Comprehensive Identification of Host Modulators of HIV-1 Replication using Multiple Orthologous RNAi Reagents

SummaryRNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene’s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1–host cell interactions

The Deubiquitylase USP5 Knockdown Reduces Semliki Forest Virus Replication in HeLa Cells

Semliki Forest Virus (SFV), a member of the Alphavirus genus in the Togaviridae family, is a small-enveloped, positive-sense single-stranded RNA (+ssRNA) virus. The virus is spread by mosquitos and can infect humans, resulting in mild febrile disease with symptoms that include fever, myalgia, arthralgia, persistent headaches and asthenia.  Virulent strains of SFV in mice cause lethal encephalitis by infecting neurons in the central nervous system. In on-going experiments in the research group using a focused siRNA screen we have investigated the role of deubiquitylases (DUBs) during SFV infection (as a model alphavirus) and monitored the effect of DUB depletion on cell viability after infection. We identified a group of DUBs that have a pro-viral effect. The DUB, USP5, from this screen was validated to determine its effect upon viral replication. Here, we show that depleted USP5 in HeLa cells resulted in SFV RNA and viral yield at 8 h post-infection being significantly reduced. In the multi-step viral growth curve assay, in the absence of USP5, similar yields of SFV were determined at 2 and 4 h post-infection. However, a significant reduction in the infectious viral particles release at 6, 8, 10 and 12 h post-infection was observed and this could be reversed by direct constraining viral replication. These results raise the potential for USP5 to play a distinct role in the replication of SFV, suggesting that USP5 may be a possible anti-viral therapy for alphavirus infection

A novel bioinformatic approach for comprehensive genome scale analysis identifies key regulators of macrophage activation.

The initiation of inflammatory cytokine transcription by bacterial ligands is a central mechanism by which the immune system activates its first line of defense. Macrophage activation by the Toll-like Receptor 4 (TLR4) pathway is initiated with receptor binding of lipopolysaccharides (LPS) and culminates in a large-scale transcriptional response of the inflammatory gene program. Advancements in genome-wide screening technologies have made it possible to interrogate the regulatory landscape of signaling pathways such as those activated by TLR4. Utilizing these high-throughput methods for the comprehensive characterization of pathway components, particularly for regulators that are involved in critical cellular processes such as transcription and translation, however, requires an approach that goes beyond the top scoring and previously characterized hits of genome-scale studies. To address this challenge, I developed the Throughput Ranking by Iterative Analysis of Genomic Enrichment (TRIAGE) method, a bioinformatic analysis model that facilitates the comprehensive identification of likely regulators by iterative sampling of pathway and network databases. I validated the TRIAGE approach by analyzing three previously published genome-wide studies of regulators of early HIV infection and viral transcription. Analysis by TRIAGE showed significantly increased overlap and identified shared novel targets across the three studies. I further developed the TRIAGE analysis method as a globally accessible web-based resource. Applying TRIAGE analysis to three genome-scale studies of LPS treatment in macrophages of mouse and human cell lines, I identified an enrichment for regulators relating to alternative splicing and protein degradation. Using short read and long read RNA-seq of ligand-stimulated macrophages I further characterized the broad transcriptional variation induced by the LPS response and the novel and known transcript variants that define different macrophage activation states. These findings define an approach for comprehensive unbiased discovery of signaling pathway regulators from genome-scale datasets and suggest a model of macrophage activation involving proteasomal removal of negative regulators and remodeling of the macrophage state via a transcriptional shift in splice variant dynamics

Host factors involved in chikungunya virus replication

In this thesis the interplay of CHIKV with cellular (host) factors involved in its replication is addressed. An in-depth understanding of the interactions between the viral proteins and those of their host is required for the elucidation of molecular mechanisms underlying viral replication. A variety of proteins and pathways involved in CHIKV replication were identified in this thesis, and the role of a selection of those is described. First, the construction and characterization of a novel synthetic CHIKV full-length cDNA clone is described in chapter 2. In chapter3 we describe the identification of dozens of cellular factors and pathways that are potentially involved in CHIKV replication. Chapter 4 describes the response of the p38 MAPK and MEK/ERK signaling pathways in CHIKV-infected human cells. Chapter 5 describes the unexpected proviral role of stress granule components G3BP1 and G3BP2 early during CHIKV infection. In chapter 6 the inhibitory effect of a RIG-I agonist (5__pppRNA) on CHIKV and DENV replication is described. A low, noncytotoxic dose of 5__pppRNA stimulates RIG-I and leads to a robust antiviral response, potentially opening up therapeutic options against these important human pathogens. Chapter 7 discusses the findings described in this thesis, and places them in a broader context.UBL - phd migration 201

Sequence-Specific Features of Short Double-Strand, Blunt-End RNAs Have RIG-I- and Type 1 Interferon-Dependent or -Independent Anti-Viral Effects

Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAsThe work was supported by the Swiss National Science Foundation (31003A_179256/1 to UFG, and 320030_205097 to JPS), the Swiss National Science Foundation SystemsX RTD InfectX (51RT 0_126008 to UFG and CvM), and the University Research Priority Program of the University of Zurich (URPP) ITINERARE – Innovative Therapies in Rare Diseases to JPS.Peer reviewe

Identification of {HNRNPK} as Regulator of Hepatitis {C} Virus Particle Production

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses