In Silico Characterization of Protein-Protein Interactions Mediated by Short Linear Motifs

Short linear motifs (SLiMs), often found in intrinsically disordered regions (IDPs), can initiate protein-protein interactions in eukaryotes. Although pathogens tend to have less disorder than eukaryotes, their proteins alter host cellular function through molecular mimicry of SLiMs. The first objective was to study sequence-based structure properties of viral SLiMs in the ELM database and the conservation of selected viral motifs involved in the virus life cycle. The second objective was to compare the structural features for SliMs in pathogens and eukaryotes in the ELM database. Our analysis showed that many viral SliMs are not found in IDPs, particularly glycosylation motifs. Moreover, analysis of disorder and secondary structure properties in the same motif from pathogens and eukaryotes shed light on similarities and differences in motif properties between pathogens and their eukaryotic equivalents. Our results indicate that the interaction mechanism may differ between pathogens and their eukaryotic hosts for the same motif

Machine-learning-based identification of factors that influence molecular virus-host interactions

Viruses are the cause of many infectious diseases such as the pandemic viruses: acquired immune deficiency syndrome (AIDS) and coronavirus disease 2019 (COVID-19). During the infection cycle, viruses invade host cells and trigger a series of virus-host interactions with different directionality. Some of these interactions disrupt host immune responses or promote the expression of viral proteins and exploitation of the host system thus are considered ‘pro-viral’. Some interactions display ‘pro-host’ traits, principally the immune response, to control or inhibit viral replication. Concomitant pro-viral and pro-host molecular interactions on the same host molecule suggests more complex virus-host conflicts and genetic signatures that are crucial to host immunity. In this work, machinelearning-based prediction of virus-host interaction directionality was examined by using data from Human immunodeficiency virus type 1 (HIV-1) infection. Host immune responses to viral infections are mediated by interferons(IFNs) in the initial stage of the immune response to infection. IFNs induce the expression of many IFN-stimulated genes (ISGs), which make the host cell refractory to further infection. We propose that there are many features associated with the up-regulation of human genes in the context of IFN-α stimulation. They make ISGs predictable using machine-learning models. In order to overcome the interference of host immune responses for successful replication, viruses adopt multiple strategies to avoid being detected by cellular sensors in order to hijack the machinery of host transcription or translation. Here, the strategy of mimicry of host-like short linear motifs (SLiMs) by the virus was investigated by using the example of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The integration of in silico experiments and analyses in this thesis demonstrates an interactive and intimate relationship between viruses and their hosts. Findings here contribute to the identification of host dependency and antiviral factors. They are of great importance not only to the ongoing COVID-19 pandemic but also to the understanding of future disease outbreaks

Confronting JC virus and Homo sapiens biological signatures.

The present report describes the peptide commonality between JC virus (JCV) and the human proteome at the heptamer level. In total, 53 viral heptapeptides occur in functionally important human proteins with potential consequences for host functions and JCV pathogenesis. A paradigmatic example of a crucial peptide match is the SGKTTLA sequence, shared by JCV LT antigen and human nicotinamide/nicotinic acid riboside kinase, an enzyme involved in myelination processes. In general, the JCV-versus-host heptapeptide overlap may result in a competition between viral sequences and identical motifs in host enzymic active sites, adhesive domains, regulatory signaling motifs, etc., thus interfering with essential reactions and posing disadvantages to the cell. Overall, this study provides a starting point for investigating the role of peptide commonality in host-pathogen interactions

Functions for Murine Norovirus Protein NS1/2 in Mice and Cells

Noroviruses are a leading cause of epidemic gastroenteritis and a major health burden worldwide. One source for outbreaks is individuals who shed virus asymptomatically and persistently. Viral persistence is a successful strategy for viruses to spread, but the mechanisms and consequences of norovirus persistent infection are unknown. In this dissertation, we sought to determine the norovirus determinant(s) of persistence and explore the functions of the associated viral molecules. To determine the viral determinants of persistent infection and tropism, we used the murine norovirus model system in mice. Using plasmid infectious clones for persistent strain CR6 and non-persistent strain CW3, we mapped the viral persistence determinant to the poorly understood non-structural gene NS1/2. The NS1 domain of NS1/2CR6 was necessary and sufficient for persistence. A single amino acid change, NS1/2D94E, conferred persistence on CW3. Viral persistence was restricted to replication and shedding in the intestine, and NS1/2 conferred intestinal tropism. In contrast, the capsid protein VP1 conferred acute replication in the spleen. Moreover, CW3 grew more rapidly in macrophages ex vivo, and this difference mapped to VP1. Therefore, NS1/2 and VP1 are the major determinants for persistence and tropism in vivo and ex vivo. To determine a molecular function of NS1/2, we characterized its interaction with the host protein Vamp-Associated Protein A (VAPA). Murine norovirus replication was delayed in Vapa-/- cells and this was rescued by exogenous VAPA. Moreover, in Vapa-/- cells, NS1/2 protein levels were decreased early during viral infection as well as with electroporated viral RNA. The interaction of murine norovirus NS1/2 with VAPA occurred in a region within the poorly conserved NS1 domain of NS1/2. Investigations in the structural basis of NS1/2-VAPA interaction revealed sequence and functional mimicry between the VAPA binding region of NS1 and the host diphenylalanine-acidic-tract (FFAT)-motif that binds VAPA. The NS1/2-FFAT-mimic interacted with VAPA similarly to bona fide host FFAT motifs. Furthermore, mutations within NS1/2 that disrupted interaction with VAPA inhibited viral replication. Thus, VAPA is a pro-norovirus host factor interacting directly with a norovirus protein that functionally mimics FFAT motifs to co-opt VAPA function. In conclusion, we mapped the norovirus determinants of persistence and tropism to NS1/2 and VP1. Furthermore, we determined that the NS1/2 interaction with VAPA enhanced murine norovirus infection. These are the first structural and functional studies to characterize NS1/2 in molecular detail. This work provides the basis for further exploration to identify the function of NS1/2 that contributes to persistent infection in mice

Alfaviiruse mittestruktuurne proteaas ja tema liitvalgust substraat: täiuslikult korraldatud kooselu reeglid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Alfaviirused (sugukond Togaviridae) on artriiti ja entsefaliiti põhjustavad RNA genoomsed viirused. Nende paljunemise strateegia aluseks on viiruse replikaasi valkude süntees ühe nn. mittestruktuurse eelvalgu P1234 kujul ning selle ajaliselt reguleeritud lõikamine valmis valkudeks nsP2 proteaasi abil. Käesoleva väitekirja aluseks olevad uurimistööd viisid nsP2 substraat-spetsiifilisust tagavate mehhanismide väljaselgitamiseni; muu hulgas kirjeldati uudset proteolüütiliste lõikamiste regulatsioonimehhanismi, mis põhineb liitvalgu erinevate regioonide vahelisel „suhtlemisel“ viiruse replikatsiooni kompleksi moodustamise käigus. Sellest saab järeldada, et P1234 lõikamise ajaline regulatsioon sõltub otseselt replikatsioonikompleksi konfiguratsioonidest, millised omakorda on määratud selle komponentide vaheliste interaktsioonide poolt. Seega tõuseb viiruse nsP2 proteaas esile kui keerulise signaalvõrgustiku keskne element, mille roll viirus infektsiooni regulatsioonis seisneb replikatsiooniga kaasnevate sündmuste „jälgimises“ ja nendele reageerimises. Viimane põhineb sellel, et kui viiruse paljunemine jõuab kindla vahe-etapini, siis kaasneb sellega lõikamiskohtade ja/või muude oluliste struktuuride „esitlemine“ proteaasile, mis reageerib toimunud muudatustele lokaalse signaalülekande, mis lõppkokkuvõttes viib replikaasi kompleksi struktuuri järjestikulistele muudatustele, käivitamisega. Kokkuvõttes, tõid läbiviidud uurimised välja asjaolu, et lisaks varem teada olnud lõikamisjärjestuste äratundmisele, omab ka makromolekulaarsete struktuuride moodustamine viiruse valkude poolt olulist (ja mitmel juhul isegi määravat) rolli viiruse proteaasi töö reguleerimisel. Veel enam, eeldati, et seesugune mitmetahuline regulatsioon võib olla paljukomponentsete proteolüütiliste süsteemide üldine omadus. Kirjeldatud avastused ja nende lahtimõtestamine omavad olulist rolli uurimistöödele, mille eesmärgiks on alfaviiruste paljunemist takistavate lähenemiste väljatöötamine. Nii võib saadud tulemuste põhjal järeldada, et lisaks proteaasi aktiivsuse otsesele mõjutamisele võib viiruse replikatsiooni takistada ka mõjutades proteolüüsi regulatsiooni tagavaid molekulide vahelised seoseid.Alphaviruses from the Togaviridae family are RNA viruses that may cause arthritic syndroms and encephalitis. The alphavirus replication strategy relies on the production of replicase proteins initially in the form of non-structural (ns) polyprotein precursor P1234, which during the course of replication becomes proteolytically processed by the virus-encoded nsP2 protease in a temporally regulated manner. The studies that constitute the basis of this thesis led to identification of the requirements for substrate specificity of nsP2 protease and revealed novel mechanism for the regulation of processing based on the specific communication between distant parts of the viral polyprotein brought together during assembly of replication complex. It was concluded that the order of alphaviral ns-polyprotein processing is mostly dependent on the configuration of the replication complex imposed by intermolecular interactions meant to guarantee timely cleavages. The alphaviral protease therefore emerges as an integral part of the sophisticated signaling mechanism, in which the regulatory task of the protease consists of monitoring the succession and completion of the events of viral infection. Once the respective replication status-induced conformational changes within replicase allow the presentation of the scissile bond and/or other essential determinants of substrate recognition like exosites, the local protease signaling is initiated, which apparently leads to further reconfiguration of the viral replication complex. Combined, the studies unveiled the decisive role played by the macromolecular assembly-dependent component of substrate recognition in addition to the sequence-dependent component, the combination of which may be expected to constitute the basis of regulation in multi-site proteolytic systems in general. Described findings and their interpretations are expected to provide with essential grounds and directions for further studies on the restriction of alphaviral replication through affecting the center of viral proteolytic activity or via intervention with its regulation by targeting intramolecular interactions

Antitarget, Anti-SARS-CoV-2 Leads, Drugs, and the Drug Discovery-Genetics Alliance Perspective

: The most advanced antiviral molecules addressing major SARS-CoV-2 targets (Main protease, Spike protein, and RNA polymerase), compared with proteins of other human pathogenic coronaviruses, may have a short-lasting clinical efficacy. Accumulating knowledge on the mechanisms underlying the target structural basis, its mutational progression, and the related biological significance to virus replication allows envisaging the development of better-targeted therapies in the context of COVID-19 epidemic and future coronavirus outbreaks. The identification of evolutionary patterns based solely on sequence information analysis for those targets can provide meaningful insights into the molecular basis of host-pathogen interactions and adaptation, leading to drug resistance phenomena. Herein, we will explore how the study of observed and predicted mutations may offer valuable suggestions for the application of the so-called "synthetic lethal" strategy to SARS-CoV-2 Main protease and Spike protein. The synergy between genetics evidence and drug discovery may prioritize the development of novel long-lasting antiviral agents

Role of LGP2 in the Innate Immune System upon Viral Infections

RIG-I-Iike receptors (RLRs) are a family of pattern recognition receptors that play an important role in the induction of cellular antiviral responses. RLRs comprise LGP2, RIG-I and MDA5. The latter two initiate antiviral signaling upon binding of viral cytoplasmic double-stranded (ds) RNA, resulting in the expression of interferons (IFNs) and IFN stimulated genes. LGP2 enhances MDA5- and represses RIG-I-mediated signaling even though in the latter case the physiological implication is less clear. Whether posttranslational modifications of LGP2 are involved in its diverse functions remains obscure. Hepatitis delta virus (HDV), a small RNA virus with a circular genome, is an important human pathogen responsible for the most severe form of viral hepatitis. HDV was shown to be sensed by MDA5 but the contribution of LGP2 to induction of the IFN response has not been explored. Hence, the aim of my thesis work was (i) to gain a deeper understanding of the role of LGP2 in regulating RLR signaling and (ii) to determine the contribution of LGP2 and its natural polymorphisms (encoding Q425R, N461S, R523Q) to sensing of HDV and other human viral pathogens. Using knockout and overexpression systems, immunocompetent lung A549 and hepatic HepaRGNTCP cells were measured for their IFN response upon viral infection and synthetic dsRNA stimulation. Mass spectrometry (MS) was performed to elucidate the impact of phosphorylation on the regulatory function of LGP2. Studies in A549 cells indicated faster RIG-I and delayed MDA5 signaling. LGP2 inhibited RIGI and strongly enhanced MDA5 signaling. RNA binding but not ATP hydrolysis was important for both LGP2 functions upon synthetic dsRNA stimulation. In HDV infected HepaRGNTCP cells LGP2 was shown to directly bind HDV RNA. Moreover, LGP2 RNA binding and ATP hydrolysis function were essential to fully activate an IFN response that impaired HDV replication. MS identified S169, S365 and S464 as differentially regulated LGP2 phosphorylation sites. Follow-up functional assays revealed enhanced RIG-I inhibition by the phosphoablative S169A substitution in LGP2. Preliminary data with an S365A/S464D LGP2 double mutation, mimicking steady-state phosphorylation at those sites, indicated delayed responsiveness of this LGP2 mutant towards HDV sensing. Investigation of the Q425R, N461S and R523Q LGP2 variants identified Q425R LGP2, which predominates in the African population, as a gain-of-function version. Q425R LGP2 enhanced basal and accelerated HDV-induced IFN signaling, thus lowering viral replication. This variant also enhanced MDA5-mediated antiviral signaling upon severe acute respiratory syndrome coronavirus type 2 infection. Mechanistically, Q425R LGP2 enhanced MDA5-RNA binding compared to wild-type LGP2. In conclusion, the results obtained during my thesis work broaden our understanding of the regulation of RLR signaling by LGP2. In the future, the gained knowledge might facilitate the development of new antiviral interventions by targeting RLRs for disease control

Circadian regulation of human immunodeficiency virus type 1 replication

Human immunodeficiency virus (HIV) causes significant health problems globally, and despite improvements in therapy there remains no cure. Viral replication is reliant on the host and many physiological processes are influenced by endogenous 24 h oscillations, called circadian rhythms. On a cellular level, circadian transcription factors generate daily oscillations in gene expression. As there is an emerging role for clock components in regulating viral replication, we studied the interplay between the circadian clock and HIV-1. Using a cellular circadian model system, we demonstrate rhythmic HIV-1 replication, which has a period of ~24 hours and is regulated by the cell-intrinsic clock. Pharmacological modulation of circadian transcription factors altered HIV-1 replication across multiple HIV-1 subtypes, indicating pan-genotypic anti-viral potential. Genetic disruption of the circadian activator BMAL1 reduced HIV-1 replication and blunted rhythmicity in transcription. In contrast, knockdown of the circadian repressor REV-ERB enhanced viral replication. We show binding of clock factors to the viral genome and reveal time differential binding of circadian nuclear receptors REV-ERB and ROR, which compete for binding to a ROR response element in the HIV-1 promoter. We demonstrate circadian regulation of HIV-1 host factors, which will influence rhythmic HIV replication. Moreover, we uncover a role for the circadian machinery in regulating latent HIV-1 infection. Bromodomain proteins and salt inducible kinases are both involved in circadian networks, and our findings indicate that they regulate reactivation from latency. Our work provides novel insights in the circadian regulation of HIV-1 replication by molecular components of the clock. Circadian modifiers with anti-viral properties could uncover novel drug targets, which may augment existing treatments and will help to inform HIV therapy and management

Immuno-Competitive Capture Mass Spectrometry, a novel unbiased approach to study endogenous protein-protein interactions

Protein-protein interactions (PPIs) are controlling the majority of biological functions and are the main driver of cellular processes observed in normal as well as pathological conditions. Such a level of controlling is only possible via a high degree of complexity; i.e. a massive number of protein-protein interactions (in the range of couple hundreds of thousands), a variety of physical and structural properties and their reversibility. Moreover, binding affinities can span from micro-molar to high pico-molar level and some proteins are acting as “hubs” by having multiple partners. This sophisticated organization and regulation of PPIs explains why their study is so challenging. No single approaches can capture the full picture and there is an urgent need for innovative platforms to study and analyze PPIs. In this thesis, a novel platform named Immuno-Competitive Capture Mass Spectrometry (ICC-MS) was developed to screen in an unbiased fashion intracellular PPIs. ICC-MS was designed to reach higher specificity compared to classical affinity purification mass spectrometry by introducing a competition step between free and capturing antibody prior to immunoprecipitation. This antibody-based label-free quantitative approach was then combined with a rigorous statistical analysis to extract the cellular interactome of proteins of interest while filtering out non-specifically binding proteins. ICC-MS was first applied to elucidate hepatitis C viral non-structural protein 5A interactome in human hepatoma cells revealing LATS kinases as potential important regulators of viral infection. The study of Glypican-2 and HtrA1 interacting partners further confirmed the ability of ICC-MS to deliver a limited number of highly confident interacting proteins being promising candidates for functional validation. Interestingly, ICC-MS can also be adapted to study interactions formed between proteins and oligonucleotides (Oligo-Competitive Capture Mass Spectrometry or OCC-MS). While it contributed to a better understanding of the mode of action of an SMN2 splicing modifier, the approach could not elucidate the role of protein interactions in antisense oligonucleotides toxicity. Taken together, this innovative approach is suitable to improve the comprehensiveness and accuracy of current protein-protein interactions databases in term of true biological interactome representation

New Insights on Molecular Mechanism of Hepatitis B Virus Covalently Closed Circular DNA Formation

The chronic factor of the Hepatitis B Virus (HBV), specifically the covalently closed circular DNA (cccDNA), is a highly stable and active viral episomal genome established in the livers of chronic hepatitis B patients as a constant source of disease. Being able to target and eliminate cccDNA is the end goal for a genuine cure for HBV. Yet how HBV cccDNA is formed from the viral genomic relaxed circular DNA (rcDNA) and by what host factors had been long-standing research questions. It is generally acknowledged that HBV hijacks cellular functions to turn the open circular DNA conformation of rcDNA into cccDNA through DNA repair mechanisms. With great efforts from the HBV research community, there have been several recent leaps in our understanding of cccDNA formation. It is our goal in this review to analyze the recent reports showing evidence of cellular factor’s involvement in the molecular pathway of cccDNA biosynthesis