The chemokine induction profile following DEN2V infection was initially assessed in various cell lines that may represent potential targets in vivo, including monocytes, liver cells and endothelial cells. We hypothesized that distinct profiles of chemokine secretion can be induced by DEN2V infection of various cell types in vitro. We found RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) and IL-8 were induced in two of the five cell lines. DEN2V infection of primary monocyte-derived dendritic cells induced RANTES and IL-8 along with macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monocyte chemoattractant protein-1 (MCP-1) but at an earlier time post infection than in the cell lines. These results showed that DEN2V infection induces distinct chemokine profiles in many cell types. In addition, monocytic-derived DCs can secrete chemokines upon infection with DEN2V. Characterization of the signaling pathways induced by DEN2V revealed that DEN2V induction of chemokines in human embryonic kidney (HEK293A) cells is mainly through the nuclear factor kappaB (NFκB) pathway, as previously reported for endothelial cells and 293T cells [4,6]. Alternatively, the liver cell line (HepG2) activated mainly activator protein (AP)-l. In addition, DENV infection can induce the activation of the interferon-stimulated response element (ISRE) driven promoter. IL-8 has been shown to have multiple effects on the immune system ranging from recruiting cells to the site of infection to countering the antiviral effects of type I interferon (IFN) [7,8]. Previous reports have shown that viral proteins can induce chemokines such as seen with IL-8 induction with the nonstructural protein 5A (NS5A) and core proteins from hepatitis C virus [9,10]. We hypothesized that protein(s) from DENV could induce chemokine production. The expression of DENV proteins was analyzed for effects on IL-8 and RANTES production in HEK293A cells. The effects of viral replication on IL-8 and RANTES induction were also analyzed using a DENV replicon that contains genes for the capsid protein and the nonstructural proteins. Transfection of plasmids expressing NS5 or the DEN2V replicon induced the expression and secretion of IL-8 but not RANTES. We attributed the lack of RANTES induction to the inability of NS5 or the DEN2V replicon to induce transcription from the ISRE driven promoter. We also found that NS5 and the DEN2V replicon induced IL-8 mainly through the CCAAT/enhancer binding protein (c/EBP) and AP-1 pathways. The profile of transcription factor activation is different from what was seen with DENV infection of HEK293A cells and suggests that the transient expression of the NS5 protein and the replication and/or translation of the DEN2V genome use different pathways than viral infection to induce IL-8. In addition, we found that the expression of prM-E, known to produce virus-like particles, could induce IL-8 secretion and activate transcription from the IL-8 promoter. As with the expression of NS5, RANTES was not induced. Analysis of the transcription factors involved in IL-8 induction using luciferase reporter constructs indicated that expression of prM-E induced transcription of IL-8 through NFκB, AP-1 and c/EBP, similar to what was seen with DEN2V infection of HEK293A cells. These results suggest that production of virions or virus-like particles induce IL-8 but that another mechanism in the viral life cycle is responsible for the induction of RANTES expression by DEN2V infection. We were also interested in the effects of drugs that have been used previously to inhibit cytokine or chemokine production on chemokine induction during DEN2V infection. We hypothesized that pharmacological inhibitors of cytokines will inhibit secretion of chemokines in DEN2V infected cells. We found that the pharmacological inhibitors SB203580 and rolipram enhanced chemokine production in a DEN2V infected liver cell line (HepG2), whereas dexamethasone had the same effect in a kidney epithelial cell line (HEK293A). We conclude that drugs that inhibit signaling pathways involved in cytokine production in other experimental systems can have variable effects on chemokine induction in different cell types during DEN2V infection. The data generated in this thesis extend our understanding of how DEN2V manipulates the host cell during viral infection to produce chemokines and perhaps enhance viral propagation and dissemination through the induction of IL-8. In addition, this study provides insight into the variable effects pharmacological drug treatment may have on disease progression during DENV infection. These results increase our understanding of DENV pathogenesis and may be helpful in finding better strategies for treatment and prevention

Systems-Biology Approaches to Discover Anti-Viral Effectors of the Human Innate Immune Response

Virus infections elicit an immediate innate response involving antiviral factors. The activities of some of these factors are, in turn, blocked by viral countermeasures. The ensuing battle between the host and the viruses is crucial for determining whether the virus establishes a foothold and/or induces adaptive immune responses. A comprehensive systems-level understanding of the repertoire of anti-viral effectors in the context of these immediate virus-host responses would provide significant advantages in devising novel strategies to interfere with the initial establishment of infections. Recent efforts to identify cellular factors in a comprehensive and unbiased manner, using genome-wide siRNA screens and other systems biology “omics” methodologies, have revealed several potential anti-viral effectors for viruses like Human immunodeficiency virus type 1 (HIV-1), Hepatitis C virus (HCV), West Nile virus (WNV), and influenza virus. This review describes the discovery of novel viral restriction factors and discusses how the integration of different methods in systems biology can be used to more comprehensively identify the intimate interactions of viruses and the cellular innate resistance

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.Agencia Nacional de Promoción Científica y Tecnológica de Argentina (ANPCyT) [2014-2888, 2015-1731, 2017-0111 to A.S. and 2015-2555, 2017-1717 to A.V.G.]; Universidad de Buenos Aires, Argentina (UBACyT) [20020170100045BA to A.S.]; NIH (NIAID) [R01.AI095175 to A.V.G.]; Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET) [PIP 11220170100171CO to C.C.G]; B.P. has been a postdoctoral fellow from CONICET from 2017 to 2019 and is currently a postdoctoral fellow at the Institute of Cell Biology in the University of Bern, Switzerland; L.B. and M.E.G.S. are recipients of doctoral fellowships from CONICET; M.F.T. is a doctoral fellowship recipient from ANPCyT; N.G. has been an undergraduate fellowship recipient from the University of Buenos Aires (2018–2020); P.M. has been a doctoral fellow from CONICET (2015–2019) and is currently a postdoctoral fellow supported by H2020-Marie Sklodowska-Curie Research and Innovation Staff Exchanges [734825-LysoMod]; R.V.D. has been a visiting post-doctoral fellow at the Srebrow lab from IMM (Lisbon, Portugal) supported by the same program. A.S., A.V.G., C.C.G., N.G.I. and L.G.G. are career investigators from CONICET.info:eu-repo/semantics/publishedVersio