Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition

Quantitative Approaches to the Genomics of Clonal Evolution

Many problems in the biological sciences reduce to questions of genetic evolution. Entire classes of medical pathology, such as malignant neoplasia or infectious disease, can be viewed in the light of Darwinian competition of genomes. With the benefit of today's maturing sequencing technologies we can observe and quantify genetic evolution with nucleotide resolution. This provides a molecular view of genetic material that has adapted, or is in the process of adapting, to its local selection pressures. A series of problems will be discussed in this thesis, all involving the mathematical modeling of genomic data derived from clonally evolving populations. We use a variety of computational approaches to characterize over-represented features in the data, with the underlying hypothesis that we may be detecting fitness-conferring features of the biology. In Part I we consider the cross-sectional sampling of human tumors via RNA-sequencing, and devise computational pipelines for detecting oncogenic gene fusions and oncovirus infections. Genomic translocation and oncovirus infection can each be a highly penetrant alteration in a tumor's evolutionary history, with famous examples of both populating the cancer biology literature. In order to exert a transforming influence over the host cell, gene fusions and viral genetic programs need to be expressed and thus can be detected via whole transcriptome sequencing of a malignant cell population. We describe our approaches to predicting oncogenic gene fusions (Chapter 2) and quantifying host-viral interactions (Chapter 3) in large panels of human tumor tissue. The alterations that we characterize prompt the larger question of how the genetics of tumors and viruses might vary in time, leading us to the study of serially sampled populations. In Part II we consider longitudinal sampling of a clonally evolving population. Phylogenetic trees are the standard representation of a clonal process, an evolutionary picture as old as Darwin's voyages on the Beagle. Chapter 4 first reviews phylogenetic inference and then introduces a certain phylogenetic tree space that forms the starting point of our work on the topic. Specifically, Chapter 4 describes the construction of our projective tree space along with an explicit implementation for visualizing point clouds of rescaled trees. The Chapter finishes by defining a method for stable dimensionality reduction of large phylogenies, which is useful for analyzing long genomic time series. In Chapter 5 we consider medically relevant instances of clonal evolution and the longitudinal genetic data sets to which they give rise. We analyze data from (i) the sequencing of cancers along their therapeutic course, (ii) the passaging of a xenografted tumor through a mouse model, and (iii) the seasonal surveillance of H3N2 influenza's hemagglutinin segment. A novel approach to predicting influenza vaccine effectiveness is demonstrated using statistics of point clouds in tree spaces. Our investigations into clonal processes may be extended beyond naturally occurring genomes. In Part III we focus on the directed clonal evolution of populations of synthetic RNAs in vitro. Analogous to the selection pressures exerted upon malignant cells or viral particles, these synthetic RNA genomes can be evolved against a desired fitness objective. We investigate fitness objectives related to reprogramming ribosomal translation. Chapter 6 identifies high fitness RNA pseudoknot geometries capable of inducing ribosomal frameshift, while Chapter 7 takes an unbiased approach to evolving sequence and structural elements that promote stop codon readthrough

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity

Activation of the Antiviral Kinase PKR and Viral Countermeasures

The interferon-induced double-stranded (ds)RNA-dependent protein kinase (PKR) limits viral replication by an eIF2α-mediated block of translation. Although many negative-strand RNA viruses activate PKR, the responsible RNAs have long remained elusive, as dsRNA, the canonical activator of PKR, has not been detected in cells infected with such viruses. In this review we focus on the activating RNA molecules of different virus families, in particular the negative-strand RNA viruses. We discuss the recently identified non-canonical activators 5′-triphosphate RNA and the vRNP of influenza virus and give an update on strategies of selected RNA and DNA viruses to prevent activation of PKR

Topology of Reticulate Evolution

The standard representation of evolutionary relationships is a bifurcating tree. However, many types of genetic exchange, collectively referred to as reticulate evolution, involve processes that cannot be modeled as trees. Increasing genomic data has pointed to the prevalence of reticulate processes, particularly in microorganisms, and underscored the need for new approaches to capture and represent the scale and frequency of these events. This thesis contains results from applying new techniques from applied and computational topology, under the heading topological data analysis, to the problem of characterizing reticulate evolution in molecular sequence data. First, we develop approaches for analyzing sequence data using topology. We propose new topological constructions specific to molecular sequence data that generalize standard constructions such as Vietoris-Rips. We draw on previous work in phylogenetic networks and use homology to provide a quantitative measure of reticulate events. We develop methods for performing statistical inference using topological summary statistics. Next, we apply our approach to several types of molecular sequence data. First, we examine the mosaic genome structure in phages. We recover inconsistencies in existing morphology-based taxonomies, use a network approach to construct a genome-based representation of phage relationships, and identify conserved gene families within phage populations. Second, we study influenza, a common human pathogen. We capture widespread patterns of reassortment, including nonrandom cosegregation of segments and barriers to subtype mixing. In contrast to traditional influenza studies, which focus on the phylogenetic branching patterns of only the two surface-marker proteins, we use whole-genome data to represent influenza molecular relationships. Using this representation, we identify unexpected relationships between divergent influenza subtypes. Finally, we examine a set of pathogenic bacteria. We use two sources of data to measure rates of reticulation in both the core genome and the mobile genome across a range of species. Network approaches are used to represent the population of S. aureus and analyze the spread of antibiotic resistance genes. The presence of antibiotic resistance genes in the human microbiome is investigated

Phleboviruses versus the Type I/III Interferon Response: How Sandfly Fever Sicilian Virus NSs Tackles Interferon Induction and PKR-Mediated Restriction

Phleboviruses (order Bunyavirales, family Phenuiviridae) are arthropod-borne viruses that are emerging globally due to the geographic expansion of long-known members and the identification of numerous novel ones. They span a wide spectrum of virulence, comprising clinically inapparent infection, febrile disease, encephalitis, up to severe haemorrhagic fever and multiorgan failure – with novel isolates including both highly virulent members and abundant others with as yet unknown disease potential. Rift Valley fever virus (RVFV), a long-known member, is highly pathogenic for humans and livestock. Thus, RVFV has been subject to extensive molecular characterization, which established the phleboviral non-structural protein NSs as antagonist of the antiviral interferon (IFN) system and main virulence factor in the mammalian host. Sandfly fever Sicilian virus (SFSV), on the other hand, was identified as causative agent of ‘sandfly fever’, a self-limited febrile disease, during World War II. Nowadays, SFSV is one of the geographically most widespread phleboviruses, causing disease mainly in immunologically naïve military troops and travellers. Although SFSV has been thoroughly characterized with regard to its clinical picture, its interaction with the mammalian host remained almost entirely elusive on the molecular level. In this work, we thus elucidated the molecular mechanisms with which the NSs protein of SFSV counteracts the interferon system. We identified that SFSV NSs dampened the induction of both type I and III interferons by specifically masking the DNA-binding domain of the transcription factor interferonregulatory factor 3 (IRF3). Despite IRF3 inhibition, however, SFSV did not fully abrogate IFN induction, leading to IFN-dependent upregulation of related transcription factor IRF7, which was not affected by SFSV NSs and fostered IFN induction. Additionally, SFSV NSs completely failed to impede IFN signalling, resulting in substantial expression of anti-phleboviral IFN-stimulated genes (ISGs). Thus, SFSV NSs appears to be a modulator rather than a full-blown antagonist of the IFN system. Further, protein kinase R (PKR) possesses a strong restrictive activity towards phleboviruses due to the phosphorylation of its substrate eukaryotic elongation factor 2α (eIF2α) and the ensuing block of protein synthesis. Surprisingly, we found that the NSs protein of SFSV conferred PKR resistance and boosted translation without affecting the activation of PKR or the phosphorylation state of eIF2α. Instead, SFSV NSs targeted eIF2B, the central regulatory hub of the integrated stress response (ISR), further downstream. Of note, as previously characterized viral PKR antagonists all act at the levels of PKR activation and eIF2α phosphorylation, targeting of eIF2B by SFSV NSs represented a novel viral evasion strategy. Interestingly, a common theme emerged during our studies: Highly virulent RVFV, on the one hand, utilizes its NSs to induce the degradation of target host factors via the proteasome, thereby acting in a catalytic mode. Furthermore, it establishes a global block of host gene expression to evade the IFN system. The NSs of mildly virulent SFSV, on the other hand, does not affect the expression levels of its host targets, but rather acts in a very specific and stoichiometric manner for both the inhibition of IFN induction and PKR evasion. Given its importance as exclusive phleboviral IFN antagonist, the NSs protein has been speculated to constitute a correlate of virulence. Our data on SFSV NSs support this hypothesis and argue for the characterization of the NSs proteins of novel phleboviruses with respect to their capacity to inhibit IFN induction, IFN signalling, and PKR activity in order to better estimate their potential to induce disease.Phleboviren (Ordnung Bunyavirales, Familie Phenuiviridae) sind Arboviren, die aufgrund der geographischen Expansion bekannter und der Identifizierung zahlreicher neuer Mitglieder global vermehrt auftreten. Sie umfassen ein breites Spektrum an Virulenz, darunter klinisch inapparente Infektionen, fiebrige Erkrankungen, Enzephalitis, bis hin zu hämorrhagischem Fieber und Multiorganversagen – wobei neue Isolate sowohl hochpathogene Mitglieder als auch zahllose andere mit soweit unbekanntem Krankheitspotential beinhalten. Das Rifttalfieber-Virus (RVFV), ein lange bekanntes Mitglied, ist hochpathogen für Mensch und Vieh. Daher wurde RVFV ausgiebig molekular charakterisiert, was das Nichtstrukturprotein NSs als Antagonist des antiviralen Interferon (IFN)-Systems und Hauptvirulenzfaktor im Säugetierwirt etabliert hat. Das sizilianische Sandfliegenfieber-Virus (SFSV) dagegen wurde während des Zweiten Weltkriegs als Erreger des „Sandfliegenfiebers”, einer selbstlimitierten fiebrigen Erkrankung, identifiziert. Heute ist es bekannt als eines der Phleboviren mit der weitesten geographischen Verbreitung und verursacht Symptome hauptsächlich in immunologisch naiven Soldaten und Reisenden. Obwohl SFSV im Hinblick auf das klinische Bild ausführlich charakterisiert wurde, ist seine Interaktion mit dem Säugetierwirt auf der molekularen Ebene fast komplett unbekannt. In dieser Arbeit haben wir daher die molekularen Mechanismen aufgeklärt, mit denen das NSs-Protein von SFSV dem IFN-System entgegenwirkt. Wir konnten zeigen, dass SFSV NSs die Induktion von Typ-I- und III IFN dämpft, indem es gezielt die DNA-Bindedomäne des Transkriptionsfaktors IRF3 verdeckt. Trotz der Inhibition von IRF3 unterband SFSV die IFN-Induktion dennoch nicht vollständig, was zu einer IFNabhängigen Hochregulation des Transkriptionsfaktors IRF7 führt, der nicht von SFSV NSs beeinträchtigt wird und die IFN-Induktion fördert. Zusätzlich versagte SFSV NSs darin, die IFN-Signaltransduktion zu behindern, woraus eine erhebliche Expression anti-phleboviraler IFN-stimulierter Gene (ISGs) resultierte. Folglich scheint SFSV NSs eher ein Modulator als ein starker Antagonist des IFN-Systems zu sein. Daneben besitzt die Proteinkinase R (PKR) aufgrund der Phosphorylierung des eukaryotischen Initiationsfaktors 2α (eIF2α), und der resultierenden Blockade der Proteinbiosynthese eine stark restriktive Aktivität gegenüber Phleboviren. Überraschenderweise fanden wir, dass das SFSV NSs eine PKR-Resistenz vermittelt und die Translation steigert, ohne jedoch die PKR-Aktivierung oder die eIF2α-Phosphorylierung zu beeinträchtigen. Vielmehr wirkt SFSV NSs auf den nachgeschalteten eIF2B-Komplex, den regulatorischen Knotenpunkt der integrierten Stressantwort. Bemerkenswerterweise hemmen alle bisher charakterisierten viralen PKR-Antagonisten die PKR-Aktivierung oder die eIF2α-Phosphorylierung, sodass die Manipulation von eIF2B durch SFSV NSs eine neue virale Evasionsstrategie darstellt. Interessanterweise zeichnete sich bei unseren Untersuchungen ein gemeinsames Muster ab: Das hochvirulente RVFV setzt sein NSs-Protein für den proteasomalen Abbau seiner Zielfaktoren ein, agiert also sozusagen katalytisch. Daneben verursacht es eine globale Blockade der Genexpresssion des Wirtes, um dem IFN-System zu entgehen. Das NSs des weniger virulenten SFSV dagegen beeinträchtigt nicht die Expressionslevel von Zielfaktoren, sondern scheint sowohl die Hemmung der IFN-Induktion als auch die PKR-Evasion auf stöchiometrische Weise zu vermitteln. In Anbetracht seiner Bedeutung als alleiniger phleboviraler IFN-Antagonist wurde spekuliert, dass das NSs-Protein ein Korrelat für die Virulenz darstellt. Unsere Daten zu SFSV NSs unterstützen diese Hypothese und sprechen für die Charakterisierung der NSs-Proteine neuartiger Phleboviren bezüglich ihrer Fähigkeit, die IFN-Induktion, die IFN-Signaltransduktion und die PKR-Aktivität zu hemmen, um ihr Krankheitspotential besser einzuschätzen

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures