University of Veterinary Medicine Hannover Institute of Virology & Research Center for Emerging Infections and Zoonoses
Virus-cell interactions of mumps viruses and mammalian cells: Entry, replication and immune evasion
THESIS
Submitted in partial fulfilment of the requirements for the degree of Doctor of Natural Sciences
Doctor rerum naturalium (Dr. rer. nat.)
awarded by the University of Veterinary Medicine Hannover
by Sarah Isabella Theresa Hüttl (Hannover)
Hannover, Germany 2020
Main supervisor: PD Nadine Krüger, PhD
Supervision group: PD Nadine Krüger, PhD Prof. Dr. Bernd Lepenies Prof. Dr. Jan Felix Drexler
1st Evaluation: PD Nadine Krüger, PhD Infection Biology Unit German Primate Center - Leibniz Institute for Primate Research, Göttingen
Prof. Dr. Bernd Lepenies Institute of Infection Immunology Research Center for Emerging Infections and Zoonoses University of Veterinary Medicine Hannover
Prof. Dr. Jan Felix Drexler Institute of Virology Charité - Universitätsmedizin Berlin
2nd Evaluation: PD Dr. Anne Balkema-Buschmann Institute of Novel and Emerging Infectious Diseases (INNT) Friedrich-Loeffler-Institut, Insel Riems
Date of submission: 08.09.2020
Date of final exam: 29.10.2020
This project was funded by grants of the German Research Foundation (DFG) to Nadine Krüger (KR 4762/2-1).
Parts of this thesis have been communicated or published previously in:
Publications: N. Krüger, C. Sauder, S. Hüttl, J. Papies, K. Voigt, G. Herrler, K. Hardes, T. Steinmetzer, C. Örvell, J. F. Drexler, C. Drosten, S. Rubin, M. A. Müller, M. Hoffmann. 2018. Entry, Replication, Immune Evasion, and Neurotoxicity of Synthetically Engineered Bat-Borne Mumps Virus. Cell Rep. 25(2):312-320.
S. Hüttl, M. Hoffmann, T. Steinmetzer, C. Sauder, N. Krüger. 2020. The amino acid at position 8 of the proteolytic cleavage site of the mumps virus fusion protein affects viral proteolysis and fusogenicity. J Virol. 94(22):e01732-20.
S. Hüttl, T. Steinmetzer, J. F. Drexler, N. Krüger. 2020. Furin-independent cleavage of human and bat-derived mumps virus F proteins. Submitted.
Oral presentations: 23/10/2019 Seminar in “Current Topics in Biomedicine” University of Veterinary Medicine Hannover, Germany Proteolytic activation and fusogenicity of human and bat-derived mumps viruses. S. Hüttl.
Poster presentations: 03/2018 28th Annual Meeting of the Society for Virology, Würzburg, Germany Human and bat-derived mumps virus N, P and L proteins are able to interact for efficient replication activity. S. Hüttl, M. Hoffmann, N. Krüger.
10/2018 National Symposium on Zoonoses Research 2018, Berlin, Germany Immunomodulatory properties of the V and SH proteins of a bat-derived mumps virus. S. Hüttl, M. Hoffmann, N. Krüger.
03/2019 29th Annual Meeting of the Society for Virology, Düsseldorf, Germany Proteolytic activation of the fusion glycoprotein of a bat-derived mumps virus. S. Hüttl, C. Sauder, S. Rubin, M. Hoffmann, N. Krüger.
10/2019 National Symposium on Zoonoses Research 2019, Berlin, Germany Proteolytic activation and fusogenicity of human and bat-derived mumps viruses. S. Hüttl, T. Steinmetzer, C. Sauder, S. Rubin, M. Hoffmann, N. Krüger.
Contents
Contents
I List of abbreviations ...... III II List of figures ...... VII III Summary ...... IX IV Zusammenfassung...... XI 1 Introduction ...... 1 1.1 Paramyxoviruses ...... 1 1.1.1 Taxonomy ...... 1 1.1.2 Morphology and genome organization ...... 1 1.2 Replication cycle of paramyxoviruses ...... 3 1.2.1 Viral attachment ...... 3 1.2.2 Virus-induced fusion ...... 4 1.2.3 Viral replication ...... 6 1.3 Host immune response and viral immune evasion ...... 8 1.3.1 Interferon pathway ...... 8 1.3.2 NF-κB signaling pathway ...... 10 1.4 Mumps virus infection ...... 12 1.4.1 The infectious disease mumps ...... 13 1.4.2 Vaccination ...... 13 1.4.3 Mumps virus outbreaks ...... 14 1.5 Bats and zoonotic viruses ...... 15 1.5.1 Zoonotic diseases and pathogens ...... 15 1.5.2 Bats as reservoir hosts of zoonotic viruses ...... 16 1.5.3 Bat-derived mumps virus ...... 17 1.6 Aims of the study ...... 18 2 Entry, replication, immune evasion and neurotoxicity of a synthetically-engineered bat-borne mumps virus ...... 21 3 The amino acid at position 8 of the proteolytic cleavage site of the mumps virus fusion protein affects viral proteolysis and fusogenicity ...... 23 4 Furin-independent cleavage of human and bat-derived mumps virus F proteins .. 25 5 Discussion ...... 37 5.1 batMuV is capable of infecting human cells and circumventing the host immune response ...... 37 5.2 A single amino acid modifies fusogenicity and proteolysis of MuV F ...... 40 5.3 Furin-independent cleavage of MuV F ...... 43 6 References ...... 47
I Contents
7 Supplementary data ...... 61 8 Appendix ...... 63 8.1 Sequences ...... 63 8.2 Primers ...... 77 8.3 Sequences of F peptides used in the FRET assay ...... 81
II List of abbreviations
I List of abbreviations
88-1961 hMuV strain MuVi/1961.USA/0.88 aa Amino acid batMuV BatPV/Epo_spe/AR1/DCR/2009, bat-derived MuV CD Cytoplasmic domain CNS Central nervous system CoV Coronavirus CPE Cytopathic effect C-terminus COOH terminus of proteins DNA Deoxyribonucleic acid EBOV Ebola virus ED Ectodomain e.g. Exempli gratia (for example) F Fusion glycoprotein Fig. Figure FP Fusion peptide FRET Fluorescence resonance energy transfer G Glycoprotein H Hemagglutinin HAT Human airway trypsin-like protease HCV Hepatitis C virus HeV Hendra virus HIV Human immunodeficiency virus hMuV Human mumps virus HN Hemagglutinin-neuraminidase HPIV Human parainfluenza virus HR Heptad repeat region IFN Interferon IFNAR Interferon alpha/beta receptor IκBα NF-κB inhibitor alpha IKK Inhibitor of NF-κB kinase IL Interleukin IL-1R Interleukin 1 receptor IRF Interferon-regulatory factor ISG Interferon-stimulated gene ISGF Interferon-stimulated gene factor
III List of abbreviations
ISRE Interferon-stimulated response elements JAK Janus kinase kDa Kilodalton L Large protein (RNA polymerase) M Matrix protein MAPK Mitogen-activated protein kinase MeV Measles virus MMR Measles, mumps and rubella vaccine mRNA Messenger RNA MuV Mumps virus MYD88 Myeloid differentiation primary response 88 N Nucleoprotein NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NiV Nipah virus NSP Non-structural protein nt Nucleotide
N-terminus NH2-terminal end of proteins ORF Open reading frame P Phosphoprotein P8 Position 8 within cleavage site of fusion glycoprotein PAMP Pathogen-associated molecular patterns PIV Parainfluenza virus rbatMuV Recombinant batMuV RdRp RNA-dependent RNA polymerase RIP Receptor-interacting protein kinase RNA Ribonucleic acid RNP Ribonucleoprotein RSV Respiratory syncytial virus SA Sialic acids SARS Severe acute respiratory syndrome SeV Sendai virus SH Small hydrophobic protein STAT Signal transducer and activator of transcription TAB TGF-ß-activated kinase and MAP3K7-binding protein Tab. Table TD Transmembrane domain TGN trans-Golgi network
IV List of abbreviations
TLR Toll-like receptor TNF Tumor necrosis factor TNFR Tumor necrosis factor receptor TMPRSS2 Transmembrane protease serine subtype 2 TRADD TNFR-associated DEATH domain TRAF TNFR-associated factor TTSP Type II transmembrane serine proteases TYK Tyrosine kinase
Nucleobases: A - adenine; C - cytosine; G - guanine; T - thymine
Amino acid 1-letter symbol 3-letter symbol Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamic acid E Glu Glutamine Q Gln Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val
V
List of figures
II List of figures
Fig. 1: Taxonomic classification of the family Paramyxoviridae...... 1 Fig. 2: Schematic structure of virus particles and organization of the RNA genome of mumps viruses...... 2 Fig. 3: Schematic structure of the fusion glycoprotein...... 5 Fig. 4: Membrane fusion process mediated by paramyxoviruses...... 6 Fig. 5: Replication cycle of paramyxoviruses...... 7 Fig. 6: Interferon pathway...... 10 Fig. 7: NF-κB signaling pathway...... 12 Fig. S1: Structural formula of leucine, proline, serine and threonine...... 61 Fig. S2: Modeling of mumps virus F protein structure...... 62
VII
Summary
III Summary
“Virus-cell interactions of mumps viruses and mammalian cells: Entry, replication and immune evasion” by Sarah Hüttl
This thesis aimed to gain deeper knowledge about the virus-host interplay between mumps viruses (MuV) and mammalian cells focusing on the viral entry, replication and interference with the host immune response.
The detection of RNA of a bat-derived MuV (batMuV) in an African epauletted fruit bat (Epomophorus sp.) implies that bats might serve as reservoir hosts for MuVs. First studies provided evidence that batMuV is likely to be infectious for humans, but due to the lack of an infectious virus isolate many questions regarding the viral replication cycle remained unclear. Here authentic recombinant batMuV was generated and used for in vitro infection studies constantly drawing a comparison with human MuV (hMuV). It was shown that batMuV can replicate in human and bat-derived cells with a higher efficiency than hMuV. Cellular factors required for viral entry seemed to be similar for both MuVs irrespective of their origin. Further, batMuV and hMuV were found to be capable of interfering with the host immune response in bat and human cells by the activity of their immunomodulatory proteins, the small hydrophobic protein (SH) and the non-structural proteins V and I. The obtained results strengthen the assumption of a zoonotic potential of batMuV.
The second objective addressed the fusogenicity and proteolysis of the MuV fusion (F) protein. F induces the fusion between viral and host cell membranes and between the membranes of infected and neighbored cells to enable the release of the viral genome into the cytoplasm of the target cell. A varying fusogenicity among MuV strains has been described and might correlate with the virulence. So far, the reason for the differences in the degree of mediating fusion as well as the mechanism behind impaired or extended fusogenicity and the impact for viral pathogenesis are incompletely understood. The amino acid residue at position 8 (P8) turned out to be a remarkable variation within the multibasic cleavage motif of F of different MuV strains. The results described here demonstrate that P8 affects the fusogenicity of MuVs by influencing the efficiency of furin-mediated proteolysis. Further, P8 might have an impact on the mode of viral spread and either facilitates virus-cell or cell-to-cell fusion suggesting that these fusion events display two distinct processes with different mechanisms and requirements.
IX Summary
Further investigations on the proteolysis of human and bat-derived MuV F proteins revealed that a furin-independent activation by type II transmembrane serine proteases (TTSPs) can occur and presumably supports the viral replication within the respiratory epithelium. Importantly, camostat mesylate, a safe and clinically applied inhibitor of TTSPs, has been identified as a potential antiviral reagent against MuV infections. These findings provide novel insights into the proteolysis and fusogenicity of MuV F and might help to identify licensed protease inhibitors as potential antiviral drugs to reduce the duration and severity of clinical signs during MuV infections.
X Zusammenfassung
IV Zusammenfassung
“Virus-Zell Interaktionen von Mumpsviren und Säugetierzellen: Eintritt, Replikation und Immunevasion” von Sarah Hüttl
Ziel dieser Arbeit war es, fundierte Kenntnisse über die Virus-Zell Interaktionen zwischen Mumpsviren und Säugetierzellen zu erhalten, wobei der Schwerpunkt auf dem Viruseintritt, der Replikation sowie der Umgehung der Immunantwort der Wirtszellen lag.
Der Nachweis von RNA eines Fledertier-assoziierten Mumpsvirus (batMuV) in einem afrikanischen Flughund (Epomophorus sp.) deutete auf Flughunde als zusätzliche Reservoirwirte für MuVs hin. Erste Studien ergaben, dass batMuV wahrscheinlich für den Menschen infektiös ist, aber aufgrund eines fehlenden Virusisolats blieben bislang viele Fragen bezüglich des viralen Replikationszyklus ungeklärt. Im Zuge dieser Arbeit wurde daher rekombinantes batMuV generiert und für in vitro Infektionsstudien herangezogen, in denen ein ständiger Vergleich mit einem humanen MuV (hMuV) gezogen wurde. Es wurde gezeigt, dass batMuV in humanen und Fledertier-Zellen effizienter repliziert als hMuV. Beide MuVs scheinen zudem ähnliche zelluläre Faktoren für den Viruseintritt zu nutzen. Weiterhin sind batMuV und hMuV in der Lage durch die Aktivität ihrer immunmodulatorischen Proteine, dem SH-Protein und der Nichtstrukturproteine V und I, in die Immunantwort des Wirts einzugreifen. Die erzielten Ergebnisse bestärken die Annahme eines zoonotischen Potentials seitens batMuV.
Der zweite Teil dieser Arbeit befasste sich mit der Fusogenität und Proteolyse des MuV- Fusionsproteins (F). F vermittelt die Fusion zwischen Virus- und Wirtszellmembranen sowie zwischen den Membranen infizierter und benachbarter Zellen, um die Freisetzung des viralen Genoms in das Zytoplasma der Zielzelle zu ermöglichen. Unter den MuV-Stämmen wurde eine variierende Fusogenität beobachtet, die möglicherweise mit der Virulenz korreliert. Die Bedeutung und der Mechanismus hinter diesen Unterschieden im Fusionsausmaß sowie deren Auswirkung auf die Pathogenese sind bislang nur unvollständig untersucht worden. Die Aminosäure an Position 8 (P8) des multibasischen Spaltmotivs von F weist eine deutliche Variation innerhalb verschiedener MuV-Stämme auf. In dieser Arbeit wird gezeigt, dass P8 Einfluss auf die Fusogenität von MuVs hat, indem es die Effizienz der Furin-vermittelten Proteolyse beeinflusst. Darüber hinaus scheint P8 einen Effekt auf die Virusausbreitung zu haben: P8 erleichtert entweder die Virus-Zell- oder die Zell-Zell-Fusion, was die Vermutung aufkommen lässt, dass diese beiden Fusionsereignisse zwei zu unterscheidende Prozesse darstellen, die nach unterschiedlichen Mechanismen ablaufen.
XI Zusammenfassung
Weitere Untersuchungen zur proteolytischen Aktivierung von humanen und Fledertier- assoziierten MuV F Proteinen zeigten, dass eine Furin-unabhängige Spaltung von F durch Typ II transmembrane Serinproteasen (TTSPs) stattfinden kann, und somit vermutlich die Replikation im respiratorischen Trakt begünstigt. Zudem wurde gezeigt, dass Camostat Mesilate, ein sicherer und bereits klinisch angewendeter TTSP-Inhibitor, ein potentielles Therapeutikum mit antiviraler Aktivität gegen MuV darstellt. Diese Erkenntnisse gewähren neue Einblicke in die Proteolyse und Fusogenität von MuV F und können zur Identifizierung von lizensierten Protease-Inhibitoren beitragen, die als potentielle antivirale Medikamente die Dauer und Schwere von MuV-Infektionen reduzieren könnten.
XII Introduction
1 Introduction 1.1 Paramyxoviruses 1.1.1 Taxonomy Paramyxoviruses belong to the order Mononegavirales which comprises enveloped viruses with a single-stranded, non-segmented and negative-sensed ribonucleic acid (RNA) genome. The family Paramyxoviridae harbors four subfamilies (Avulavirinae, Metaparamyxovirinae, Orthoparamyxovirinae, Rubulavirinae), as well as three genera that are not assigned to a viral subfamily (Fig. 1). Paramyxoviruses have a broad host range and can be found in mammalian and avian species, fish and reptiles (Clark et al. 1979; Mitchell et al. 2011; Hyndman et al. 2013). Depending on the virus species, paramyxovirus infections result in respiratory diseases, systemic infections or infections of the central nervous system (CNS) (Sherrini et al. 2014; Rubin et al. 2015; Rendon-Marin et al. 2019; Liu et al. 2020). Well-known species representatives are the zoonotic henipaviruses Hendra (HeV) and Nipah (NiV) virus as well as the human pathogenic measles (MeV) and mumps (MuV) virus.
Fig. 1: Taxonomic classification of the family Paramyxoviridae. Human and veterinary medicine relevant species are listed exemplarily.
1.1.2 Morphology and genome organization The RNA genome of paramyxoviruses contains at least six open reading frames (ORFs) encoding six structural proteins from the 3´ to the 5´ terminus: nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), attachment glycoprotein (hemagglutinin-neuraminidase (HN), hemagglutinin (H) or glycoprotein (G)) and viral RNA-
1 Introduction dependent RNA polymerase (RdRp, large protein L) (Fig. 2). All viruses of the genus Orthorubulavirus and one avulavirus (avian paramyxovirus type 6) code for an additional structural protein, the small hydrophobic protein (SH) (Samuel et al. 2010; Baker et al. 2013). The genome size of paramyxoviruses varies between 13,000 and 19,000 nucleotides (nt) and follows the rule of six (Kolakofsky et al. 1998).
Typically, paramyxovirus particles are spherical shaped but filamentous or pleomorphic forms can also occur (Chua et al. 2001; Alayyoubi et al. 2015). A phospholipid bilayer forms the envelope of paramyxoviruses and is derived from the host cell membrane (Fig. 2). The M protein lines the inner coat of the viral envelope and is required for the rigidity and structure of viral particles. During viral replication, M is responsible for the assembly of newly synthesized viral proteins within and budding of virions from infected cells (Tanabayashi et al. 1990; Schmitt et al. 2002; Wang et al. 2010; Battisti et al. 2012). The viral RNA genome is encapsidated by N forming the ribonucleoprotein (RNP). Through the binding of P to N and L, the RNA transcriptase complex is built. The viral envelope contains two glycoproteins mediating the viral entry process: The F protein and the attachment glycoprotein which are described in more detail in the following chapters. Also the SH protein, that is present in some paramyxoviruses, is incorporated into the envelope and expressed on the viral surface.
Fig. 2: Schematic structure of virus particles and organization of the RNA genome of mumps viruses. F: fusion glycoprotein, HN: hemagglutinin-neuraminidase, L: large protein (RNA-dependent RNA polymerase), M: matrix protein, N: nucleoprotein, P: phosphoprotein, SH: small hydrophobic protein, UTR: untranslated region.
The SH protein seems to be not essential for viral replication, but it might be involved in determining virulence and immunogenicity of paramyxoviruses as SH is capable of modulating the host cell immune response (Bukreyev et al. 1997; Tripp et al. 1999; Whitehead et al. 1999;
2 Introduction
Jin et al. 2000; Ling et al. 2008). For some viruses including MuV, J paramyxovirus, parainfluenza virus 5 (PIV5, former simian virus 5) and respiratory syncytial virus (RSV) it has been demonstrated that SH interferes with the tumor necrosis factor (TNF)-α pathway resulting in the reduced nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κb) activation and translocation and decreased cell apoptosis (Bukreyev et al. 1997; Fuentes et al. 2007; Carter et al. 2010; Franz et al. 2017; Abraham et al. 2018; Kruger et al. 2018). A variety of different non-structural proteins (NSP) exist among all paramyxovirus species. These proteins are not part of the viral particles but are produced within the infected cells and have beneficial effects on viral replication and/or infectivity. The NSPs of paramyxoviruses are probably involved in the evasion of the host immune response and the manifestation of virulence (Yoneda et al. 2010; Mathieu et al. 2012). The most frequent and well-studied NSP is the V protein which interferes with the interferon (IFN)-α pathway (Andrejeva et al. 2004; Xu et al. 2012b). The NSPs C and I share a similar function and inhibit the IFN-mediated immune response (Yokota et al. 2011; Mathieu et al. 2012; Odkhuu et al. 2014; Nishie et al. 2015). For most paramyxoviruses, the function of their NSPs has not been investigated in detail.
1.2 Replication cycle of paramyxoviruses The first step of the viral replication cycle is the entry process that comprises the attachment and binding of viruses to their host cells and the release of the viral genome into the cytoplasm. In the case of enveloped viruses, a fusion between the membranes of the virus particle and the target cell has to occur to release the genetic information into the host cell. The entry of paramyxoviruses is mediated by their surface glycoproteins one of whom is responsible for the attachment and the other for the fusion process.
1.2.1 Viral attachment Attachment glycoproteins of paramyxoviruses are type II membrane proteins that are integrated into the viral envelope as tetramers consisting of two dimers. Each monomer is composed of an N-terminal cytoplasmic domain (CD), followed by a transmembrane domain (TD) and the ectodomain (ED) comprising a stalk region that forms a four helix bundle and probably represents the interaction site with F and a globular head domain which consist of a six-bladed ß-propeller and mediates the binding to cellular receptors and - depending on the virus species - exhibits enzymatic activity (Deng et al. 1995; Crennell et al. 2000; Lawrence et al. 2004; Melanson et al. 2004; Colf et al. 2007; Lee et al. 2008; Bose et al. 2011; Yuan et al. 2011; El Najjar et al. 2014). The attachment glycoproteins are responsible for the binding of viruses to the target cell membrane - either by binding to sialic acids on cell surface-expressed macromolecules or to specific cellular receptors.
3 Introduction
Depending on their binding and enzymatic activity, the attachment glycoproteins of paramyxoviruses are divided into three classes: HN proteins which are expressed by avula- and rubulaviruses attach to sialic acids and are therefore also able to catalyze the cleavage of these neuramic acid derivates to avoid self-aggregation and to enable the release of new virions (Villar et al. 2006). Besides this neuraminidase activity, HN proteins are able to hemagglutinate red blood cells. The H protein which is expressed by morbilliviruses lacks the neuraminidase activity and binds to specific protein receptors e.g. SLAMs or CD46 (Masse et al. 2004; Seki et al. 2020). The glycoprotein G is expressed by henipaviruses, binds to specific receptors such as Ephrin-B2/-B3 and lacks both, neuraminidase and hemagglutination activity (Xu et al. 2012a; Johnson et al. 2015).
Besides binding to cellular receptors, the attachment glycoproteins are further involved in activating the F proteins for membrane fusion. The interaction between the viral glycoproteins is highly specific and occurs only between closely related viruses. According to the “stalk exposure model” of Bose et al., the receptor binding of the attachment protein leads to rearrangements within the head domain, causing a disturbed connection of head and stalk which in turn exposes an F-interaction site in the stalk domain (Bose et al. 2011; Bose et al. 2014).
The HN protein of MuVs has a molecular weight of 74 - 80 kDa and a size of 582 amino acids (aa) (Mahon 2003). HN is the main target of neutralizing antibodies that bind to the head domain where they disturb the binding of HN to cellular receptors and its enzymatic activity (Orvell et al. 1997a; Mahon 2003). It has been shown that MuV HN proteins bind to α2,3-, α2,6- and α2,8-linked sialic acids with different preference depending on the MuV strain (Brostrom et al. 1971; Leprat et al. 1979; Hosaka et al. 1998; Santos-Lopez et al. 2009; Kubota et al. 2016; Kubota et al. 2019).
1.2.2 Virus-induced fusion The fusion glycoprotein F of paramyxoviruses is a type I membrane protein which is integrated in the viral envelope as a homotrimer. The F protein consists of an N-terminal ED which includes the hydrophobic fusion peptide (FP) and three heptad repeat regions (HRA, HRB, HRC) followed by the TD and the CD at the C-terminal end (Fig. 3). The F protein plays an important role in the viral fusion process by promoting the fusion between viral and host cell membranes (Lamb 1993; Samal 2011). Further, F mediates cell-to-cell fusions: infected cells fuse with neighbored (uninfected) cells and as a result, giant multinucleated cells, so called syncytia, are formed (Wolinsky et al. 1978; McCarthy et al. 1980). Viral spread by release of virions as well as by syncytium formation is therefore possible.
4 Introduction
F is synthesized as the fusion-inactive precursor F0 that has to undergo proteolysis and conformational changes to obtain the ability to mediate fusion. The proteolytic cleavage is performed by cellular proteases that recognize a specific cleavage motif directly upstream of the FP. The cleavage motif and thereby also the cellular protease responsible for F cleavage differ among the paramyxovirus species. Many paramyxovirus F proteins such as these of MuV, PIV5 or MeV are processed intracellularly by furin at a multibasic cleavage site within the trans-Golgi network (TGN) (Molloy et al. 1999; Thomas 2002). In contrast to this, F0 precursors of Sendai virus (SeV) or human parainfluenza virus 1 (HPIV1) are first integrated into newly synthesized viral particles before cleavage at a monobasic cleavage site occurs extracellularly by proteases such as transmembrane protease serine subtype II (TMPRSS2) or secreted trypsin-like proteases expressed in the respiratory tract (Hidaka et al. 1984; Kido et al. 1992; Ambrose et al. 1995). In case of henipaviruses, the cleavage involves clathrin- mediated endocytosis of F0 that is first transported to the cell surface. Within the endosome, the cysteine protease cathepsin L (or B) cleaves after a single basic aa residue of F requiring a low pH (Diederich et al. 2005). In the following, the cleavage of paramyxovirus F is described in more detail. The proteolysis results in the cleavage of F0 into the disulfide-linked F1 and F2 subunits (Fig. 3). As a consequence, the FP is present at the new N-terminal end of the F1 subunit to be exposed towards the host cell membrane (Lamb et al. 2006).
Fig. 3: Schematic structure of the fusion glycoprotein. F0: fusion-inactive F precursor, F1/2: disulfide-linked subunits of proteolytically cleaved and fusion-active F protein, FP: fusion peptide, HRA/HRB/HRC: heptad repeat regions A/B/C, TD: transmembrane domain, CD: cytoplasmic domain. Scissor indicates multibasic cleavage site as found in mumps virus F: arginine-X-(arginine/lysine/X)-arginine.
Second, an interaction of F with the corresponding attachment glycoprotein is necessary to induce conformational changes of F that enable the exposed FP to get in contact with the target cell membrane (Stone-Hulslander et al. 1997; Yao et al. 1997; Plemper et al. 2011; Porotto et al. 2011; Chang et al. 2012; Porotto et al. 2012a; Porotto et al. 2012b). This interaction of F and H/HN/G is often incompletely understood but F always undergoes
5 Introduction irreversible conformational changes at the end of which a six-helix bundle of its HRs is formed (Zhu et al. 2003; Markosyan et al. 2009; Smith et al. 2009). In the pre-hairpin intermediate conformation, the FP of F is exposed and able to contact the host cell membrane: HRA converts into an unstable helical trimeric coiled coil and extends towards the target membrane in which FP is integrated while HRB, also present as coiled α-helices, remains anchored in the viral membrane (Fig. 4). Rearrangements continue as HRB rotates about 180° to bring HRA and HRB into closer contact. A “zipping process” (principle of zipper) follows in which HRA and HRB are anti-parallel orientated and gradually merge, the six-helix bundle starts to be formed and the outer membranes start to fuse resulting in a hemifusion. Next, the inner membranes fuse as well and with the fully completed formation of a stable six-helix bundle structure of F the post-fusion state is reached. Finally, an expanding fusion pore is formed through which the viral RNA genome is released into the cytoplasm. Probably, the actin of the cytoskeleton helps to expand the fusion pore (Smith et al. 2009; Aguilar et al. 2016; Azarm et al. 2020).
Fig. 4: Membrane fusion process mediated by paramyxoviruses. Attachment protein (yellow: head, purple: stalk), fusion protein F (red: fusion peptide FP, blue: HRA and stalk including HRB, green: transmembrane domain, orange: cytoplasmic domain), HRA/B: heptad repeat region A/B. Modified after Aguilar et al., 2016.
1.2.3 Viral replication The viral replication process of paramyxoviruses takes place in the host cell cytoplasm and is mediated by the RNA transcriptase complex consisting of N, P and L proteins (Fig. 5). Once the viral genome has been released into the cytoplasm of the host cell, the negative-sensed RNA genome serves as template for the RdRp during the transcription as well as the replication process (Lamb et al. 2001). The transcription results in the synthesis of positive-stranded messenger RNAs (mRNA) which are further translated by host cell enzymes. After protein synthesis, the viral proteins are transported to the surface of the infected cell to assemble with the replicated negative-sensed copy RNAs. During their transport to the cell surface, viral glycoproteins are further modified in cellular compartments. These modifications include e.g. the addition of carbohydrates to and the folding of the viral proteins in the endoplasmic
6 Introduction reticulum (ER) and Golgi apparatus (Watanabe et al. 2019). Also the proteolytic activation of the F proteins by cellular proteases occurs during the cellular transport within different compartments, depending on the virus species and in particular on the proteolytic cleavage motif. Regarding the replication, transcription of the viral RNA genome into a full-length positive-stranded anti-genome template is necessary. The anti-genome is further used by the RdRp to synthesize full-length copies of the negative-sensed RNA genome (Pickar et al. 2014). Finally, the M protein-mediated assembly of newly synthesized viral proteins and RNA has to take place to form new infectious viral particles. The budding process is the last step of the viral replication cycle and describes the release of newly synthesized virions from the host cell membrane. For those paramyxoviruses, that are capable of binding to sialic acids present on the cellular membrane, the neuraminidase activity of the HN proteins is required to disrupt the binding of virions to the producer cell and to avoid self-aggregation (Takimoto et al. 2004; Harrison et al. 2010; Aguilar et al. 2011).
Fig. 5: Replication cycle of paramyxoviruses. The shown intracellular cleavage of the fusion protein F by cellular proteases within the trans-Golgi network occurs e.g. for mumps viruses. Modified after Aguilar et al., 2011.
7 Introduction
1.3 Host immune response and viral immune evasion To guarantee a successful infection and its establishment several barriers have to be overcome by the virus - one of these is the immune response of the host. To evade or antagonize the host immunity, viruses have developed several strategies. These strategies include (i) the secretion of ligand or receptor imitates, virus-encoded enzymes such as neuraminidase, proteases or polymerase, (ii) the interference with apoptosis or the complement system, (iii) the induction of cell death of immune cells, (iv) the escape or hiding via latency and (v) the intervention with signaling pathways by interaction with cytokines, chemokines or other involved proteins resulting in the inhibition of antiviral gene expression (Finlay et al. 2006). The efficiency of circumventing the antiviral host defense decisively defines the virulence and pathogenicity of a virus. However, many viral immunomodulating proteins are yet not identified and mechanisms behind some strategies remain elusive. In the following, signaling pathways are addressed in which the immunomodulatory proteins of MuVs, the glycoprotein SH and the NSPs V and I, are involved.
1.3.1 Interferon pathway During the type I IFN signaling pathway, the cytokines IFN-α or IFN-ß bind to a heterodimeric receptor which is composed of the subunits IFN alpha/beta receptor 1 (IFNAR-1) and IFNAR- 2 and expressed on the cell surface resulting in the activation of the Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway. The tyrosine kinase 2 (TYK2) and JAK1 are intracellularly recruited to the receptor and activate the dimerizing STAT1/2 by phosphorylation. Next, STAT1/STAT2 binds to the IFN-regulatory factor 9 (IRF9) forming the IFN-stimulated gene factor 3 (ISGF3) complex which translocates into the nucleus where it binds to IFN-stimulated response elements (ISRE) inducing the expression of IFN-stimulated genes (ISG) (Platanias 2005; García-Sastre 2017). ISGs interfere with the viral replication cycle e.g. by inhibiting viral entry, uncoating and release of new virions or by destroying single- stranded RNA (Haller et al. 1998; Stremlau et al. 2006; Hovanessian 2007; Sauter et al. 2010; Huang et al. 2011). Viruses can intervene in the IFN-mediated JAK-STAT pathway at different points to disturb the further signaling (Fig. 6) (Nan et al. 2017). For example, the vaccinia virus expresses a soluble IFNAR analogue that catches IFN (Alcamí et al. 2000) and influenza A virus H5N1 downregulates the IFNAR expression (Jia et al. 2010). The inhibition of the phosphorylation of STATs by suppressing the phosphorylation activity of JAK1 and TYK2 or the dephosphorylation of STATs are described for many viruses such as hepatitis C virus (HCV), RSV, West Nile virus and dengue virus (Guo et al. 2005; Ho et al. 2005; Senft et al. 2010; Kumthip et al. 2012). In addition, the proteasomal degradation of STATs is a common strategy as observed for HCV, RSV or Zika virus (Lin et al. 2005; Elliott et al. 2007; Dar et al. 2017).
8 Introduction
The interference via miRNAs targeting JAK1, IRF9 or IFNARs is e.g. known for HCV or RSV (Mukherjee et al. 2015; Zhang et al. 2016). The ISGF3 complex represents an additional target for viral proteins: its translocation into the nucleus can be reduced by Ebola virus (EBOV) (Reid et al. 2006) or its binding to ISRE is prevented by HCV (de Lucas et al. 2005). With regard to paramyxoviruses, their NSPs encoded by the P gene interfere with the STAT signaling in different ways: MeV V and C proteins inhibit the phosphorylation of STAT1 by binding to the IFNAR-JAK1-STAT1 complex (Yokota et al. 2003; Caignard et al. 2007) and SeV C protein binds to STAT1 thus suppressing its activity (Oda et al. 2015). The V proteins of MuV, PIV5 and Newcastle disease virus degrade STAT1 via the ubiquitin-proteasome pathway whereas the V protein of HPIV2 is responsible for STAT2 degradation (Didcock et al. 1999; Kubota et al. 2001; Parisien et al. 2001; Andrejeva et al. 2002; Huang et al. 2003; Precious et al. 2005). Nuclear translocation of STATs is also disturbed during a NiV and HeV infection as their V proteins bind and restrain the transcription factors in the cytoplasm (Rodriguez et al. 2003). It has further been stated that the V proteins of orthorubulaviruses not only restrict IFN signaling but also the IFN induction by the inhibition of IRF3 which mediates the expression of IFN-α and IFN-ß (He et al. 2002; Komatsu et al. 2004; Lu et al. 2008; Xu et al. 2012b). Moreover, the MuV V protein can bind to the receptor for activated C kinase 1 (RACK1) and thereby prevent its function as adaptor protein promoting the association of STAT1 to IFNAR (Kubota et al. 2002). In addition, MuV V can further antagonize the IFN-ß- induced phosphorylation of STAT1 and STAT2 (Kubota et al. 2005). As shown by Rosas- Murrieta et al. MuV V protein can block IFN-α-induced cell apoptosis by modulating the activity of certain caspases as well as reduce STAT1 phosphorylation (Rosas-Murrieta et al. 2010; Rosas-Murrieta et al. 2011). As MuV V and I are expressed from the P gene by RNA editing both NSPs share an identical aa sequence upstream of the RNA editing site. MuV V has a length of 224 aa, whereas MuV I consists of 171 aa, 155 of which are identical to the N- terminus of V (Paterson et al. 1990; Vidal et al. 1990). Consequently, MuV I is also able to interfere with the IFN-β-mediated immune response in a similar fashion as MuV V (Kruger et al. 2018).
9 Introduction
Fig. 6: Interferon pathway. Intervention of some exemplary viruses at different sites in the pathway is shown. DENV: dengue virus, EBOV: Ebola virus, IFN: interferon, IFNAR: IFN receptor, IRF: IFN-regulatory factor, ISGF: IFN-stimulated gene factor, ISRE: IFN-stimulated response element, HCV: hepatitis C virus, HeV: Hendra virus, HPIV: human parainfluenza virus, JAK: Janus kinase, MeV: measles virus, MuV: mumps virus, NiV: Nipah virus, RSV: respiratory syncytial virus, SeV: Sendai virus, STAT: signal transducer and activator of transcription, TYK: tyrosine kinase, VACV: vaccinia virus, WNV: West Nile virus, ZIKV: Zika virus. Modified after Platanias et al., 2005.
1.3.2 NF-κB signaling pathway The nuclear transcription factor NF-κB can be activated by several pathways including the TNF-, interleukin (IL)- or pathogen-associated molecular patterns (PAMPs)-induced pathway (Fig. 7) (Moynagh 2005; Xia et al. 2005). After the binding of TNF-α to TNF receptor type 1 (TNFR) occurred, the receptor-interacting protein kinase (RIP) and TNFR-associated factor (TRAF) are recruited and form a complex with the adapter protein TNFR type 1-associated DEATH domain protein (TRADD). It leads to the activation and complex formation of mitogen-activated protein kinase kinase kinase 7 (MAP3K7, also called TGF-ß-activated kinase 1 (TAK1)) and TAK1-binding protein (TAB) by which in turn the phosphorylation of inhibitor of NF-κB kinase (IKK) is induced. Thus, the IKK
10 Introduction complex phosphorylates the inhibitory IκBα protein which is bound to NF-κB, a heterodimer consisting of the subunits p50 and p65. As a result, IκBα dissociates and is degraded whereas NF-κB is activated. The released NF-κB can translocate into the cell nucleus where it initiates gene transcription; the expression of cytokines, chemokines, growth or transcription factors or adhesion molecules induces an inflammatory response and cell survival (CUSABIO 2020). Viral single- or double-stranded RNA or deoxyribonucleic acid (DNA) or glycoproteins of the viral envelope can serve as PAMPs that bind to Toll-like receptors (TLR). By binding of dsRNA to TLR3, the adaptor protein TIR-domain-containing adapter-inducing IFN-ß (TRIF) recruits RIP. The binding of ssRNA to TLR8 however involves the adaptor myeloid differentiation factor 88 (MyD88) and leads to the formation of a complex composed of ILR-associated kinase (IRAK)-TRAF proteins and a subsequent complex of MAP3K7-TAB. In the end of both pathways, IκBα is phosphorylated and degraded followed by the nuclear translocation of activated NF-κB (Thwaites et al. 2014). The same pathway starting with MyD88 signaling can be triggered by the binding of IL-1ß to IL-1 receptor type 1 (IL-1R1). MuV SH has been shown to interfere with the TNF-α induction and/or signaling (Wilson et al. 2006). It has been demonstrated that the NF-kB activation is reduced in the presence of viral SH probably due to a reduced phosphorylation of IκBα as well as of the p65 subunit of the NF- κB complex during the TNF-α-induced pathway. It is additionally assumed that SH is able to impair NF-κB activation via IL-1R- and TLR3-mediated pathways (Franz et al. 2017). It has further been shown that MuV SH prevents the nuclear translocation of p65 (Kruger et al. 2018). Besides a decreased NF-kB activation, TNF-α production as well as cell apoptosis were reduced in the presence of MuV SH (Xu et al. 2011). A similar inhibitory role of SH in TNF-α production and the associated cell death was also assumed for bovine RSV and J paramyxovirus (Taylor et al. 2014; Abraham et al. 2018).
11 Introduction
Fig. 7: NF-κB signaling pathway. IκBα: NF-κB inhibitor alpha, IKK: inhibitor of NF-κB kinase, IL: interleukin, IL-1R: IL-1 receptor, IRAK: ILR-associated kinase, MuV: mumps virus, MyD88: myeloid differentiation factor 88, NF-κB: nuclear transcription factor (subunits p50 and p65), PAMP: pathogen-associated pattern, TLR: Toll-like receptor, TNF: tumor necrosis factor, TNFR: TNF receptor, RIP: receptor-interacting protein kinase, TRADD: TNFR type 1- associated DEATH domain protein, TRAF: TNFR-associated factor, TRIF: TIR-domain- containing adapter-inducing IFN-ß.
1.4 Mumps virus infection MuVs belong to the subfamily Rubulavirinae within the family Paramyxoviridae. Rubulaviruses are divided into the two genera Ortho- and Pararubulavirus. Whereas the genus Orthorubulavirus comprises different human pathogenic viruses including MuV as well as porcine and simian rubulaviruses, all members of the genus Pararubulavirus have their origin in bat species.
MuVs are further classified into 12 genotypes (A - N, excluding E and M) based on the sequence identity of the SH proteins (Yeo et al. 1993; Afzal et al. 1997; Orvell et al. 1997b; Wu et al. 1998; Tecle et al. 2002). Each genotype contains different MuV strains that can be distinguished by variations in their protein sequences, mainly in the F and HN genes. Most
12 Introduction currently circulating MuV strains and isolates in Europe, the USA and Canada belong to genotype G (Gouma et al. 2014; Jin et al. 2015; Veneti et al. 2018a; Barrabeig et al. 2019). Further, there is a correlation between MuV genotypes and their global occurrence: Besides genotype G, MuVs of the genotypes H, D, C and J are present in West Europe, the USA and Canada. A mixture of the genotypes C, F, G, H and I is circulating in Asia. With regard to South Africa, South America and Australia, the occurrence of MuV genotypes is more consistent with only one predominant genotype (B, K and J, respectively) (WHO 2012a; Cui et al. 2014; Cui et al. 2017; Willocks et al. 2017; Zengel et al. 2017; Gouma et al. 2018; Barrabeig et al. 2019).
1.4.1 The infectious disease mumps The highly contagious disease mumps (parotitis epidemica) is the result of MuV infections. Mumps is transmitted among humans by direct or indirect droplet infection with an infectivity rate up to 45% and an incubation period of two to four weeks (Levitt et al. 1970; CDC 2015). Basically, mumps is a childhood disease, but during the last years several outbreaks among adolescents and adults have been reported (Veneti et al. 2018a; Carol et al. 2019; Ferenczi et al. 2020). MuV infections mainly result in mild symptoms such as fever, headache, joint pain and parotitis as the most characteristic clinical sign. One-third of mumps infections are asymptomatic but the virus can still be further transmitted (CDC 2015). In rare events, severe and even fatal complications including pancreatitis, orchitis, deafness or myocarditis can occur; the CNS is involved in 50% of cases and meningitis or encephalitis have been observed (Russell et al. 1958; Vuori et al. 1962; Beard et al. 1977; Falk et al. 1989; Ozkutlu et al. 1989; Taii et al. 2008; Rubin et al. 2015). The risk of such complications increases with the age of the infected individuals. However, the mortality rate of mumps is with one death per 10,000 cases still very low (Galbraith et al. 1984; Clemmons et al. 2012).
1.4.2 Vaccination Given that no specific antiviral drug is available, vaccination is even more essential to prevent a MuV infection in the first place. Currently, a combined measles, mumps and rubella (MMR) vaccine, called MMR-Vax in Europe and MMR-II in the US, is worldwide primarily used and contains the live attenuated Jeryl Lynn strain (genotype A) as mumps component which stimulates an effective immune response in roughly 90% of cases (Plotkin et al. 2013; CDC 2019a). All MuV strains and isolates belong to one single serotype meaning that antibodies directed against one particular strain are capable of neutralizing the remaining strains, although the efficiency might differ depending on the strain (WHO 2012b). The vaccination is recommended to be obtained in two doses during early childhood: the first dose should be given at the age of 12 to 15 months and the second dose should be administered at the age of four to six years (CDC 2015). According to a bulletin of “Die Ständige Impfkommission am
13 Introduction
Robert Koch-Institut” from 2001, German children should be vaccinated with the first dose earliest at 11 months and with the second one latest at 23 months (RKI 2020). There have been several issues in the history of developing a safe and efficient MuV vaccine. In Canada in the 1970s, the MMR vaccine “Trivirix” produced with the Urabe AM9 strain came onto the market and replaced the so far used Jeryl Lynn strain containing vaccine. In 1988, Trivirix was withdrawn due to the incidence of vaccine-associated meningitis and the administration of the Urabe AM9 vaccine strain (Bonnet et al. 2006). Nevertheless, the same vaccine was simultaneously administered under the name “Pluserix” in the United Kingdome because the safe MMR vaccine formulated with the Jeryl Lynn strain was too expensive. In 1992, the production and sale of Pluserix has been ceased. However, Urabe AM9 is still a compound of an available vaccine called Trimovax Mérieux. The reason for the neurological complications has been assigned to the fact that the Urabe AM9 vaccine contained a mixture of viruses harboring differences in the HN gene that affected virulence (Brown et al. 1996; Afzal et al. 1998). Similarly, a risk of meningitis following vaccination has been presumed for the vaccine strains Leningrad-3 or Leningrad-Zagreb that are primarily administered in Russia or India and Eastern Europe (da Silveira et al. 2002; Plotkin et al. 2013; Wakefield et al. 2017; Tolzin 2019). Further, it has been documented that the Leningrad-3 vaccine strain has been transmitted from recently vaccinated individuals to contact persons resulting in clinical infections (Atrasheuskaya et al. 2006). Besides safety issues, in particular neurological complications, the increased frequency of mumps outbreaks in vaccinated populations raises the question whether new vaccines against MuV infections are needed.
1.4.3 Mumps virus outbreaks Even though MuV vaccines are available, there are still mumps virus outbreaks which lately increase in number (Rubin et al. 2012; Isaac et al. 2017; Fields et al. 2019; Lau et al. 2019). Mostly, places gathering plenty of people such as hospitals, schools or universities are hot spots of infection. From 2015 to 2017, multiple larger outbreaks in Arkansas, Iowa and Illinois have been reported (Albertson et al. 2016; Donahue et al. 2017; CDC 2020). In total, more than 9,000 mumps cases were registered during this time span, the largest outbreak which was reported in Arkansas resulted in 3,000 cases. From 2009 to 2010, New Jersey (mainly New York City) listed 3,000 mumps cases among two-dose vaccinated students (Dayan et al. 2008; Fiebelkorn et al. 2013b; Rota et al. 2013; CDC 2020). More than 6,500 people were infected mostly in several states in the Midwest in 2006 despite a vaccination coverage of 63% (Dayan et al. 2008). In Norway from 2015 to 2016, 230 MuV infections were confirmed among students; the outbreak started with the infection of two international travelling students (Veneti et al. 2018b). Recently, in March 2019, outbreaks were observed in two universities in Nottingham, England, which expanded until June to more than 300 stated cases in the East
14 Introduction
Midlands. Until end of 2019, a total of 5,000 cases across England were registered, the highest number of cases since 2009 (BBC 2019; England 2019). Most current, a small outbreak with 20 cases occurred in Phoenix, Arizona (PublicHealth 2019). General observations during recent MuV outbreaks were that either the vaccination rate has been declined in the affected area or that among vaccinated but infected people, the majority has obtained only one vaccine shot. Still, many cases occurred even in two-dose vaccine covered populations. Therefore, the impact of a third vaccine dose is increasingly discussed. It could be shown that in a population that is mainly composed of people with two-dose vaccination, a third dose may decline the risk of MuV infection and help in outbreak control (Ogbuanu et al. 2012; Fiebelkorn et al. 2013a; Nelson et al. 2013). However, the number of administered vaccine doses might not be the only concern. A shift in the circulating MuVs might be hold responsible as well: While the vaccine strain Jeryl Lynn belongs to the genotype A, most circulating MuVs are genotype G or H viruses (Rubin et al. 2008; Jin et al. 2015; Gouma et al. 2018). Although all MuVs belong to one serogroup, the efficiency of cross-neutralization varies depending on the currently circulating strain leading to infection (Zengel et al. 2017). There are also speculations on the decrease of protective antibody levels over time leading to a waning immunity in vaccinated populations questioning again the effectiveness of the vaccination (Lewnard et al. 2018; Su et al. 2020). Another explanation for mumps outbreaks in vaccinated populations was given by immunological investigations suggesting that naturally occurring MuV infections but not MuV vaccination results in the generation of memory B cells and CD8+ T cells (de Wit et al. 2018; Rasheed et al. 2019). Therefore, the development of a new vaccine should be considered.
1.5 Bats and zoonotic viruses 1.5.1 Zoonotic diseases and pathogens The term zoonoses describes infectious diseases which can be transmitted from animals to humans (zooanthroponose) or vice versa (anthropozoonose) and are caused by viruses, bacteria, parasites or prions (WHO 2020). About 61% of human infections and 75% of the emerging diseases are caused by zoonotic pathogens (Taylor et al. 2001). It is assumed that one third of the viruses that can infect livestock and domestic animals can also be transmitted to humans and in total, 200 zoonotic virus species might exist (Cleaveland et al. 2001; Taylor et al. 2001; Woolhouse et al. 2005; Woolhouse et al. 2012). Important zoonotic diseases are e.g. tuberculosis, Lyme disease (Lyme borreliosis), malaria, leishmaniosis, acquired immune deficiency syndrome and influenza. Zoonotic pathogens can be found in saliva, blood, urine or feces of infected individuals. Therefore, the transmission can either occur by direct contact to infected people or animals or - depending on the agent’s tenacity - indirect via contaminated areas, water, food or other objects. In addition, spread of infection by living vectors such as ticks or mosquitos has been reported for several pathogens (CDC 2017).
15 Introduction
1.5.2 Bats as reservoir hosts of zoonotic viruses Bats and flying foxes are the natural reservoir hosts for many zoonotic viruses, especially for emerging and re-emerging viruses, hence playing an important role in virus transmission (Calisher et al. 2006). As a typical feature of reservoir hosts, virus-harboring bats develop no or only mild clinical signs (Mandl et al. 2015). A variety of unique characteristics make bats advantageous hosts for viruses and other pathogens. Bats are geographically widespread and due to their ability to fly, they can cover quite large distances. During active flight, their body temperature and metabolism are increased speculating an effect on the immune system such as DNA damage repair and a reduced sensing of exogenous and endogenous DNA to avoid inflammatory responses induced by an accumulation of cytosolic self-DNA (Zhang et al. 2013; O'Shea et al. 2014; Ahn et al. 2016; Xie et al. 2018). Given that bats show a high tolerance to viral infections in form of reduced clinical illness due to a reduced inflammation, a from other mammals deviating innate immune system is discussed (Banerjee et al. 2017; Pavlovich et al. 2018; Banerjee et al. 2020). Their long lifespan enables a long persistence of a chronic infection (Luis et al. 2013). During torpor and/or hibernation the bat’s energy metabolism is minimized leading to a reduced virus replication what in turn dampens the risks of clinical disease for bats. In return, the immune system of hibernating bats is also almost shut down supporting the persistence of the virus infection (Sulkin 1974; Calisher et al. 2006). In addition, their colonial behavior including roosting results in a rapid intra- and interspecies dissemination of viruses (Luis et al. 2013). Until 2006, 66 virus species belonging to the genera Alpha-, Flavi-, Henipa-, Lyssa-, Orbi-, Orthoreo-, Phlebo-, Orthobunya- and Orthorubulavirus were detected in bat samples (Calisher et al. 2006). These include e.g. rabies virus that was found in many different bat species worldwide, or severe acute respiratory syndrome coronavirus (SARS-CoV) being isolated from Rhinolophus bats (Piraccini 2016; Sun 2019). Flying foxes of the genus Pteropus were identified as reservoir hosts of the henipaviruses HeV, NiV and Cedar virus (Hooper et al. 2000; Marsh et al. 2012; Roberts et al. 2017; Tsang 2020). Further, in numerous bat species the chikungunya and rift valley fever virus which are both transmitted by mosquitos, as well as the Japanese encephalitis virus were detected (Fagre et al. 2019). In a more recent study, bats might also be revealed as reservoir hosts for polyomaviruses (Tan et al. 2020). In addition, the circulation of the Bombali ebolavirus belonging to the genus Ebolavirus within free-tailed bats was reported (Goldstein et al. 2018; Forbes et al. 2019). The amount of novel viruses detected in bats increased significantly during the last years. However, in most cases no virus isolate but viral RNA was derived from the bat samples. The detection of several highly diverse paramyxoviruses in African fruit bats might indicate that most paramyxoviruses have their origin in bats (Drexler et al. 2009; Baker et al. 2012; Drexler et al. 2012; Baker et al. 2013).
16 Introduction
1.5.3 Bat-derived mumps virus In 2009, the discovery of the RNA of a MuV-related virus (BatPV/Epo_spe/AR1/DCR/2009, GenBank accession no.: HQ660095.1, bat-derived MuV, batMuV) in the spleen of an African flying fox of the genus Epomophorus in the Democratic Republic of Congo queried the assumption that humans are the only reservoir host for MuVs (Drexler et al. 2012). The batMuV is phylogenetically closely related to human MuVs (hMuV) as shown by Bayesian interference. Its genome is comprised of 15,378 nt and shares an identical organization with the viral genome of hMuVs. Further, the aa sequence homology for all proteins of human and bat- derived MuVs is noticeable high and ranges between maximal 72.6% identity for the P proteins and 94.2% for the L proteins. One exception is the SH protein which is also hypervariable among hMuV strains and shares only 38.6% similarity between hMuV and batMuV (Drexler et al. 2012). A serological relatedness has been demonstrated by the reactivity of bat sera against hMuV as well as by the cross-reactivity of neutralizing antibodies directed against hMuV strains that were capable of detecting and neutralizing batMuV (Drexler et al. 2012; Kruger et al. 2015; Katoh et al. 2016; Kruger et al. 2016; Beaty et al. 2017; Kruger et al. 2018). Due to the cross- reactivity, it is impossible to distinguish between batMuV and hMuV only on the basis of serological tests. The sequencing of the SH protein would provide information required to differentiate between hMuV and batMuV; however, RNA isolation and sequencing are not performed as part of routine diagnostics. Thus, it is not known whether humans have already been infected with batMuV. First investigations on the biological characterization of batMuV focused on the surface glycoproteins F and HN. Transfection studies revealed that batMuV F and HN are able to mediate cell-to-cell fusion in cell lines originated from human, bat, non-human primate or rodent species. hMuV and batMuV F and HN proteins can interact with each other to mediate fusions underlining the close relatedness between these viruses. A similar functionality of human and bat-derived MuV glycoproteins was further proven by showing that batMuV HN utilizes sialic acids for binding to target cells and exhibits neuraminidase activity similar to its human counterpart (Kruger et al. 2015). In a follow-up study, recombinant chimeric MuVs in which the ORFs of batMuV F and/or HN were cloned into the genome of a hMuV strain were generated. The batMuV glycoproteins have been incorporated into the human backbone strain and chimeric viruses were able to mediate entry into different mammalian cell lines confirming the functionality of batMuV F and HN and their close relatedness to hMuV. Moreover, neutralizing antibodies directed against hMuVs were capable of inhibiting infection of all chimeric MuVs expressing batMuV glycoproteins highlighting the serological relatedness of MuVs (Kruger et al. 2016). So far, only the entry process of batMuV has been investigated and the remaining steps of the viral replication cycle are unclear.
17 Introduction
1.6 Aims of the study The overall objective of this thesis was to gain a better understanding of the virus-host interactions between mumps viruses (MuVs) and mammalian cells with focus on the viral entry process, replication and interference with the host immunity. Besides different human MuV strains (hMuV), a bat-derived MuV strain (batMuV) has been included as bats might serve as an additional or even the reservoir host for these viruses.
One aim of the study was to investigate and compare the interactions of batMuV and hMuV with human and bat-derived cells with regard to viral entry, replication and evasion of the host immune response. Recombinant viruses were generated and used for the infection of cell lines derived from different organs of human, non-human primate and bat origin to gain information whether batMuV and hMuV are able to mediate entry into and to replicate in cells of potential host species. In addition, cellular factors required for viral attachment and fusion were identified by analyzing the sialic acid-dependent binding of MuV hemagglutinin-neuraminidase (HN) and the dependence of the MuV fusion glycoprotein (F) on proteolytic activation by the cellular protease furin, respectively. To assess how batMuV and hMuV interfere with the host immunity in bat and human cells, the effect of the immunomodulatory proteins V, I and SH on the interferon or NF-kB signaling pathway was investigated.
The second part of this thesis focused on the proteolysis and fusogenicity of the MuV F protein. To be able to mediate fusion and facilitate viral entry and spread of infection, MuV F has to be proteolytically cleaved by cellular proteases, most likely by the protease furin that recognizes the multibasic cleavage motif R-X-(R/K/X)-R. Following cleavage, MuV F can induce the fusion between viral and cellular membranes or between the membranes of infected and neighbored cells resulting in the formation of syncytia (multinucleated giant cells). It has been shown that the extent of syncytium formation varies among MuV strains. However, the mechanisms of the differentiated fusogenicity and its importance for viral infectivity are so far unknown. Sequence analysis identified one amino acid within the cleavage site that varies depending on the MuV strain raising the question whether this particular amino acid residue (P8) might affect proteolysis and fusogenicity. The role of P8 has been investigated by quantifying the amount and size of F-induced syncytia following overexpression of modified F proteins and by measuring the furin-mediated cleavage rate of peptides in which P8 has been altered. Viral pseudotypes have been used as well as an internalization assay with a recombinant hMuV has been performed to focus on the fusion that occurs during the viral entry process between viral and cellular membranes. Recombinant viruses that only differ in P8 have been generated to focus on viral replication, spread of infection within the cell layer and virus-induced cytopathic effects.
18 Introduction
Further, the involvement of other cellular proteases besides furin in the proteolysis of MuV F has been investigated. Besides direct contact to infected persons, MuV can be transmitted by droplets or aerosols with the oropharynx as the primary entry site. As shown for many other viruses infecting the respiratory tract e.g. influenza and coronaviruses, extracellular proteases play an important role in activating the viral glycoproteins. By the infection of a cell line expressing certain extracellular proteases, the usage of protease inhibitors and by quantifying the cleavage rate of MuV F peptides by recombinant membrane-associated proteases, it was verified whether MuV F can be cleaved by additional proteases others than furin.
19
Manuscript I
2 Entry, replication, immune evasion and neurotoxicity of a synthetically-engineered bat-borne mumps virus
Nadine Krüger1,2,#, Christian Sauder3, Sarah Hüttl1,2, Jan Patrick Papies4, Kathleen Voigt1,2, Georg Herrler1, Kornelia Hardes5, Torsten Steinmetzer5, Claes Örvell6, Jan Felix Drexler4, Christian Drosten4, Steven Rubin3, Marcel Alexander Müller4, Markus Hoffmann7
1 Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany 2 Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany 3 Food and Drug Administration (FDA), Center for Biologics Evaluation and Research (CBER), Silver Spring, MD, USA 4 Institute of Virology, Charité - Universitätsmedizin Berlin, Berlin, Germany 5 Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany 6 Division of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden 7 Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany
# Corresponding author: Nadine Krüger, [email protected]
State of publication: published, Cell Rep. Cell Rep. 2018 Oct 9; 25(2):312-320.e7. doi: 10.1016/j.celrep.2018.09.018. Available at: https://www.cell.com/cell-reports/pdf/S2211-1247(18)31448-7.pdf
Authors contribution (SH): Scientific design: - Investigation, data collection: 20% Evaluation, formal analysis: 10% Scientific writing: 10%
21 Manuscript I
Abstract Bats harbor a plethora of viruses with an unknown zoonotic potential. In-depth functional characterization of such viruses is often hampered by a lack of virus isolates. The genome of a virus closely related to human mumps viruses (hMuV) was detected in African fruit bats, batMuV. Efforts to characterize batMuV were based on directed expression of the batMuV glycoproteins or use of recombinant chimeric hMuVs harboring batMuV glycoprotein. Although these studies provided initial insights into the functionality of batMuV glycoproteins, the host range, replication competence, immunomodulatory functions, virulence, and zoonotic potential of batMuV remained elusive. Here, we report the successful rescue of recombinant batMuV. BatMuV infects human cells, is largely resistant to the host interferon response, blocks interferon induction and TNF-a activation, and is neurotoxic in rats. Anti-hMuV antibodies efficiently neutralize batMuV. The striking similarities between hMuV and batMuV point at the putative zoonotic potential of batMuV.
22 Manuscript II
3 The amino acid at position 8 of the proteolytic cleavage site of the mumps virus fusion protein affects viral proteolysis and fusogenicity
Sarah Hüttla,b, Markus Hoffmannc, Torsten Steinmetzerd, Christian Saudere, Nadine Krügera,b,c,# a Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany b Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany c Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany d Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany e Food and Drug Administration (FDA), Center for Biologics Evaluation and Research (CBER), Silver Spring, MD, USA
# Corresponding author: Nadine Krüger, [email protected]
State of publication: published, J. Virol. J. Virol. 2020 Oct 27; 94(22):e01732-20. doi: 10.1128/JVI.01732-20. Available at: https://jvi.asm.org/content/94/22/e01732-20.long
Authors contribution (SH): Scientific design: 25% Investigation, data collection: 75% Evaluation, formal analysis: 75% Scientific writing: 50%
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Abstract The mumps virus (MuV) fusion protein (F) plays a crucial role for the entry process and spread of infection by mediating fusion between viral and cellular membranes as well as between infected and neighboring cells, respectively. The fusogenicity of MuV F differs depending on the strains and might correlate with the virulence; however, it is unclear which mechanisms contribute to the differentiated fusogenicity. The cleavage motif of MuV F is highly conserved among all strains except the amino acid residue at position 8 (P8) that shows a high variability with a total of four amino acid variants (leucine (L), proline (P), serine (S), threonine (T)). We demonstrate that P8 affects the proteolytic processing and the fusogenicity of MuV F. The expression of L or S at P8 resulted in a slower proteolysis of MuV F by furin and a weaker ability to mediate cell-to-cell fusion. However, virus-cell fusion was more efficient for F proteins harboring L or S at P8, suggesting that P8 contributes to the mechanism of viral spread: P and T enable a rapid spread of infection by cell-to-cell fusion, whereas viruses harboring L or S at P8 spread by the release of infectious viral particles. Our study provides novel insights into the fusogenicity of MuV F and its influence on the mechanisms of virus spread within infected tissues. Assuming a correlation between MuV fusogenicity and virulence, sequence information on the amino acid residue at P8 might be helpful to estimate the virulence of circulating and emerging strains.
Importance Mumps virus (MuV) is the causative agent of the highly infectious disease mumps. Mumps is mainly associated with mild symptoms, but severe complications such as encephalitis, meningitis or orchitis can also occur. There is evidence that the virulence of different MuV strains and variants might correlate with the ability of the fusion protein (F) to mediate cell-to- cell fusion. However, the relation between virulence and fusogenicity or the mechanisms responsible for the varying fusogenicity of different MuV strains are not fully understood. Here we focused on the amino acid residue 8 (P8) of the proteolytic cleavage site of MuV F, because this amino acid residue shows a striking variability depending on the genotypes of MuV. P8 has an effect on the proteolytic processing and fusogenicity of MuV F and might thereby determine the route of virus spread within infected tissues.
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4 Furin-independent cleavage of human and bat-derived mumps virus F proteins
Sarah Hüttla,b, Torsten Steinmetzerc, Jan Felix Drexlerd, Nadine Krügera,b,e,# a Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany b Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany c Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany d Institute of Virology, Charité - Universitätsmedizin Berlin, Berlin, Germany e Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Göttingen, Germany
# Corresponding author: Nadine Krüger, [email protected]
State of publication: submitted
Authors contribution (SH): Scientific design: 50% Investigation, data collection: 75% Evaluation, formal analysis: 75% Scientific writing: 75%
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Abstract Proteolytic cleavage of the mumps virus (MuV) fusion (F) protein by cellular proteases is an indispensable requirement for F-mediated fusion between viral envelope and host cell membrane to enable viral entry. Furin has been described to be responsible for the proteolysis of MuV F. So far, it is unknown whether other proteases are capable of cleaving MuV F in addition to furin. In this study, we demonstrate that besides furin also type II transmembrane serine proteases (TTSPs) allow proteolytic activation of human and bat-derived MuV F and may support MuV spread in the upper respiratory tract. Viral entry and infectivity was blocked by camostat mesylate, an inhibitor of TTSPs. The antiviral activity of camostat mesylate in combination with furin inhibitors was more potent than the administration of the inhibitors alone, indicating that furin-independent activation of MuV F has to be taken into account with regard to therapeutic treatments of mumps.
Impact statement Mumps virus (MuV) primarily targets the upper respiratory tract epithelium and is transmitted by aerosols and respiratory droplets. So far, the ubiquitous expressed cellular protease furin has been proven to be capable of cleaving the MuV fusion (F) protein. Here we demonstrate that MuV F can also be activated by type II transmembrane serine proteases (TTSPs) that are expressed on the surface of the respiratory epithelium. TTSPs have been described to represent an attractive target for the treatment of respiratory infections and inhibitors of TTSPs might also be effective against MuV infections.
Data summary The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.
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Introduction Mumps viruses (MuV) belong to the genus Orthorubulavirus within the family Paramyxoviridae. The highly-infectious disease mumps is mainly known to cause mild symptoms but severe disease progressions including neurological disorders can also occur (1-5). The viral entry process of MuV is mediated by two surface glycoproteins integrated in the viral envelope: The hemagglutinin-neuraminidase (HN) initiates viral attachment by binding to sialic acids on the cell surface whereas the fusion protein (F) promotes the fusion of the viral and host cell membranes (6-9). MuV F is synthesized as a fusion-incompetent F0 precursor protein that has to be activated by proteolytic cleavage mediated by cellular proteases and by protein- interactions between F and HN that induce irreversible conformational changes of F to enable the exposed fusion peptide to get in contact with the host cell membrane (10, 11). All MuV strains, including the bat-derived MuV (batMuV) that has been detected in an African fruit bat (12), harbor a multibasic cleavage motif that can be cleaved by furin, a calcium-dependent proprotein convertase containing a subtilisin-like protease domain (13-17). Whether additional proteases besides furin play a role in MuV F cleavage has not been investigated so far.
It is assumed that MuV primarily infects the upper respiratory tract epithelium followed by spread to regional lymph nodes and viremia (18-21). Several respiratory viruses are proteolytically activated by proteases expressed in the respiratory tract, raising the question whether also MuV F might be activated by proteases others than furin. Here we focused on the furin-independent cleavage of MuV F by type II transmembrane serine proteases (TTSPs). The TTSPs comprise a group of numerous trypsin-like serine proteases that are mainly distributed in respiratory or gastrointestinal epithelial cells (22). They play a crucial role in the activation of fusion-mediating glycoproteins of influenza A viruses and coronaviruses, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and can target mono- and multibasic cleavage motifs (23-27). Therefore, TTSPs are a suitable target of antiviral treatment of respiratory infections. A clinically applied inhibitor of trypsin-like serine proteases including the group of TTSPs, camostat mesylate, has been shown to inhibit the proteolytic activation of the SARS-CoV-2 spike protein and is currently evaluated in clinical studies to be used as an antiviral treatment for patients infected with SARS-CoV-2 (27, 28).
We demonstrate that MuV F can be cleaved in a furin-independent manner enabling efficient viral replication even in the presence of furin inhibitors. Viral infectivity was most efficiently blocked when furin inhibitors and camostat mesylate were applied in combination. Fluorescence resonance energy transfer (FRET)-based assays revealed that the TTSP matriptase is capable of processing the cleavage motif of MuV F. Taken into account that mumps has to be considered as a re-emerging disease and that batMuV exhibits important
27 Manuscript III requirements to infect humans, antiviral therapeutics against MuV infections are needed. Similar to other respiratory infections, TTSPs represent a suitable target for antiviral therapeutics such as camostat mesylate.
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Main text Furin-independent cleavage of MuV F The dependence of human and bat-derived MuV F on the proteolytic cleavage by cellular proteases has been investigated by evaluating viral replication of recombinant human MuV (strain 88-1961, r88) (29) and batMuV (rbatMuV) (13) in the presence of inhibitors of furin (MI- 1148, 10 µM) (30) and trypsin-like serine proteases (camostat mesylate, 100 µM) (31-33). In Vero76 cells, a cell line that lacks sufficient expression of TTSPs such as TMPRSS2, TMPRSS4, HAT, DESC1 and MSPL (34-36), the inhibition of cellular furin resulted in a significant decrease of viral infectivity whereas no antiviral effect was observed for camostat mesylate (Fig. 1). In contrast, viral replication was only slightly affected following inhibition of furin in Caco-2 cells, a cell line that is known to express TTSPs such as TMPRSS2, TMPRSS4 and matriptase besides furin (36-39). Further, MuV infectivity in Caco-2 cells was reduced by camostat mesylate, but a strong inhibition of replication was only achieved if the furin inhibitor and camostat mesylate were administered in combination (Fig. 1). Conclusively, human and batMuV F can be cleaved by furin and trypsin-like serine proteases depending on their availability. Therefore, TTSPs can compensate MuV F cleavage when furin is inhibited whereas furin, in turn, can undertake the proteolysis during the inhibition or absence of TTSPs.
Matriptase is capable of cleaving MuV F To identify whether a particular TTSP is involved in the proteolysis of MuV F, peptides consisting of the cleavage motif of human and batMuV F and the donor-quencher pair Dabcyl/Edans (Fig. 2a) were incubated with recombinant proteases and the proteolytic cleavage rate was quantified by measuring the fluorescence signal. Assay conditions for each protease are given in Tab. 1. As expected, both peptides were cleaved very efficiently by furin. Further, also matriptase was able to cleave both peptides whereas no proteolysis was obtained for HAT and DESC1 (Fig. 2b). Given that TMPRSS2 gained importance due to its involvement in the activation of the SARS-CoV-2 spike protein (27, 40), it was further investigated whether TMPRSS2 might also play a role for the cleavage of MuV F. As no suitable protease with enzymatic activity was available for FRET assays, the antiviral effect of camostat mesylate in Vero cells stably expressing TMPRRS2 (27) was investigated. For r88 and rbatMuV, infectivity in Vero-TMPRSS2 cells depended on the processing of MuV F by furin as inhibition of furin alone already completely abolished viral replication whereas a minor inhibitory effect was observed for camostat mesylate (Fig. 2c). In sum, we demonstrate that the F protein of human and batMuV can be cleaved in a furin- independent manner, probably by the TTSP matriptase, which also prefers substrates with two arginine residues in P1 and P4 positions (41). Besides TTSPs, also GPI-anchored trypsin-like serine proteases such as prostasin might be able to process the MuV F proteins. Prostasin is
29 Manuscript III also expressed in Caco-2 cells and most likely involved in the proteolytic activation of certain influenza A and influenza B virus as well as infectious bronchitis virus hemagglutinins (42).
The usage of alternative proteases has to be taken into account with respect to drug development and antiviral treatment. Given that mumps has to be considered as a re-emerging disease (43-45) and that batMuV seems to be capable of infecting humans (13), antiviral therapeutics against MuV infections are needed. Our findings reveal furin and TTSPs as potential therapeutical targets for the treatment of clinical MuV infections. Camostat mesylate is a clinically proven and safe drug which is currently considered as antiviral treatment for COVID-19 patients (46). It is unknown whether the administration of camostat mesylate alone would sufficiently inhibit MuV replication efficient enough to prevent severe clinical manifestations or if a combined antiviral therapy is required. However, no furin inhibitors are approved for antiviral treatment in humans but some promising candidates are currently in development (47).
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Author statements Authors and contributors Conceptualization, S.H., N.K.; investigation, S.H., N.K.; formal analysis, S.H., N.K.; resources, T.S., J.F.D.; writing - original draft preparation, S.H., N.K.; writing - review and editing, all authors; funding acquisition, N.K. All authors have read and agreed to the published version of the manuscript.
Conflicts of interest The authors declare that there are no conflicts of interest.
Funding information This research was funded by grants of the German Research Foundation (DFG) to N.K. (grant number KR-4762/2-1). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Ethical approval -
Consent for publication -
Acknowledgements We are grateful to Karl-Klaus Conzelmann (LMU Munich) for providing BSR-T7/5 cells. This research was funded by the German Research Foundation (DFG), grant number KR-4762/2- 1. Part of this work was performed by S.H. in partial fulfillment of the requirements for the doctoral degree from University of Veterinary Medicine Hannover.
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Figures and tables Table 1 Protease Supplier Concentration Buffer pH Furin R&D 1.0 nM 50 mM HEPES, 154 mM 7.4
Systems NaCl, 2 mM CaCl2, 2% Ethanol Matriptase R&D 0.4 nM 50 mM TRIS, 50 mM NaCl, 9.0 Systems 0.01% (v/v) Tween 20 HAT R&D 1.0 nM 50 mM TRIS, 154 mM NaCl, 7.5 Systems 0.05% (v/v) Brij 35 DESC1 Enzo 0.8 nM 50 mM TRIS, 154 mM NaCl, 8.0 0.05% (v/v) Brij 35
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Fig. 1: Antiviral activity of protease inhibitors. Vero76 and Caco-2 cells were infected with r88 and rbatMuV at an MOI of 0.1. Prior and 1 h p.i., cells were incubated with no inhibitor (DMSO), 10 µM of furin inhibitor MI-1148, 100 µM of camostat mesylate or combinations of these inhibitors. Supernatants were collected at 2 d p.i. and viral titers were quantified by titration on Vero76 cells. The graphs show mean and SD of three independent trials. Statistical significance compared to mock treated controls was analyzed by paired, two-tailed Student´s t-test with 95% confidence intervals (p ≤ 0.5, *; p ≤ 0.1, **).
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Fig. 2: Cleavage of MuV F by TTSPs. (a) Schematic illustration of FRET peptides. Red arrows indicate cleavage site. (b) FRET peptides were incubated without proteases (mock), furin, matriptase, HAT or DESC1 for 30 min. Proteolytic cleavage was quantified by measuring the fluorescence signal using an excitation wavelength of 340 nm and an emission wavelength of 535 nm and is given as relative fluorescence units (RFU). The graphs show means and SD of three independent trials. Statistical significance compared to mock treated controls was tested by paired, two-tailed Student´s t-test with 95% confidence intervals (*: p ≤ 0.05; **: p ≤ 0.01; *** p ≤ 0.005). (c) Vero-TMPRSS2 cells were infected with r88 and rbatMuV at an MOI of 0.1. At 1 h p.i., cells were incubated with 10 µM of the furin inhibitor MI-1148, 100 µM of camostat mesylate or both inhibitors in combination. Supernatants were collected at 2 d p.i. and viral titers were determined by titration on Vero76 cells. The graph shows mean and SD of three independent trials. Statistical significance compared to mock treated controls was analyzed by one-way ANOVA and Dunnett´s multiple comparison test (*: p ≤ 0.05).
36 Discussion
5 Discussion MuV infections result in the highly contagious disease mumps that mainly causes mild symptoms such as fever, headache or the characteristic parotitis (Beard et al. 1977; CDC 2015). Nevertheless, severe disease progressions characterized by pancreatitis, orchitis or neurological disorders including encephalitis, meningitis or hydrocephalus can also occur (Russell et al. 1958; Falk et al. 1989; Taii et al. 2008). A live attenuated vaccine is available but more recently, an increase in MuV outbreaks has been observed during which an age postponement was stated and the administration rate, dose and effectiveness of current vaccines were queried (CDC 2019b). Therefore, mumps is still a global threat to human health and should no longer be categorized as childhood disease. Further, with the occurrence of batMuV, a virus that has been detected in African fruit bats and shares a great phylogenetic relatedness to hMuV (Drexler et al. 2009), the disease might be an even greater risk in Africa where the population’s vaccination status is already low (WHO 2012a). Certainly, it cannot be excluded that a worldwide spread of batMuV or other so far unknown bat-borne orthorubulaviruses has passed unnoticed or will be observed in the future. Given the re- emergence of MuV and the detection of bats as an animal host of these viruses, studies focusing on the characterization of batMuV, virus-host interactions, vaccine efficiency and the development of potential antiviral treatments are urgently needed.
In this study, it was sought to depict and clarify the virus-host interplay between MuVs and mammalian cell lines whereby the viral entry, replication and modulation of the host immunity were paramount. One focus of this thesis was laid on the characterization of batMuV to identify cellular factors that are required for efficient viral entry and replication enabling the estimation of the zoonotic potential of batMuV. The second aim was to specify the fusogenicity and proteolytic cleavage of MuV F as fusogenicity might contribute to virulence of MuVs. New insights into the mechanisms of the differently pronounced fusogenicity and the proteolytic cleavage of MuV F might be helpful to assess the virulence of MuV strains and to develop antiviral therapeutics.
5.1 batMuV is capable of infecting human cells and circumventing the host immune response First investigations of batMuV revealed that the surface glycoproteins F and HN are functionally related to their human counterparts as batMuV HN shows hemagglutination and neuraminidase activity and the expression of both glycoproteins is obligatory to mediate cell- to-cell fusions in human and bat-derived cell lines (Kruger et al. 2015; Kruger et al. 2016). In addition, the function of the N, P and L proteins is similar to that of the corresponding hMuV proteins suggesting that the replication mechanism of batMuV is comparable to that of hMuV
37 Discussion
(Hüttl 2017). Nevertheless, these findings were based on transfection studies in which particular proteins were expressed in cell culture systems or on recombinant chimeric MuVs. Results of the latter varied regarding the phenotype of infection depending on the used hMuV backbone strain and further, interrelationships with other virus- or host cell-encoded proteins involved in viral entry or replication should not be disregarded or underestimated (Katoh et al. 2016; Kruger et al. 2016; Beaty et al. 2017). Consequently, to ensure a more accurate characterization of batMuV including a more reliable comparison with hMuV and to assess whether batMuV is able to infect human cells, recombinant batMuV (rbatMuV) was generated to allow the analysis in a viral context.
A successful infection of human, non-human primate and bat cells by rbatMuV was reported (Kruger et al. 2018). However, the replication efficiency differed depending on the cell line. For instance, rbatMuV replicated especially well in cells derived from fruit bats belonging to the same subfamily (Epomophorinae) as the reservoir host of batMuV. Surprisingly, the highest titer was obtained for the human cell line Caco-2 which is derived from a colorectal adenocarcinoma. Furthermore, the extent of rbatMuV-induced cell-to-cell fusion and the resulting cytopathic effect (CPE) correlated with the replication efficiency. Very strong syncytium formation was observed in cells derived from fruit bats of the genus Epomops and Hypsignathus whereas less or no cell-to-cell fusion was detected in the other cell lines. Given that rbatMuV efficiently infected cells of human origin it is very likely that batMuV has a zoonotic potential. Replication of hMuV in cells of bat origin was less efficient compared to batMuV probably due to an adaptation of hMuVs to the human host. The viral entry process of batMuV depended on the same cellular requirements as hMuV: The attachment of HN to sialic acids on the cell surface and the proteolytic cleavage of MuV F by the cellular protease furin. Conclusively, human cells provide all cellular factors to enable the entry of batMuV followed by an efficient viral replication.
MuVs are capable of circumventing the host immune response by the immunomodulatory functions of the V and SH proteins that interfere with the IFN or NF-kB signaling pathway, respectively (Kubota et al. 2002; Andrejeva et al. 2004; Kubota et al. 2005; Wilson et al. 2006; Rosas-Murrieta et al. 2010; Xu et al. 2011; Xu et al. 2012b; Franz et al. 2017). The replication of rbatMuV and hMuV was only slightly affected by IFN-α stimulation of human and bat cells indicating that these viruses can interfere with antiviral immunity in both host species. MuV V interference with the IFN pathway occurs, in particular, by the inhibition of STAT signaling via proteasomal degradation or reduced phosphorylation of STATs (Kubota et al. 2001; Kubota et al. 2005). Further, it could be demonstrated that the induction of IFN-ß in human cells was inhibited by bat und human MuV V and I, a gene product of the P gene that is expressed after
38 Discussion
RNA editing and shares 91% aa identity with V (Paterson et al. 1990). Whereas it has already been examined in previous studies how paramyxovirus V proteins affect the IFN induction, nothing has been known about the function of MuV I. It is suggested that V interacts with the transcription factor IRF3. V proteins of MuV, HPIV2 and PIV5 can impede the phosphorylation and activation of IRFs by serving as alternative substrates of the TBK1/IKKε kinases what reduces the IFN expression (Lu et al. 2008). According to Andrejeva et al., the binding of V to the melanoma differentiation-associated gene 5 (mda-5) protein might also be decisively involved in the suppression of IFN induction (Andrejeva et al. 2004). Interestingly, the inhibition of IFN-ß induction was stronger for MuV I than V and the inhibitory effect of hMuV I was higher compared to batMuV I probably due to a better adaptation of hMuV to the human cells. Still, it is elusive whether MuV I is just wrongly expressed or also has a function different from V. Even though the aa sequence of batMuV and hMuV SH is not conserved, a similar functionality of these proteins was observed: Both SH proteins could inhibit TNF-α-inducible NF-κb activation and translocation of NF-κb p65 from the cytoplasm to the nucleus in human and bat- derived cells. Probably, MuV SH dampened the phosphorylation of IκBα by IKK. As a result, IκBα cannot be degraded and remains bound to NF-kB inhibiting its release and activation (Franz et al. 2017). These observations allow two assumptions: Either batMuV is already adapted to the human immune system or the mechanisms of IFN- and NF-κb-mediated immunity in humans and bats resemble each other to such a large extend that no adaptation to the host species is required. Studies on the immune system of bats are still rare impeding the comparison between antiviral signaling pathways in humans and bats. Despite the incompletely characterized antiviral responses in bat cells, some identified principles show similarities to these in cells of human origin (Banerjee et al. 2020). In bats, infections induced by single-stranded RNA viruses can be sensed by TLR8 (Cowled et al. 2011). Then unidentified adaptor proteins and kinases are activated resulting in the activation and nuclear translocation of IRF3 or NF-κb (Banerjee et al. 2019). The former induces the expression of antiviral cytokines such as type I IFNs that stimulate the JAK-STAT signaling pathway in neighboring cells finally resulting in the expression of ISGs (Banerjee et al. 2020). NF-κb promotes the inflammatory cytokine production which is restricted though to a lower level in bat cells compared to human ones (Banerjee et al. 2017). Nevertheless, it should be noted that bat species-specific differences such as a unique repertoire and expression profile of ISGs have already been described (De La Cruz-Rivera et al. 2018). Moreover, the aa sequence of the TLR8 extracellular ligand binding domain differs not only within bats but between human and bats what might point out to a varying recognition of viral RNA (Schad et al. 2016).
39 Discussion
Taken together, batMuV can infect and efficiently replicate in human cells and shows a successful evasion of the host immune response as described and observed for hMuVs. These observations strengthen the presumption that batMuV might be zoonotic as well as pathogenic in a comparable manner to hMuVs. The potential health threat of batMuV to humans should not be underestimated. But referring to the existing cross-reaction of anti-hMuV antibodies, vaccination against hMuV might also be effective in preventing clinical infections mediated by batMuV (Kruger et al. 2015; Kruger et al. 2016).
5.2 A single amino acid modifies fusogenicity and proteolysis of MuV F MuV strains and isolates display varying fusogenic properties but the reason for this is so far elusive (Merz et al. 1983a). It is assumed that the fusogenicity might correlate with the virulence of MuVs: highly (neuro)virulent strains tend to be highly fusion-active whereas no or only a weak fusion activity has been stated for less pathogenic strains (Wolinsky et al. 1978; Merz et al. 1981; Merz et al. 1983a; Merz et al. 1983b; Love et al. 1986; Lemon et al. 2007). This hypothesis has been strengthened by recent studies: The mutation of the aa residue 91 of MuV F (Ala à Thr) resulted in the attenuation of the highly neurovirulent strain 88-1961 in vivo and a decreased fusogenicity in vitro (Malik et al. 2007b). It is further suggested that the fusogenicity might negatively correlate with the neuraminidase activity of the HN protein (Merz et al. 1981). Moreover, it has been discussed that single aa residues of F modify viral fusogenicity and might thereby play a role in virus pathogenesis (Tanabayashi et al. 1993; Malik et al. 2007a; Malik et al. 2007b; Yoshida et al. 2010). However, these observations were obtained for particular MuV strains and it is not clear whether they represent general mechanisms that apply to all MuV strains and isolates. To clarify the reasons for and mechanisms behind the varying fusogenicity and replication efficiency among MuVs, the activation and functionality of MuV F were investigated in order to identify viral markers that might be used to estimate viral fusogenicity. Given that MuV fusogenicity might correlate with virulence, such markers may be helpful to evaluate the virulence of circulating MuV strains or the safety of attenuated vaccine strains.
To get into the fusion-active conformation, the processing of the fusion-inactive precursor F0 by cellular proteases is required. The proteolytic cleavage occurs at a specific cleavage motif within the F protein that is recognized by cellular proteases. The cleavage motif of MuV F contains a multibasic furin motif and is highly conserved among all MuV strains, except the aa residue at P8 where a total of four different variants (L, P, S or T) can be found depending on the genotypes of MuV. Even though P8 is not directly located within the furin binding pocket, it might affect the interaction of furin and MuV F (Tian 2009). Indeed, P8 affects the cleavage efficiency by furin. A fluorescence resonance energy transfer (FRET)-based assay in which
40 Discussion peptides corresponding to the aa residues P8 - P1’ were incubated with soluble furin was performed showing that peptides harboring P and T at P8 were faster cleaved by furin than peptides with an S or L at P8 (Hüttl et al. 2020a). Possibly, the cleavage products of L and S might have an inhibitory effect on furin dampening the processing. However, given that S and T - two aa with polar charged side chains - have very similar structures that differ only in a single methyl-group (Fig. S1), it seems rather unlikely that this simple alkyl group may influence the cleavage efficiency to such an extent. Another explanation could be that L and S bind stronger to furin, thereby preventing an easy release of the cleavage products and the binding of new substrates. This is partly contradicted by the observation that a higher concentration of furin did still not result in the same number of processed peptides for all four variants. To reveal the exact position of P8 within the cleavage motif and its interaction with furin, crystal structures of uncleaved MuV F0 in complex with furin are required. However, the generation of such crystal complexes is challenging and so far, there are no crystal structures of uncleaved MuV F available. For other viral glycoproteins such as MeV F, it has been shown by crystallization that the electron density drops off at P4 to P7 suggesting that the aa residues upstream of these positions form only weak bonds (Dahms et al. 2018; Van Lam van et al. 2019). The assumption that aa residues in the flanking region P14 - P7 tend to undergo a weak interaction with furin is also mentioned by Tian et al. (Tian et al. 2011; Tian et al. 2012). Although P8 might not be in direct contact with furin, it is conceivable that P8 supports or disadvantages the formation of α-helix or ß-sheet structures within MuV F (Fig. S2). The cyclic side chain conformation of P often disturbs α-helix structures placing P more likely at the edge of ß- sheets. S and T are also rarely present in helices whereas L is often found in helical structures (Berg 2007). Furthermore, S and T commonly undergo posttranslational modifications in the form of phosphorylation or glycosylation (Tian et al. 2012). Secondary structures and posttranslational modifications might affect the protein structure and thereby influence the accessibility of the cleavage site for the corresponding protease.
Besides affecting proteolytic cleavage, P8 had an influence on the extent and severity of F- induced cell-to-cell fusion and virus-induced CPE. MuV F proteins derived from different strains exhibited the highest fusogenicity when P or T were present at P8. In contrast, F proteins harboring L or S at P8 were clearly less fusogenic. These observations derived from the over- expression of MuV F and HN were confirmed by infection studies using modified recombinant MuVs. In the viral context, fusogenicity correlated with the occurrence of virus-induced CPE that was most severe for MuVs harboring T at P8, whereas nearly no CPE was observed for the less fusogenic viruses harboring L at P8. The reliance of MuV F activation on furin was not affected by the aa residue at P8.
41 Discussion
In contrast, F-induced fusion between cellular and viral membranes was more efficient for P8 L and S and a less efficient viral entry was observed for P and T although these variations of F were highly effective in inducing cell-to-cell fusion. These observations led to the conclusion that virus-cell and cell-to-cell fusions are two distinct processes which most likely underlie different mechanisms and requirements. The differentiation into two varying fusion processes has already been considered for SeV, herpesvirus, mouse hepatitis CoV as well as human immunodeficiency virus type 1 (HIV-1) (Henis et al. 1989; Konopka et al. 1995; Pleskoff et al. 1995; Hernandez et al. 1996; de Haan et al. 2004). For example, the inhibition of furin prevented the cleavage of the spike protein of mouse hepatitis CoV and thereby resulted in a reduced cell-to-cell fusion but a nearly unaffected virus-cell fusion (de Haan et al. 2004). A derivative of amphotericin B was shown to prevent the entry of HIV-1 but not its syncytium formation whereas the inhibiting effect of a synthetic tripeptide was the other way around speculating that mechanism and requirements of HIV-1 entry and syncytium formation might not be identical (Konopka et al. 1995; Pleskoff et al. 1995). So far, the mechanisms behind the kinds of viral fusion have not been elucidated. On the other hand, the differences observed for MuV F-induced cell-to-cell and virus-cell fusion could be a result of the impaired proteolytic cleavage of L and S. The expression of P or T at P8 led to a faster cleavage by furin compared to L and S. Normally, the furin-mediated F proteolysis occurs in the TGN and fusion-active F1/2 is expressed on the plasma membrane and later incorporated into new synthesized virions (Waxham et al. 1987; Seidah et al. 1998). In the case of P8 P and T, the fast processing enables the expression of high quantities of cleaved F proteins on the cell surface that can directly induce cell-to-cell fusion. Given the assumption that F proteins carrying L or S at P8 are slower cleaved, more uncleaved F0 might be present at the cell surface what, in turn, reduces the ability to mediate subsequent cell-to- cell fusion. As a consequence, uncleaved F0 is incorporated into viral particles. Such F proteins are probably processed by surface-expressed furin during viral entry. Furin-mediated F proteolysis during the entry process has been observed for other viruses such as RSV and human papillomaviruses (Richards et al. 2006; Krzyzaniak et al. 2013).
The contrary abilities to mediate fusion between cells or between virus and plasma membrane might also play a role for the spread of infection. MuVs can spread within a host either by the release of infectious viral particles or directly from infected to neighboring cells by syncytium formation. Based on the results obtained regarding the fusogenicity of MuV F, P and T at P8 would preferentially led to viral spread from cell to cell whereas P8 L and S most likely spread by the release of infectious viral particles. This assumption has been strengthened by the fact that MuVs harboring L or S at P8 displayed higher released viral titers compared to the intracellularly retained titers. In contrast, intracellular titers of MuV expressing P or T at P8
42 Discussion were comparable high to the corresponding released viral titers enabling a spread of infection from cell to cell. Both ways of viral spread provide their advantages but also some disadvantages. The release of virions from infected cells enables a wide distribution into different organs by using blood or lymphatic vessels but simultaneously exposes the virus to the host immune system and confronts it with biochemical or mechanical barriers. Direct cell- to-cell spread is a faster route of infection. Besides, it is discussed to be a facilitated, more targeted and efficient way that even contributes to avoid the host immune response (Sattentau 2008; Casartelli et al. 2010; Jolly et al. 2010; Mothes et al. 2010; Cifuentes-Munoz et al. 2018). In sum, the aa residue at P8 of MuV F affects the cell-to-cell and virus-cell fusion of MuVs as well as MuV F proteolysis by furin and might thereby influence the spread of infection. Whether the differentiated fusogenicity correlates with in vivo virulence has to be proven in a suitable animal model. With respect to evaluating neurovirulence of MuV strains, the intracranial inoculation of newborn rats enables to differentiate between neurovirulent and attenuated strains (Rubin et al. 2000; Rubin et al. 2005). This model could be used to investigate the neurovirulent potential of modified MuVs depending on the aa residue at P8. Once a direct link between P8 and neurovirulence and/or attenuation has been established, this specific aa residue may be used as a marker to estimate the virulence of circulating and the attenuation of vaccine strains.
5.3 Furin-independent cleavage of MuV F The cellular protease furin is a serine endoprotease, more specific a proprotein convertase of the subtilisin-like family, that recognizes a multibasic cleavage motif with the sequence R-X- (R/K/X)-R (Waxham et al. 1987; Molloy et al. 1992; Walker et al. 1994; Molloy et al. 1999). Furin is ubiquitously expressed and primarily localized in the TGN, endosomes or the plasma membrane, constantly circling from the TGN to the cell surface (Seidah et al. 1998). Its enzymatic activity has a broad pH spectrum but is highly calcium-dependent (Thomas 2002). Furin is a key player in many cellular processes and involved in regulating cell growth and differentiation as well as in processing growth factors, hemostasis factors or precursors of matrix metalloproteinases (Takahashi et al. 1995; Molloy et al. 1999; Kang et al. 2002; Urban et al. 2013). Besides MuV F, many viral surface-expressed glycoproteins such as these of HIV, influenza viruses or EBOV are activated by furin as well (Stieneke-Grober et al. 1992; Volchkov et al. 1998; Schneck et al. 2020).
Type II transmembrane serine proteases (TTSPs) build the largest group of membrane- anchored trypsin-like serine proteases that contains a total of 17 proteases (Hooper et al. 2001; Antalis et al. 2011). TTSPs are predominantly found in respiratory or gastrointestinal epithelial cells. Some of them are known to process and activate fusion-mediating glycoproteins of
43 Discussion several viruses of the respiratory tract including influenza A viruses and CoVs (Bertram et al. 2010; Bertram et al. 2013; Shirato et al. 2013; Shen et al. 2017; Hoffmann et al. 2020b). Especially the protease TMPRSS2 attracted attention as it is responsible for the proteolytic activation of the spike proteins of SARS-CoV and the recently emerged SARS-CoV-2 (Bertram et al. 2011; Hoffmann et al. 2020a). TMPRSS2 is widely distributed in several tissues but mainly in epithelial cells of the respiratory or gastrointestinal tract (Bugge et al. 2009; Yamaya et al. 2015). Given that the epithelium of the upper airways most likely represents the primary infection site of MuV, extracellular proteases expressed in the respiratory tract might play a role in the cleavage of MuV F (Henle et al. 1948; Foy et al. 1971; Rubin et al. 2015).
This thesis focused on the identification of additional proteases that are able to cleave human and bat-derived MuV F and to identify potential antiviral drugs. Consistent with previous publications, it could be shown that hMuV and batMuV replication in cell lines such as Vero76 cells that do not sufficiently express TTSPs relies on the proteolytic activation of MuV F by furin (Shi et al. 2017; Kruger et al. 2018; Hüttl et al. 2020a; Hüttl et al. 2020b; Wang et al. 2020). However, in Caco-2 cells, a cell line that expresses high levels of TTSPs such as TMPRSS2, TMPRSS4 and matriptase (Bertram et al. 2010; Kleine-Weber et al. 2018), inhibition of furin affected MuV replication only slightly. Further, viral replication was reduced by camostat mesylate, an inhibitor of a broad spectrum of trypsin-like serine proteases, but a significant decrease of replication was only observed by simultaneous inhibition of furin and TTSPs. Conclusively, furin as well as TTSPs can cleave hMuV and batMuV F and the availability of a protease defines the protease choice. Therefore, TTSPs can compensate MuV F proteolysis during furin inhibition whereas furin, in turn, can undertake the cleavage when TTSPs are inhibited or absent. Hence, the identification of a certain TTSP that is responsible for the furin-independent cleavage of MuV F was of major interest. Caco-2 cells are derived from the epithelium of the human colon. TTSPs of the same subfamily as TMPRSS2 including TMPRSS3, TMPRSS4 and TMPRSS13 (mosaic serine protease large form (MSPL)) or others such as TMPRSS11D (human airway trypsin-like protease, HAT), TMPRSS11E (DESC1) and matriptases are present in the intestinal tract and might therefore be expressed by Caco-2 cells (Bugge et al. 2009). By performing a FRET-based assay the cleavage of hMuV and batMuV F peptides by specific proteases was investigated. Further, MuV infectivity was analyzed in camostat mesylate-treated Vero cells that constitutively express TMPRSS2. In fact, it could be shown that matriptase efficiently processes F peptides of hMuV and batMuV whereas TMPRSS2, HAT and DESC1 were not capable of activating MuV F. This is in line with the findings of the inhibitor assay as matriptase is expressed in Caco-2 cells and known to be sensitive to camostat mesylate treatment (Wang et al. 2009; Buzza et al. 2010; Nimishakavi et al. 2015;
44 Discussion
Bardou et al. 2016). Moreover, matriptase is described to preferably cleave sequence motifs with arginine residues at P1 and P4 (Takeuchi et al. 2000), both residues are present in the cleavage motifs of all MuV F proteins. Hemagglutinins of influenza viruses are already found to be processed by matriptase in human respiratory epithelial cells (Oberst et al. 2003; Beaulieu et al. 2013).
Due to their involvement in many different diseases resulting in severe or even life threatening pathologic manifestations, proteases are potential drug targets. Especially their inhibition is a promising therapeutic approach. In case of many viral infections, the focus of antiviral treatment is laid on inhibitors of cellular as well as virus-encoded proteases aimed to prevent viral protein activation and thereby viral replication (Patick et al. 1998). The findings obtained in this thesis reveal furin and TTSPs, most likely matriptase, as potential therapeutic targets for the treatment of clinical MuV infections. So far, furin inhibitors are not approved as antiviral therapeutics but ongoing studies are aimed at this (Couture et al. 2015). Camostat mesylate is a licensed drug for the treatment of e.g. pancreatitis and currently considered for antiviral treatment of COVID-19 (Sai et al. 2010; McKee et al. 2020). It still needs to be clarified whether MuV replication can be dampened to a level associated with no severe clinical signs by the sole use of camostat mesylate or whether a combined therapeutic agent including furin inhibitors and camostat mesylate is required. Nevertheless, the usage of alternative proteases by a virus could make drug development more challenging as a combined antiviral therapy could affect the efficiency of each single drug and increase the risk of side effects.
45
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Xu, K., Broder, C. C., and Nikolov, D. B. 2012a. 'Ephrin-B2 and ephrin-B3 as functional henipavirus receptors', Semin Cell Dev Biol, 23(1): 116-23. Xu, P., Li, Z., Sun, D., Lin, Y., Wu, J., Rota, P. A., and He, B. 2011. 'Rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the SH ORF in the pathogenesis of mumps virus', Virology, 417(1): 126-36. Xu, P., Luthra, P., Li, Z., Fuentes, S., D'Andrea, J. A., Wu, J., Rubin, S., Rota, P. A., and He, B. 2012b. 'The V protein of mumps virus plays a critical role in pathogenesis', J Virol, 86(3): 1768-76. Yamaya, M., Shimotai, Y., Hatachi, Y., Lusamba Kalonji, N., Tando, Y., Kitajima, Y., Matsuo, K., Kubo, H., Nagatomi, R., Hongo, S., Homma, M., and Nishimura, H. 2015. 'The serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells', Pulm Pharmacol Ther, 33(66-74. Yao, Q., Hu, X., and Compans, R. W. 1997. 'Association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces', J Virol, 71(1): 650-6. Yates, P. J., Afzal, M. A., and Minor, P. D. 1996. 'Antigenic and genetic variation of the HN protein of mumps virus strains', J Gen Virol, 77 ( Pt 10)(2491-7. Yeo, R. P., Afzal, M. A., Forsey, T., and Rima, B. K. 1993. 'Identification of a new mumps virus lineage by nucleotide sequence analysis of the SH gene of ten different strains', Arch Virol, 128(3-4): 371-7. Yokota, S., Okabayashi, T., and Fujii, N. 2011. 'Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest', Virology, 414(1): 74-82. Yokota, S., Saito, H., Kubota, T., Yokosawa, N., Amano, K., and Fujii, N. 2003. 'Measles virus suppresses interferon-alpha signaling pathway: suppression of Jak1 phosphorylation and association of viral accessory proteins, C and V, with interferon-alpha receptor complex', Virology, 306(1): 135-46. Yoneda, M., Guillaume, V., Sato, H., Fujita, K., Georges-Courbot, M. C., Ikeda, F., Omi, M., Muto-Terao, Y., Wild, T. F., and Kai, C. 2010. 'The nonstructural proteins of Nipah virus play a key role in pathogenicity in experimentally infected animals', PLoS One, 5(9): e12709. Yoshida, N., and Nakayama, T. 2010. 'Leucine at position 383 of fusion protein is responsible for fusogenicity of wild-type mumps virus in B95a cells', Intervirology, 53(4): 193-202. Yuan, P., Swanson, K. A., Leser, G. P., Paterson, R. G., Lamb, R. A., and Jardetzky, T. S. 2011. 'Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk', Proc Natl Acad Sci U S A, 108(36): 14920-5. Zengel, J., Phan, S. I., Pickar, A., Xu, P., and He, B. 2017. 'Immunogenicity of mumps virus vaccine candidates matching circulating genotypes in the United States and China', Vaccine, 35(32): 3988-94. Zhang, G., Cowled, C., Shi, Z., Huang, Z., Bishop-Lilly, K. A., Fang, X., Wynne, J. W., Xiong, Z., Baker, M. L., Zhao, W., Tachedjian, M., Zhu, Y., Zhou, P., Jiang, X., Ng, J., Yang, L., Wu, L., Xiao, J., Feng, Y., Chen, Y., Sun, X., Zhang, Y., Marsh, G. A., Crameri, G., Broder, C. C., Frey, K. G., Wang, L. F., and Wang, J. 2013. 'Comparative analysis of bat genomes provides insight into the evolution of flight and immunity', Science, 339(6118): 456-60. Zhang, Y., Yang, L., Wang, H., Zhang, G., and Sun, X. 2016. 'Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR-29a-mediated inhibition of interferon-α receptor', Biochem Biophys Res Commun, 478(3): 1436-41. Zhu, J., Li, P., Wu, T., Gao, F., Ding, Y., Zhang, C. W., Rao, Z., Gao, G. F., and Tien, P. 2003. 'Design and analysis of post-fusion 6-helix bundle of heptad repeat regions from Newcastle disease virus F protein', Protein Eng, 16(5): 373-9.
60 Supplementary data
7 Supplementary data
Fig. S1: Structural formula of leucine, proline, serine and threonine. The four amino acids that can be found at position 8 of the cleavage site of the MuV F protein are shown as their skeletal formula. Based on the IUPAC name the structural formulae were generated by using the online tool “Strukturformelzeichner” (http://www.strukturformelzeichner.de).
61 Supplementary data
Fig. S2: Modeling of mumps virus F protein structure. The complete human MuV F protein structure has been predicted via computer-based modelling. The shown part focusses on the furin cleavage motif (colored amino acid (aa) residues at P14 - P8’). The aa residue at P8 (position 95 of chain A) as only variable is highlighted as ball-stick model (red). Based on the FASTA sequence of human MuV F, a pdb format was generated by using the SWISS-MODEL server of the Biozentrum of the University of Basel (https://swissmodel.expasy.org/interactive). The modelling of the molecular 3D structure was performed with the UCSF Chimera 1.14 program.
62 Appendix
8 Appendix 8.1 Sequences batMuV F Strain: Bat Paramyxovirus Epo_spe/AR1/DRC/2009 GenBank accession number: HQ660095.1 (complete genome) Reference: Drexler et al., 2012 Cleavage motif
Nucleotide sequence: ATGAGAAAAACTCTAGCTATTGGTTTGGGTTTTATGATCTTTTCATTATCAGTATGTATAAATATCAATACTCTA CAACAAGTTGGTTATATTAAACAGCAGGTTAGACAATTAAGCTACTATTCACAGAGTTCAAGTGCATATATTGTG GTTAAGCTTCTACCAAATATCAAGCCAAGTAATGGGAATTGTGAATTTAATAGTGTAGCCCAATACAACAAGACA TTGAGTAACCTACTTCTTCCAATCGCAGAGAATATTAACAGCATAGCACCGCCCTCATCAAGTAGTCGGCGCAGG AAAAGGTTTGCAGGTATTGCTATCGGAATTGCTGCCCTTGGCGTCGCAACTGCAGCACAAGTTACAGCTGCAGTT TCATTGGTGCAAGCCCAAACGAATGCACGTGCAATTGCAACGCTTAAGAATTCTATTCAGGCCACTAACAAAGCA ATTTTTGAAGTGAAAGAAGGAACTCAACAGCTAGCCATTGCAGTACAAGCAATTCAAGATCACATCAATACAATC ATGAATACACAATTAAACAACATGTCTTGTCAAATTCTCGATAATCAGCTTGCAACATCGTTAGGATTGTATTTA ACAGAACTAACAACAGTTTTCCAACCTCAATTAGTTAATCCGGCACTATCACCTATCAGCATTCAAGCTTTAAGA TCTTTGCTAGGCAGTATGACACCTGCAGTGGTTCAAGCGGCACTATCAACTTCGATATCTGCTGCAGAGATTTTG AGTGCCGGCCTGATGGAAGGTCAGATAGTTGCAGTTTTTTTAGATCAAATGCAAATGATAGTTAAAGTTAATGTT CCGACTATCGTTACTCAATCTAATGCACTAGTAATAGATTTCCACTCCATATCAAGCTTTATCAATAATCAGGAG TCTTTGATCCAGTTACCTAGCAGAATTTTAGAAATCGGAAGTGAGCAATGGAGTTATCCGGCTGCTAACTGCAAG TTGACAAGAAATCATATATTCTGTCAATATAATGAAGCAGAGATAATGAGTCTAGAGTCAAAGCAATGCTTAGCT GGCAATATTAGTGCATGTGTGTTCACACCTATAGCAGGAAGTTATATGAGGAGGTTTGTTGCATTGGATGGAACT ATTGTTGCAAATTGCCGGAGTCTCACATGCCTTTGCAAAGAACCATCTTATCCTATCTATCAGCCCGACCATCAT GCAGTCACAACAATTGATTTAACAATATGCAAAACATTGTCTCTGGATGGATTAGACTTCAGTATTGTCTCATTA AGCAACGTTACTTATGCTGAGAATTTAACAATTTCATTATCTCAAACAATCAATACTCAACCTATAGATATATCA ACTGAGCTTAGTAGGGTCAACGCATCTCTCCAAAATGCTGTCAATTACATAAAGGAGAGCAATAATCAATTGCAA TCAGTCAGTGTTAATTCAAAGTTAGGAGCTATAGTAGTGGCATCATTAGTGCTTAGCATATTATCAATTATCATT TCTCTCTTGTTTTGTTGCTGGGCTTATATAGCTACTAAAGAGATTAGACGAATTAATTTCAGAACTAATCATATT AACACTGTATCAAGCAGTGTTGATGACCTTGTGAGATACTAA
Amino acid sequence: MRKTLAIGLGFMIFSLSVCININTLQQVGYIKQQVRQLSYYSQSSSAYIVVKLLPNIKPSNGNCEFNSVAQYNKT LSNLLLPIAENINSIAPPSSSSRRRKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIATLKNSIQATNKA IFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLVNPALSPISIQALR SLLGSMTPAVVQAALSTSISAAEILSAGLMEGQIVAVFLDQMQMIVKVNVPTIVTQSNALVIDFHSISSFINNQE SLIQLPSRILEIGSEQWSYPAANCKLTRNHIFCQYNEAEIMSLESKQCLAGNISACVFTPIAGSYMRRFVALDGT IVANCRSLTCLCKEPSYPIYQPDHHAVTTIDLTICKTLSLDGLDFSIVSLSNVTYAENLTISLSQTINTQPIDIS TELSRVNASLQNAVNYIKESNNQLQSVSVNSKLGAIVVASLVLSILSIIISLLFCCWAYIATKEIRRINFRTNHI NTVSSSVDDLVRY
63 Appendix batMuV HN Strain: Bat Paramyxovirus Epo_spe/AR1/DRC/2009 GenBank accession number: AFH96016.1 Reference: Drexler et al., 2012
Nucleotide sequence: ATGGAGCCATCAAAATTATTCACAGTTTCGGATGGTACAACATTCACACCCGGGTCTGCTCAAAACTCTGTTGGT AAGAAAACTTTTCGTGTCTGCTTCCGAATATTGGTTTTATCTATGCAACTTGCTACTTTGATACTTATTATTGTA ACACTCGGGGAGCTTATAAGGATGATCAACGACCAAGGTCTAAGTAATCAATTGAATTCAGCAATAGACAAGATC AAAGATGCTGCTGCTTTGATAGCATCAGCTATGGGGGTGATGAACCAGGTTATTTACGGTATAACTGTGTCTCTA CCTCTTCAAATCGAAGGAAACCAGAATCAATTATTATCTACTATTTCTACAATTTGCTCTAATAATAAACAATTT TCCAATTGCTCGGCAGCTATACCTCTACTTAATGATGTTAGGTTTATCAATGGTATCAATAAGTTTATCATTGAG GATCAAAATACTCATGGTTTTTCTATTGGAAGTCCACTTAACATGCCAAGCTTTATCCCAACTGCAACTTCACCA ACCGGGTGCACCAGAATTCCATCCTTTTCTTTGGGCAATACACACTGGTGTTATACTCACAATGTCATCAATGCA AATTGCAAGGATCATACCTCTTCAAATCAATATGTTTCTATGGGAATTCTCGTGCAAACTGCATCCGGGTATCCA ATGTTCAAAACCTTAAAAATCCAGTATCTGAGTGATGGATTAAATCGTAAGAGTTGCTCGATTGCTACTGTCCCT GATGGTTGTGCAATGTATTGCTATGTTTCAACTCAATTAGAGACGGATGATTATGCAGGTRCTAGTCCACCTACT CAAAAAATCATTATATTATTTTACAATGATACTATGATAGAAAGAACAATAACACCTACCAATTTGGAGGGAAAC TGGGCTACCATAGTACCTGGGGTGGGGAGTGGTGTGTATTTTGAGAATAAATTGATTTTCCCAGCCTATGGGGGG GTTCTACCAAATAGCTCACTGGGGATTAAGTTAACTAATGAATTTTTCCGACCTGTTAACCCATATAATCCATGC TCTGGATCAGACCAAGATTTGAATCAGCGTGCACTTCAATCGTACTTCCCAAATTATTTTTCAAGCCGAAGAATA CAAAGTGCATTCTTACTCTGTACCTGGAGCCAAATTTTAGTAACAAACTGTGAGTTAGTGGTCCCGTCAGACAAT CAGGTAATGATGGGTGCAGAAGGGCGGGTATTATTGATTAATAATAGACTAATCTATTATCAGAGGAGCACAAGC TGGTGGCCATATGAGCTGCTCTACGAAATGTCATTTACGCTTGCTGAGTCTGGTCAAACACTTGTGAACCTGTCT TGGATACCAATATACTCATTCACTCGTCCTGGGTCAGGTGATTGCAGTGGGCTAAATGTTTGTCCAACTGTCTGC GTGTCCGGAGTCTACTTGGATGCTTGGCCCCTCTCTCAATACACCTTGCAGTCAGGGGTGAACAGAAATTTCTAC TTTACAGGAGCCCTCCTTAATACAAGCACAATGAGAATTAATCCAACATTATATGTTTCTGCATTAAGTAATCTC AAGACCTCATCTTCATATGGAACTCAGGGATTGATTGCTGCTTATACCACCACCACTTGCTTTCAAGACACAGGT GACTCTAGTGTGTATTGTGTATATATCATGGAACTGGCGTCAAATATTGTGGGAGAATTCCAGATTTTACCTGTA CTAACAAGATTGACTATCACATGA
Amino acid sequence: MEPSKLFTVSDGTTFTPGSAQNSVGKKTFRVCFRILVLSMQLATLILIIVTLGELIRMINDQGLSNQLNSAIDKI KDAAALIASAMGVMNQVIYGITVSLPLQIEGNQNQLLSTISTICSNNKQFSNCSAAIPLLNDVRFINGINKFIIE DQNTHGFSIGSPLNMPSFIPTATSPTGCTRIPSFSLGNTHWCYTHNVINANCKDHTSSNQYVSMGILVQTASGYP MFKTLKIQYLSDGLNRKSCSIATVPDGCAMYCYVSTQLETDDYAGXSPPTQKIIILFYNDTMIERTITPTNLEGN WATIVPGVGSGVYFENKLIFPAYGGVLPNSSLGIKLTNEFFRPVNPYNPCSGSDQDLNQRALQSYFPNYFSSRRI QSAFLLCTWSQILVTNCELVVPSDNQVMMGAEGRVLLINNRLIYYQRSTSWWPYELLYEMSFTLAESGQTLVNLS WIPIYSFTRPGSGDCSGLNVCPTVCVSGVYLDAWPLSQYTLQSGVNRNFYFTGALLNTSTMRINPTLYVSALSNL KTSSSYGTQGLIAAYTTTTCFQDTGDSSVYCVYIMELASNIVGEFQILPVLTRLTIT-
64 Appendix
88-1961 F Strain: 88-1961 GenBank accession number: AF467767.2 (complete genome) Reference: (Amexis et al. 2003)
Nucleotide sequence: ATGAAGGTTTCTTTAGTTACTTGCTTGGGCTTTGCAGTCTTTTCATTTTCCATATGTGTGAATATCAATATCTTG CAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTGAGCTATTACTCACAAAGTTCAAGCTCCTACATAGTG GTCAAGCTTTTACCGAATATCCAACCCACTGATAACAGCTGTGAATTCAAGAGTGTAACTCAATACAATAAGACC TTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAATAATATTGCATCGCCCTCACCTGGATCAAGACGTCAT AAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCACTCGGTGTTGCAACCGCAGCACAAGTAACCGCCGCTGTC TCATTAGTTCAAGCACAGACAAATGCACGCGCAATAGCGGCGATGAAAAATTCAATACAGGCAACTAATCGAGCA GTCTTCGAAGTGAAAGAAGGCACCCAACAGTTAGCTATAGCGGTACAAGCAATACAGAACCACATCAATACTATT ATGAACACCCAATTGAACAATATGTCCTGTCAGATTCTTGATAACCAGCTTGCAACCTCCCTGGGATTATACCTA ACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCCTTGAGG TCTTTGCTTGGAAGTATGACACCTGCAGTGGTTCAAGCAACATTATCTACTTCAATTTCTGCTGCTGAAATACTA AGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTGGATGAGATGCAGATGATAGTTAAGATAAATATT CCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAATCAGGAA TCCATAATTCAATTACCAGACAGGATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCAAAAAATTGTAAG TTGACAAGACACAACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCTTAGAATCAAAACTATGCCTTGCA GGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGATGGAACA ATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAGTCCATCTTATCCTATATACCAACCTGACCATCAT GCAGTCACGACCATTGATCTAACCGCATGTCAGACGTTGTCCCTAGACGGATTGGATTTCAGCATTGTCTCTCTA AGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGACATATCA ACTGAACTGATCAAGGTCAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAACTCCAA TCTGTGAGTATAAATTCTAAAATCGGAGCTATAATCATAGCAGCCTTAGtTTTGAGCATCCTGTCAATGATCATT TCACTGTTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAGATCAGAAGAATCAACTTCAAAACAAATCATATC AACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAA
Amino acid sequence: MKAFSVICLGFAVFSSSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKSISQYNKT LSNLLLPIAENINNIASPSPGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFVNNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY
65 Appendix
88-1961 HN Strain: 88-1961 GenBank accession number: AF467767.2 (complete genome) Reference: Amexis et al., 2003
Nucleotide sequence: ATGGAACCCTCAAAACTCTTCACAATATCAGACAATGCCACCTTTGCACCTGGGCCTGTTATCAATGCGGCTGAC AAGAAGACATTCCGAACCTGCTTCCGAATATTGGTACTGTCTGTACAAGCTGTTACCCTTATATTAGTTATTGTC ACTTTAGGTGAGCTTGTGAGGATGATCAATGATCAAGGCTTGAGCAATCAGTTGTCTTCAATTACAGACAAGATA AGAGAGTCAGCTACTATTATTGCATCTGCTGTGGGAGTAATGAATCAAGTTATTCACGGGGTAACGGTATCCTTA CCCCTACAAATTGAGGGAAATCAAAATCAATTGTTATCCACAATTGCCACGATCTGTACAAGCAAAAAACAAGCC TCAAACTGCTCTACAAACGTCCCCTTAGTTAATGACCTTAGGTTTATAAACGGGATCAATAAATTCATCATTGAA GATTATGCAACTCATGAGTTCTCTATCGGCCATCCACTCAACATGCCTAGCTTTATCCCAACTGCAACTTCACCC AATGGTTGCACAAGAATTCCATCCTTTTCTCTAGGCAAGACACACTGGTGCTACACACATAATGTAATTAATGCC AACTGCAAGGATCATACTTCGTCTAACCAATATGTTTCCATGGGGATTCTCGTTCAGACCGCGTCAGGATATCCT ATGTTCAAAACCTTGAAGATCCAATACCTCAGTGATGGCCTGAATCGGAAAAGCTGCTCAATTGCAACAGTCCCC GATGGATGCGCAATGTACTGTTACGTCTCAACTCAACTTGAAACCGACGACTATGCGGGGTCCAGCCCACCTACC CAGAAACTTACCCTGTTATTCTATAATGAYACCGTCACAGAAAGGACAATTTCTCCATCTGGTCTTGAAGGGAAT TGGGCTACTTTGGTGCCAGGAGTAGGGAGTGGAATATATTTCGAGAATAAATTGATCTTCCCTGCATATGGGGGT GTCTTGCCCAATAGTACACTCGGAGTTAAATCAGCAAGAGAATTTTTTCGGCCTGTTAATCCATATAACCCATGT CCAGGACCACAACAAGATCTAGATCAGCGGGCTTTGAGATCATACTTCCCCAGTTACTTCTCTAATCGAAGAGTG CAGAGTGCATTTCTTGTCTGTGCCTGGAATCAGATCCTAGTTACAAATTGCGAGCTAGTTGTCCCCTCAAACAAT CAGACACTGATGGGTGCAGAAGGAAGAGTTTTATTGATCAATAATCGACTATTATATTATCAGAGAAGTACCAGC TGGTGGCCGTATGAACTCCTCTATGAGATATCATTCACATTTACAAACTCTGGTTTATCATCTGTGAACATGTCC TGGATACCTATATATTCATTCACTCGTCCTGGTTCAGGCAACTGTAGCGGTGAAAATGTGTGCCCAACTGCTTGT GTGTCAGGGGTTTATCTTGATCCCTGGCCATTAACTCCATACAACCACCAATCAGGCATTAACAGAAATTTCTAT TTCACAGGCGCACTATTAAATTCAAGCACAACCAGAGTAAATCCTACCCTTTATGTCTCTGCCCTTAATAATCTT AAAGTACTAGTCCCATATGGTACTCAAGGACTGTTTGCCTCGTACACCACAACCACCTGCTTTCAAGACACCGGT GATGCTAGTGTGTATTGTGTTTATATTATGGAACTAGCATCGAATATCGTTGGAGAATTCCAAATTCTACCTGTG CTAACCAGATTGACTATCACTTGA
Amino acid sequence: MEPSKLFTISDNATFAPGPVINAADKKTFRTCFRILVLSVQAVTLILVIVTLGELVRMINDQGLSNQLSSITDKI RESATIIASAVGVMNQVIHGVTVSLPLQIEGNQNQLLSTIATICTSKKQASNCSTNVPLVNDLRFINGINKFIIE DYATHEFSIGHPLNMPSFIPTATSPNGCTRIPSFSLGKTHWCYTHNVINANCKDHTSSNQYVSMGILVQTASGYP MFKTLKIQYLSDGLNRKSCSIATVPDGCAMYCYVSTQLETDDYAGSSPPTQKLTLLFYNXTVTERTISPSGLEGN WATLVPGVGSGIYFENKLIFPAYGGVLPNSTLGVKSAREFFRPVNPYNPCPGPQQDLDQRALRSYFPSYFSNRRV QSAFLVCAWNQILVTNCELVVPSNNQTLMGAEGRVLLINNRLLYYQRSTSWWPYELLYEISFTFTNSGLSSVNMS WIPIYSFTRPGSGNCSGENVCPTACVSGVYLDPWPLTPYNHQSGINRNFYFTGALLNSSTTRVNPTLYVSALNNL KVLVPYGTQGLFASYTTTTCFQDTGDASVYCVYIMELASNIVGEFQILPVLTRLTIT-
66 Appendix
Urabe AM9 F Strain: Urabe AM9 vaccine strain GenBank accession number: - Reference: provided by C. Sauder
Nucleotide sequence: ATGAAGGCTTTTTTAGTTACTTGCTTAAGCTTTGCAGTCTTTTCATCTTCTGTATGTGTGAATATCAACATCTTG CAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCACAAAGTTCAAGCTCCTACATAGTG GTCAAGCTTTTACCGAATATCCAACCCATTGATAACAGCTGTGAATTTAAGAGTGTAACTCAATACAATAAGACC TTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAACAATATTGCATCGCCCTCATCTGGGTCAAGACGGCAT AAAAGGTTTGCTGGTATTGCTATTGGCATTGCTGCGCTCGGTGTTGCGACCGCAGCACAAGTAACTGCCGCTGTC TCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCGGCGATGAAAAATTCAATACAAGCAACTAATCGAGCA GTCTTCGAAGTGAAGGAAGGCACTCAACAGTTAGCTATAGCGGTACAAGCAATACAAGACCACATCAATACTATT ATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTTCCTAGGATTATACCTA ACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCCTTGAGG TCTTTGCTTGGAAGTATGACGCCTGCAGTGGTCCAAGCAACATTATCTACTTCAATCTCTACTGCTGAAATACTA AGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATAAATATT CCAACTATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAATCAGGAA TCCATAATCCAATTACCAGACAGAATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAAAATTGTAAG TTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGCCTTGCA GGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTACGGCACTGGATGGAACA ATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAATCCATCTTATCCTATATACCAACCTGACCATCAT GCAGTCACGACCATTGATCTAACCGCATGTCAAACACTGTCCCTAGACGGATTGGATTTCAGCATTGTCTCTCTA AGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGACATATCA ACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAGCTCCAA TCTGTGAGTGTAAACTCCAAAATCGGAGCTATAATTGTAGCAGCCTTAGTTTTGAGCATTCTGTCAATTATCATT TCGCTATTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGAATCAACTTCAAAACAAATCATATC AATACAATATCAAGTAGTGTCGATGATCTCATTAGGTACTAA
Amino acid sequence: MKAFLVTCLSFAVFSSSVCVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPIDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSSGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATFLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISTAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFAALDGT IVANCRSLTCLCKNPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY
67 Appendix
Urabe AM9 HN Strain: Urabe AM9 vaccine strain GenBank accession number: X93181.1 Reference: (Yates et al. 1996)
Nucleotide sequence: AAAGAAAAAATCAAGCCAGAACAAGCTTAGGATCACAATACAACACAGAACCCCAGCTGCCATCATAACTGTTCT CTGGCCGCTCGAAAGATGGAGCCCTCAAAACTCTTCACAATGTCAGACAATGCCACCTTTGCACCTGGACCTTTT ATCAATGCGGCAGACAAGAAGACGTTCCGAACCTGCTTCCGAATATTGGTACTGTCTGTACAAGCTGTTACCCTT ATATTAGTTATTGTCACTTTAGGTGAGCTTGTGAGGATGATCAATGATCAAGGCTTGAGCAATCAGTTGTCTTCA ATTGCAGACAAGATAAGAGAGTCAGCTACTATGATTGCATCTGCTGTGGGAGTAATGAATCAAGTTATTCACGGA GTAACGGTATCCTTACCCCTACAAATTGAGGGAAACCAAAATCAATTGTTATCCACACTTGCCACAATCTGTACA GGCAAAAAACAAGTCTCAAACTGCTCTACAAACATCCCCTTAGTTAATGACCTTAGGTTTATAAATGGGATCAAT AAATTCATCATTGAAGATTATGCAACTCATGATTTCTCTATCGGCCATCCACTCAACATGCCTAGCTTTATCCCA ACTGCAACTTCACCCAATGGTTGCACAAGAATTCCATCCTTTTCTCTAGGTAAGACACACTGGTGCTACACACAT AATGTAATTAATGCCAACTGCAAGGATCATACTTCGTCTAACCAATATATTTCCATGGGGATACTCGTTCAGACC GCGTCAGGGTATCCTATGTTCAAAACCTTAAAAATCCAATATCTCAGTGATGGCCTTAATCGGAAAAGCTGCTCA ATTGCAACAGTCCCTGATGGATGCGCAATGTACTGTTACGTCTCAACTCAACTTGAAACCGACGACTATGCGGGG TCCAGCCCACCTACCCAGAAACTTACCCTGTTATTCTATAATGATACCGTCACAGAAAGGACAATATCTCCAACT GGTCTTGAAGGGAATTGGGCTACTTTGGTTCCAGGAGTGGGGAGTGGAATATATTTCGAGAATAAATTGATTTTT CCTGCATATGGGGGTGTCTTGCCCAATAGTACACTCGGAGTTGAATCAGCAAGAGAATTTTTCCGGCCTGTTAAT CCATATAATCCATGTTCAGGACCACAACAAGATTTAGATCAGCGTGCTTTGAGATCATACTTCCCAAGTTACTTC TCTAATCGAAGAGTACAGAGTGCATTTCTTGTCTGTGCCTGGAATCAGATCCTAGTTACAAATTGCGAGCTAGTT GTCCCCTCAAACAATCAGACACTGATGGGTGCAGAAGGAAGAGTTTTATTGATCAATAATCGACTATTATATTAT CAGAGAAGTACCAGCTGGTGGCCGTATGAACTCCTCTATGAGATATCATTCACATTTACAAACTCTGGTCAATCA TCTGTGAATATGTCCTGGATACCTATATATTCATTCACTCGTCCTGGTTCAGGCAACTGCAGTGGTGAAAATGTG TGCCCAACTGCTTGTGTGTCAGGGGTTTATCTTGATCCCTGGCCATTAACTCCATATAGCCACCAATCAGGCATT AACCGAAATTTCTATTTCACAGGCGCACTATTAAATTCAAGCACAACTAGAGTAAATCCTACCCTTTATGTCTCT GCCCTTAATAATCTTAAAGTACTAGCCCCATATGGTAATCAGGGACTGTTTGCCTCGTACACCACAACCACCTGC TTTCAAGATACCGATGATGCTAGTGTGTATTGTGTTTATATTATGGAACTAGCATCGAATATCGTTGGAGAATTC CAAATTCTACCTGTGCTAACCAGATTGACCATCACTTGAGTCATAGTGAATGCAGTGGGAGGCCCTATGGGCGTG CTTCAATCTTTATCGATTATTAAG
Amino acid sequence: MEPSKLFTMSDNATFAPGPFINAADKKTFRTCFRILVLSVQAVTLILVIVTLGELVRMINDQGLSNQLSSIADKI RESATMIASAVGVMNQVIHGVTVSLPLQIEGNQNQLLSTLATICTGKKQVSNCSTNIPLVNDLRFINGINKFIIE DYATHDFSIGHPLNMPSFIPTATSPNGCTRIPSFSLGKTHWCYTHNVINANCKDHTSSNQYISMGILVQTASGYP MFKTLKIQYLSDGLNRKSCSIATVPDGCAMYCYVSTQLETDDYAGSSPPTQKLTLLFYNDTVTERTISPTGLEGN WATLVPGVGSGIYFENKLIFPAYGGVLPNSTLGVESAREFFRPVNPYNPCSGPQQDLDQRALRSYFPSYFSNRRV QSAFLVCAWNQILVTNCELVVPSNNQTLMGAEGRVLLINNRLLYYQRSTSWWPYELLYEISFTFTNSGQSSVNMS WIPIYSFTRPGSGNCSGENVCPTACVSGVYLDPWPLTPYSHQSGINRNFYFTGALLNSSTTRVNPTLYVSALNNL KVLAPYGNQGLFASYTTTTCFQDTDDASVYCVYIMELASNIVGEFQILPVLTRLTIT
68 Appendix
ZK-BN F Strain: MuVi/ZK-BN/H-0001 GenBank accession number: KM519599 Reference: Krüger et al., 2015
Nucleotide sequence: ATGAAGGCTTTTTCAGTTACTTGCTTGGGCTTCGCAGTCTTTTCATCTTCTATATGTGTGAACATCAACATCTTG CAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCCCAAAGTTCAAGCTCCTACATAGTG GTCAAACTTTTACCGAATATCCAACCCCCTGATGACAGCTGTGAATTTAAGAGTATAAGTCAATACAATAAGACC TTGAGTAATTTGCTTCTTCCAATTGCAGAGAATATAAACAATATTGCATCGCCCTCACCTGGGTCAAGACGTCAC AAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCGCTTGGTGTTGCAACAGCAGCACAAGTAACTGCCGCTGTC TCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCAGCGATGAAAAATTCAATACAGGCAACTAATCGGGCA GTATTTGAAGTGAAGGAAGGCACCCAACAGTTAGCCATAGCAGTACAAGCTATACAAGACCACATCAACACTATT ATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTCCCTAGGATTATACCTA ACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCCTTGAGG TCCTTGCTTGGGAGTATGACTCCCGCAGTGGTTCAAGCAGCATTATCTACTGCAATTTCTGCTGCTGAAATACTT AGCGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATAAATATT CCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTGTTAATAATCAGGAA TCCATAATTCAATTGCCAGACAGGATCTTAGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAGAATTGTAAG TTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGCCTTGCA GGCAATATAAGTGCCTGTGTGTTCTCACCTATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGATGGAACA ATTGTTGCAAACTGTCGAAGCCTAACGTGTCTATGCAAAAGTCCATCTTATCCTATATACCAACCTGACCATCAT GCGGTCACGACCATTGATCTAACCGCGTGTCAAACATTGTCCCTAGACGGATTGGACTTTAGCATTGTCTCTCTA AGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGACATATCA ACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAACTCCAA TCCGTGAGTGTAAATTCCAAAATCGGAGCTATAATTGTAGCAGCTTTAGTCTTGAGCATCCTGTCAATTATCATT TCACTATTATTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGGATCAACTTCAAAACAAATCATATC AACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAA
Amino acid sequence: MKAFSVTCLGFAVFSSSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKSISQYNKT LSNLLLPIAENINNIASPSPGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFVNNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
69 Appendix
ZK-BN HN Strain: MuVi/ZK-BN/H-0001 GenBank accession number: KM519600 Reference: Krüger et al., 2015
Nucleotide sequence: ATGGAACCCTCGAAACTCTTCACAATATCAGACAATGCCACCTTTGCACCTGGGCCTGCCATCAATGCGGCTGAC AAGAAGACATTCCGAACCTGTTTCCGAATATTGGTACTATCTGTACAAGCTGTTACCCTTATATTAGTCATTGTC ACTTTAGGTGAGCTCGTGAGGATGATCAATGATCAAGGCTTGAGCAATCAGTTGTCTTCAATTACAGACAAGATA AGAGAGTCAGCTATTATGATTGCATCTGCTGTGGGAGTAATGAATCAAGTTATTCACGGGGTAACGGTATCCTTA CCCCTACAAATTGAGGGAAACCAAAATCAATTGTTATCCACGATTGCCACGATTTGTACAAGCAAAAAACAAGCC TCAAACTGCTCTACAAACGTCCCCTTAGTTAATGACCTTAGGTTTATAAACGGGATCAATAAATTCATCATTGAA GATTATGCAACTCATGAGTTCTCTATCGGCCATCCACTCAACATGCCTAGCTTTATCCCAACTGCAACTTCACCC AATGGTTGCACAAGAATTCCATCCTTTTCTCTAGGCAAGACACACTGGTGCTACACACATAATGTAATTAATGCC AACTGCAAGGATCATACTTCGTCTAACCAATATGTTTCCATGGGGATTCTCGTTCAGACCGCATCAGGGTATCCT ATGTTCAAAACCTTGAAAATCCAATATCTCAGTGATGGCCTGAATCGGAAAAGCTGCTCAATTGCAACAGTCCCC GATGGATGCGCAATGTACTGTTACGTCTCAACTCAACTTGAAACCGACGACTATGCGGGGTCCAGCCCACCTACC CAGAAACTTACCCTGTTATTCTATAATGACACCGTCACAGAAAGGACAATTTCTCCATCTGGTCTTGAAGGGAAT TGGGCTACTTTGGTGCCAGGAGTAGGGAGTGGAATATATTTCGAGAATAAATTGATCTTTCCTGCATATGGGGGT GTCTTGCCCAATAGTACACTCGGAGTTAAATCAGCAAGAGAATTTTTTCGACCTGTTAATCCATATAACCCATGT CCAGGGCCACAACAAGATCTAGATCAGCGGGCTTTGAGATCATACTTCCCCAGTTACTTCTCTAATCGAAGAGTG CAGAGTGCATTTCTTGTCTGTGCCTGGAATCAGATCCTAGTTACAAATTGCGAGCTAGTTGTCCCCTCAAACAAT CAGACACTGATGGGTGCAGAAGGAAGAGTTTTATTGATCAATAATCGACTATTATATTATCAGAGAAGTACCAGC TGGTGGCCGTATGAACTCCTCTATGAGATATCATTTACATTTACAAACTCTGGTTTATCATCTGTGAATATGTCC TGGATACCTATATATTCATTCACTCGTCCTGGTTCAGGCAACTGTAGCGGTGAAAACGTGTGCCCAACTGCTTGT GTGTCAGGGGTTTATCTTGATCCCTGGCCATTAACTCCATACAGCCACCAATCAGGCGTTAACAGAAATTTTTAT TTCACAGGCGCCCTATTAAATTCAAGCACAACCAGAGTAAATCCTACCCTTTATGTCTCTGCCCTTAATAATCTT AAAGTACTAGCCCCATATGGTACTCAAGGACTGTTTGCCTCGTACACCACAACCACCTGCTTTCAAGACACCGGT GATGCTAGTGTGTATTGTGTTTATATTATGGAACTAGCATCGAATATCGTTGGAGAATTCCAAATTCTACCTGTG CTAACCAGATTGACTATCACTTGA
Amino acid sequence: MEPSKLFTISDNATFAPGPAINAADKKTFRTCFRILVLSVQAVTLILVIVTLGELVRMINDQGLSNQLSSITDKI RESAIMIASAVGVMNQVIHGVTVSLPLQIEGNQNQLLSTIATICTSKKQASNCSTNVPLVNDLRFINGINKFIIE DYATHEFSIGHPLNMPSFIPTATSPNGCTRIPSFSLGKTHWCYTHNVINANCKDHTSSNQYVSMGILVQTASGYP MFKTLKIQYLSDGLNRKSCSIATVPDGCAMYCYVSTQLETDDYAGSSPPTQKLTLLFYNDTVTERTISPSGLEGN WATLVPGVGSGIYFENKLIFPAYGGVLPNSTLGVKSAREFFRPVNPYNPCPGPQQDLDQRALRSYFPSYFSNRRV QSAFLVCAWNQILVTNCELVVPSNNQTLMGAEGRVLLINNRLLYYQRSTSWWPYELLYEISFTFTNSGLSSVNMS WIPIYSFTRPGSGNCSGENVCPTACVSGVYLDPWPLTPYSHQSGVNRNFYFTGALLNSSTTRVNPTLYVSALNNL KVLAPYGTQGLFASYTTTTCFQDTGDASVYCVYIMELASNIVGEFQILPVLTRLTIT-
70 Appendix
88-1961 F P8-L GGATCCATGAAGGTTTCTTTAGTTACTTGCTTGGGCTTTGCAGTCTTTTCATTTTCCATATGTGTGAATATCAAT ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTGAGCTATTACTCACAAAGTTCAAGCTCCTAC ATAGTGGTCAAGCTTTTACCGAATATCCAACCCACTGATAACAGCTGTGAATTCAAGAGTGTAACTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAATAATATTGCATCGCCCTCACTTGGATCAAGA CGTCATAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCACTCGGTGTTGCAACCGCAGCACAAGTAACCGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGCGCAATAGCGGCGATGAAAAATTCAATACAGGCAACTAAT CGAGCAGTCTTCGAAGTGAAAGAAGGCACCCAACAGTTAGCTATAGCGGTACAAGCAATACAGAACCACATCAAT ACTATTATGAACACCCAATTGAACAATATGTCCTGTCAGATTCTTGATAACCAGCTTGCAACCTCCCTGGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCTTTGCTTGGAAGTATGACACCTGCAGTGGTTCAAGCAACATTATCTACTTCAATTTCTGCTGCTGAA ATACTAAGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTGGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAAT CAGGAATCCATAATTCAATTACCAGACAGGATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCAAAAAAT TGTAAGTTGACAAGACACAACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCTTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAGTCCATCTTATCCTATATACCAACCTGAC CATCATGCAGTCACGACCATTGATCTAACCGCATGTCAGACGTTGTCCCTAGACGGATTGGATTTCAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGATCAAGGTCAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAA CTCCAATCTGTGAGTATAAATTCTAAAATCGGAGCTATAATCATAGCAGCCTTAGtTTTGAGCATCCTGTCAATG ATCATTTCACTGTTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAGATCAGAAGAATCAACTTCAAAACAAAT CATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAAGTCGAC
MKVSLVTCLGFAVFSFSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPTDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSLGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQNHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHNIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELIKVNASLQNAVKYIKESNHQLQSVSINSKIGAIIIAALVLSILSMIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
88-1961 F P8-S GGATCCATGAAGGTTTCTTTAGTTACTTGCTTGGGCTTTGCAGTCTTTTCATTTTCCATATGTGTGAATATCAAT ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTGAGCTATTACTCACAAAGTTCAAGCTCCTAC ATAGTGGTCAAGCTTTTACCGAATATCCAACCCACTGATAACAGCTGTGAATTCAAGAGTGTAACTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAATAATATTGCATCGCCCTCATCTGGATCAAGA CGTCATAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCACTCGGTGTTGCAACCGCAGCACAAGTAACCGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGCGCAATAGCGGCGATGAAAAATTCAATACAGGCAACTAAT CGAGCAGTCTTCGAAGTGAAAGAAGGCACCCAACAGTTAGCTATAGCGGTACAAGCAATACAGAACCACATCAAT ACTATTATGAACACCCAATTGAACAATATGTCCTGTCAGATTCTTGATAACCAGCTTGCAACCTCCCTGGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCTTTGCTTGGAAGTATGACACCTGCAGTGGTTCAAGCAACATTATCTACTTCAATTTCTGCTGCTGAA ATACTAAGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTGGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAAT CAGGAATCCATAATTCAATTACCAGACAGGATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCAAAAAAT TGTAAGTTGACAAGACACAACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCTTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAGTCCATCTTATCCTATATACCAACCTGAC CATCATGCAGTCACGACCATTGATCTAACCGCATGTCAGACGTTGTCCCTAGACGGATTGGATTTCAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGATCAAGGTCAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAA CTCCAATCTGTGAGTATAAATTCTAAAATCGGAGCTATAATCATAGCAGCCTTAGtTTTGAGCATCCTGTCAATG ATCATTTCACTGTTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAGATCAGAAGAATCAACTTCAAAACAAAT CATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAAGTCGAC
71 Appendix
MKVSLVTCLGFAVFSFSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPTDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSSGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQNHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHNIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELIKVNASLQNAVKYIKESNHQLQSVSINSKIGAIIIAALVLSILSMIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
88-1961 F P8-T GGATCCATGAAGGTTTCTTTAGTTACTTGCTTGGGCTTTGCAGTCTTTTCATTTTCCATATGTGTGAATATCAAT ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTGAGCTATTACTCACAAAGTTCAAGCTCCTAC ATAGTGGTCAAGCTTTTACCGAATATCCAACCCACTGATAACAGCTGTGAATTCAAGAGTGTAACTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAATAATATTGCATCGCCCTCAACTGGATCAAGA CGTCATAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCACTCGGTGTTGCAACCGCAGCACAAGTAACCGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGCGCAATAGCGGCGATGAAAAATTCAATACAGGCAACTAAT CGAGCAGTCTTCGAAGTGAAAGAAGGCACCCAACAGTTAGCTATAGCGGTACAAGCAATACAGAACCACATCAAT ACTATTATGAACACCCAATTGAACAATATGTCCTGTCAGATTCTTGATAACCAGCTTGCAACCTCCCTGGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCTTTGCTTGGAAGTATGACACCTGCAGTGGTTCAAGCAACATTATCTACTTCAATTTCTGCTGCTGAA ATACTAAGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTGGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAAT CAGGAATCCATAATTCAATTACCAGACAGGATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCAAAAAAT TGTAAGTTGACAAGACACAACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCTTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAGTCCATCTTATCCTATATACCAACCTGAC CATCATGCAGTCACGACCATTGATCTAACCGCATGTCAGACGTTGTCCCTAGACGGATTGGATTTCAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGATCAAGGTCAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAA CTCCAATCTGTGAGTATAAATTCTAAAATCGGAGCTATAATCATAGCAGCCTTAGtTTTGAGCATCCTGTCAATG ATCATTTCACTGTTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAGATCAGAAGAATCAACTTCAAAACAAAT CATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAAGTCGAC
MKVSLVTCLGFAVFSFSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPTDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSTGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQNHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHNIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELIKVNASLQNAVKYIKESNHQLQSVSINSKIGAIIIAALVLSILSMIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
72 Appendix
Urabe AM9 F P8-L GGATCCATGAAGGCTTTTTTAGTTACTTGCTTAAGCTTTGCAGTCTTTTCATCTTCTGTATGTGTGAATATCAAC ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCACAAAGTTCAAGCTCCTAC ATAGTGGTCAAGCTTTTACCGAATATCCAACCCATTGATAACAGCTGTGAATTTAAGAGTGTAACTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAACAATATTGCATCGCCCTCACTTGGGTCAAGA CGGCATAAAAGGTTTGCTGGTATTGCTATTGGCATTGCTGCGCTCGGTGTTGCGACCGCAGCACAAGTAACTGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCGGCGATGAAAAATTCAATACAAGCAACTAAT CGAGCAGTCTTCGAAGTGAAGGAAGGCACTCAACAGTTAGCTATAGCGGTACAAGCAATACAAGACCACATCAAT ACTATTATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTTCCTAGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCTTTGCTTGGAAGTATGACGCCTGCAGTGGTCCAAGCAACATTATCTACTTCAATCTCTACTGCTGAA ATACTAAGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACTATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAAT CAGGAATCCATAATCCAATTACCAGACAGAATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAAAAT TGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTACGGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAATCCATCTTATCCTATATACCAACCTGAC CATCATGCAGTCACGACCATTGATCTAACCGCATGTCAAACACTGTCCCTAGACGGATTGGATTTCAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAG CTCCAATCTGTGAGTGTAAACTCCAAAATCGGAGCTATAATTGTAGCAGCCTTAGTTTTGAGCATTCTGTCAATT ATCATTTCGCTATTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGAATCAACTTCAAAACAAAT CATATCAATACAATATCAAGTAGTGTCGATGATCTCATTAGGTACTAAGTCGAC
MKAFLVTCLSFAVFSSSVCVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPIDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSLGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATFLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISTAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFTALDGT IVANCRSLTCLCKNPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
Urabe AM9 F P8-P GGATCCATGAAGGCTTTTTTAGTTACTTGCTTAAGCTTTGCAGTCTTTTCATCTTCTGTATGTGTGAATATCAAC ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCACAAAGTTCAAGCTCCTAC ATAGTGGTCAAGCTTTTACCGAATATCCAACCCATTGATAACAGCTGTGAATTTAAGAGTGTAACTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAACAATATTGCATCGCCCTCACCTGGGTCAAGA CGGCATAAAAGGTTTGCTGGTATTGCTATTGGCATTGCTGCGCTCGGTGTTGCGACCGCAGCACAAGTAACTGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCGGCGATGAAAAATTCAATACAAGCAACTAAT CGAGCAGTCTTCGAAGTGAAGGAAGGCACTCAACAGTTAGCTATAGCGGTACAAGCAATACAAGACCACATCAAT ACTATTATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTTCCTAGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCTTTGCTTGGAAGTATGACGCCTGCAGTGGTCCAAGCAACATTATCTACTTCAATCTCTACTGCTGAA ATACTAAGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACTATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAAT CAGGAATCCATAATCCAATTACCAGACAGAATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAAAAT TGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTACGGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAATCCATCTTATCCTATATACCAACCTGAC CATCATGCAGTCACGACCATTGATCTAACCGCATGTCAAACACTGTCCCTAGACGGATTGGATTTCAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAG CTCCAATCTGTGAGTGTAAACTCCAAAATCGGAGCTATAATTGTAGCAGCCTTAGTTTTGAGCATTCTGTCAATT ATCATTTCGCTATTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGAATCAACTTCAAAACAAAT CATATCAATACAATATCAAGTAGTGTCGATGATCTCATTAGGTACTAAGTCGAC
73 Appendix
MKAFLVTCLSFAVFSSSVCVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPIDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSPGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATFLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISTAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFTALDGT IVANCRSLTCLCKNPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
Urabe AM9 F P8-T GGATCCATGAAGGCTTTTTTAGTTACTTGCTTAAGCTTTGCAGTCTTTTCATCTTCTGTATGTGTGAATATCAAC ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCACAAAGTTCAAGCTCCTAC ATAGTGGTCAAGCTTTTACCGAATATCCAACCCATTGATAACAGCTGTGAATTTAAGAGTGTAACTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAAAACATAAACAATATTGCATCGCCCTCAACTGGGTCAAGA CGGCATAAAAGGTTTGCTGGTATTGCTATTGGCATTGCTGCGCTCGGTGTTGCGACCGCAGCACAAGTAACTGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCGGCGATGAAAAATTCAATACAAGCAACTAAT CGAGCAGTCTTCGAAGTGAAGGAAGGCACTCAACAGTTAGCTATAGCGGTACAAGCAATACAAGACCACATCAAT ACTATTATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTTCCTAGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCTTTGCTTGGAAGTATGACGCCTGCAGTGGTCCAAGCAACATTATCTACTTCAATCTCTACTGCTGAA ATACTAAGTGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACTATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTATTAATAAT CAGGAATCCATAATCCAATTACCAGACAGAATCTTGGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAAAAT TGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCCATAGCAGGGAGTTATATGAGGCGATTTACGGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGTCTAACGTGTCTATGCAAGAATCCATCTTATCCTATATACCAACCTGAC CATCATGCAGTCACGACCATTGATCTAACCGCATGTCAAACACTGTCCCTAGACGGATTGGATTTCAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAG CTCCAATCTGTGAGTGTAAACTCCAAAATCGGAGCTATAATTGTAGCAGCCTTAGTTTTGAGCATTCTGTCAATT ATCATTTCGCTATTGTTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGAATCAACTTCAAAACAAAT CATATCAATACAATATCAAGTAGTGTCGATGATCTCATTAGGTACTAAGTCGAC
MKAFLVTCLSFAVFSSSVCVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPIDNSCEFKSVTQYNKT LSNLLLPIAENINNIASPSTGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATFLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQATLSTSISTAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFINNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFTALDGT IVANCRSLTCLCKNPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
74 Appendix
ZK-BN F P8-L GGATCCATGAAGGCTTTTTCAGTTACTTGCTTGGGCTTCGCAGTCTTTTCATCTTCTATATGTGTGAACATCAAC ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCCCAAAGTTCAAGCTCCTAC ATAGTGGTCAAACTTTTACCGAATATCCAACCCCCTGATGACAGCTGTGAATTTAAGAGTATAAGTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAGAATATAAACAATATTGCATCGCCCTCACTTGGATCAAGA CGTCATAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCGCTTGGTGTTGCAACAGCAGCACAAGTAACTGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCAGCGATGAAAAATTCAATACAGGCAACTAAT CGGGCAGTATTTGAAGTGAAGGAAGGCACCCAACAGTTAGCCATAGCAGTACAAGCTATACAAGACCACATCAAC ACTATTATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTCCCTAGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCCTTGCTTGGGAGTATGACTCCCGCAGTGGTTCAAGCAGCATTATCTACTGCAATTTCTGCTGCTGAA ATACTTAGCGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTGTTAATAAT CAGGAATCCATAATTCAATTGCCAGACAGGATCTTAGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAGAAT TGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCTATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGCCTAACGTGTCTATGCAAAAGTCCATCTTATCCTATATACCAACCTGAC CATCATGCGGTCACGACCATTGATCTAACCGCGTGTCAAACATTGTCCCTAGACGGATTGGACTTTAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAA CTCCAATCCGTGAGTGTAAATTCCAAAATCGGAGCTATAATTGTAGCAGCTTTAGTCTTGAGCATCCTGTCAATT ATCATTTCACTATTATTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGGATCAACTTCAAAACAAAT CATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAAGTCGAC
MKAFSVTCLGFAVFSSSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKSISQYNKT LSNLLLPIAENINNIASPSLGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFVNNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
ZK-BN F P8-S GGATCCATGAAGGCTTTTTCAGTTACTTGCTTGGGCTTCGCAGTCTTTTCATCTTCTATATGTGTGAACATCAAC ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCCCAAAGTTCAAGCTCCTAC ATAGTGGTCAAACTTTTACCGAATATCCAACCCCCTGATGACAGCTGTGAATTTAAGAGTATAAGTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAGAATATAAACAATATTGCATCGCCCTCATCTGGGTCAAGA CGTCACAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCGCTTGGTGTTGCAACAGCAGCACAAGTAACTGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCAGCGATGAAAAATTCAATACAGGCAACTAAT CGGGCAGTATTTGAAGTGAAGGAAGGCACCCAACAGTTAGCCATAGCAGTACAAGCTATACAAGACCACATCAAC ACTATTATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTCCCTAGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCCTTGCTTGGGAGTATGACTCCCGCAGTGGTTCAAGCAGCATTATCTACTGCAATTTCTGCTGCTGAA ATACTTAGCGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTGTTAATAAT CAGGAATCCATAATTCAATTGCCAGACAGGATCTTAGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAGAAT TGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCTATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGCCTAACGTGTCTATGCAAAAGTCCATCTTATCCTATATACCAACCTGAC CATCATGCGGTCACGACCATTGATCTAACCGCGTGTCAAACATTGTCCCTAGACGGATTGGACTTTAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAA CTCCAATCCGTGAGTGTAAATTCCAAAATCGGAGCTATAATTGTAGCAGCTTTAGTCTTGAGCATCCTGTCAATT ATCATTTCACTATTATTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGGATCAACTTCAAAACAAAT CATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAAGTCGAC
75 Appendix
MKAFSVTCLGFAVFSSSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKSISQYNKT LSNLLLPIAENINNIASPSSGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFVNNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
ZK-BN F P8-T GGATCCATGAAGGCTTTTTCAGTTACTTGCTTGGGCTTCGCAGTCTTTTCATCTTCTATATGTGTGAACATCAAC ATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCCCAAAGTTCAAGCTCCTAC ATAGTGGTCAAACTTTTACCGAATATCCAACCCCCTGATGACAGCTGTGAATTTAAGAGTATAAGTCAATACAAT AAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAGAATATAAACAATATTGCATCGCCCTCAACTGGATCAAGA CGTCATAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCGCTTGGTGTTGCAACAGCAGCACAAGTAACTGCC GCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCAATAGCAGCGATGAAAAATTCAATACAGGCAACTAAT CGGGCAGTATTTGAAGTGAAGGAAGGCACCCAACAGTTAGCCATAGCAGTACAAGCTATACAAGACCACATCAAC ACTATTATGAACACCCAATTGAACAATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTCCCTAGGATTA TACCTAACAGAATTAACAACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTATACAAGCC TTGAGGTCCTTGCTTGGGAGTATGACTCCCGCAGTGGTTCAAGCAGCATTATCTACTGCAATTTCTGCTGCTGAA ATACTTAGCGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAGATA AATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTTTGTTAATAAT CAGGAATCCATAATTCAATTGCCAGACAGGATCTTAGAGATCGGGAATGAACAATGGAGCTATCCAGCTAAGAAT TGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGGCTGAGCCTAGAATCAAAACTATGC CTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCTATAGCAGGGAGTTATATGAGGCGATTTGTAGCACTGGAT GGAACAATTGTTGCAAACTGTCGAAGCCTAACGTGTCTATGCAAAAGTCCATCTTATCCTATATACCAACCTGAC CATCATGCGGTCACGACCATTGATCTAACCGCGTGTCAAACATTGTCCCTAGACGGATTGGACTTTAGCATTGTC TCTCTAAGCAACATCACTTACGCTGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGAC ATATCAACTGAACTGAGTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAA CTCCAATCCGTGAGTGTAAATTCCAAAATCGGAGCTATAATTGTAGCAGCTTTAGTCTTGAGCATCCTGTCAATT ATCATTTCACTATTATTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGGATCAACTTCAAAACAAAT CATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAAGTCGAC
MKAFSVTCLGFAVFSSSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKSISQYNKT LSNLLLPIAENINNIASPSTGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARAIAAMKNSIQATNRA VFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELTTVFQPQLINPALSPISIQALR SLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVKINIPTIVTQSNALVIDFYSISSFVNNQE SIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAERLSLESKLCLAGNISACVFSPIAGSYMRRFVALDGT IVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLTACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDIS TELSKVNASLQNAVKYIKESNHQLQSVSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHI NTISSSVDDLIRY-
76 Appendix
8.2 Primers Primers for cloning of expression plasmids Primer Sequence (5‘-3‘) 88-1961 F pCG1 for CTTGGATCCATGAAGGTTTCTTTAG 88-1961 F pCG1 rev CTTGTCGACTTAGTACCTGATGAGATC 88-1961 F-HA pCG1 rev CTTGTCGACTTAAGCATAATCAGGAACATCATAAGGATAGTACCTG ATGAGATC 88-1961 HN pCG1 for CTTGGATCCATGGAGCCCTCGAAATTC 88-1961 HN pCG1 rev CTTGTCGACTCAAGTGATAGTCAATCTAG 88-1961 HN-FLAG pCG1 rev CTTGTCGACTCATTTATCATCATCATCTTTATAATCAGTGATAGTC AATCTAG Urabe AM9 F pCG1 for CTTGGATCCATGAAGGCTTTTTTAGTTAC Urabe AM9 F pCG1 rev CTTGTCGACTTAGTACCTAATGAGATC Urabe AM9 F-HA pCG1 rev CTTGTCGACTTAAGCATAATCAGGAACATCATAAGGATAGTACCTA ATGAGATC Urabe AM9 HN pCG1 for CTTGGATCCATGGAGCCCTCAAAAC
Urabe AM9 HN pCG1 rev CTTGTCGACTCAAGTGATGGTCAATCTGG
Urabe AM9 HN-FLAG pCG1 rev CTTGTCGACTCATTTATCATCATCATCTTTATAATCAGTGATGGTC AATCTGG
ZK-BN F pCG1 for AAGGCCGGATCCATGAAGGCTTTTTCAG
ZK-BN F pCG1 rev AAGGCCGTCGACTTAGTACCTGATGAGATC
ZK-BN F-HA pCG1 rev AAGGCCGTCGACTCAAGCGTAATCCGGGACGTCGTACGGATAGTAC CTGATGAGATCATCGACACT
ZK-BN HN pCG1 for AAGGCCGGATCCATGGAACCCTCGAAACTC
ZK-BN HN pCG1 rev AAGGCCGTCGACTTAAGTGATAGTCAATCTGGTTAGCAC ZK-BN HN-FLAG pCG1 rev AAGGCCGTCGACTTATTTATCATCATCATCTTTATAATCAGTGATA GTCAATCTGGTTAGCAC
88-1961 P8-L hyb for CATCGCCCTCACTTGGATCAAGACG 88-1961 P8-L hyb rev ATGACGTCTTGATCCAAGTGAGGGCG 88-1961 P8-S hyb for CATCGCCCTCATCTGGATCAAGACG 88-1961 P8-S hyb rev GACGTCTTGATCCAGATGAGGGC 88-1961 P8-T hyb for CATCGCCCTCAACTGGATCAAGACG 88-1961 P8-T hyb rev ATGACGTCTTGATCCAGTTGAGGGCG
77 Appendix
Urabe AM9 F P8-P hyb for GCATCGCCCTCACCTGGGTCAAG Urabe AM9 F P8-P hyb rev CCGTCTTGACCCAGGTGAGGGCG Urabe AM9-ZK-BN F P8-L hyb for GCATCGCCCTCACTTGGGTCAAGACG Urabe AM9-ZK-BN F P8-L hyb rev GACGTCTTGACCCAAGTGAGGGCGATGC Urabe AM9-ZK-BN F P8-T hyb for GCATCGCCCTCAACTGGGTCAAGACG Urabe AM9-ZK-BN F P8-T hyb rev GACGTCTTGACCCAGTTGAGGGCGATGC ZK-BN F P8-S hyb for GCATCGCCCTCATCTGGGTCAAGACG ZK-BN F P8-S hyb rev GACGTCTTGACCCAGATGAGGGCGATGC
Primers for cloning of recombinant MuVs r88 F for CCTCCAGGAGGATCAATAATCAG r88 F rev GTCACGTCACCCTGCCGTTGCAC r88 HN for CTCCAGTTAGGACAAGTCGCGATCGC r88 HN rev GTATCTCATTTAGGCCCGCC r88 frag 7 for GTTCTGCCTCACTCAAGTTTCGAG r88 frag 8 for GGAGTATCCTGGAGTCCATTCAGAAAGTC r88 frag 8 rev CTTGTATAGCTTGTACCGCTATAGC r88 frag 9 for GCACAAGTAACTGCCGCTGTCTCATTAG r88 frag 9 rev GATTCTAGGCTCAGCCTCTCTGCCTC r88 frag 10 for TGCCAGACAGGATCTTGGAGATCG r88 frag 11 for CGGAGCTATAATTGTAGCAGCTTTAGTC r88 frag 11 rev GGTAACAGCTTGTACAGACAGTAC r88 frag 12 rev TGAACATAGGATATCCTGACGCGGTCTG r88 frag 13 for GACACACTGGTGCTACACACATAATGT r88 frag 13 rev GTACTTCTCTGATAATATAATAGTCGA r88 frag 14 for CTTGTCTGTGCCTGGAATCAGATCCTAG r88 frag 15 rev GAGGTACGTTAATGCTTTGTGCTC r88 frag 16 for GACAGGTGAGTTAACAGATTTAG r88 frag 17 rev CATGATTGGCTAGCAATGATTCCGC r88 frag 18 for TGTGCATCTTACTCTCTCAAGGAGAAG r88 frag 18 rev ATATATCATCATTGAGAGCTGTATC r88 frag 19 for CGAATGGATACATTTAAGGCTCATGCG r88 frag 20 rev ACTCTGGGCATGACTACCTCACGGTC r88 frag 21 for TACACATTAATGGAGCGGAGTGTT r88 frag 21 rev GTAAGATTAACACGAGTGGAAGCTAAC r88 frag 22 for GCATCTGTATCACTTAAATCAGC
78 Appendix r88 frag 23 rev CTGTAAGAGATGCTGATTCGGATGAAC r88 frag 24 for CTGCTTAATGCTATTCGCGATTCTG r88 frag 25 rev CAACAATCAATCTGAGTATTGTA r88 frag 26 for CGGAGATGGCTAGACTCATAACAATTC
rbat F for GATTAGTAATCATGAGAAAAACTCTAGCTATTGG rbat F rev CACCATGTCAGGATTAGTATCTCACAAGGTCATC rbat frag 1a for ACCAAGGGGAAAATGAAGATGGGATATTAA rbat frag 1b for GATCCTGTTCCTCTATTATTTATAAGGAGC rbat frag 1 rev GCGAACCGTCGGTCAGCAGAGCC rbat frag 2 for ACCATGTGATGAGATAGAACAATTCC rbat frag 2 rev CTGTCCTTTGTTCAGGTGTCAAGCC rbat frag 3 for GAGAAATTACACCTATGCTCGGCCA rbat frag 4 for GTAACTACCCCGTCCCAAAG rbat frag 4 rev GAATCTAGCTGTGGCTGTGATAGGTGAG rbat frag 5 for GCCCAGAAACAGACAATCCAAGAGG rbat frag 5 rev GGGAATCTGAGTCTGGAGG rbat frag 6 rev GATCAGTCAGCATACTTTTTGCCA rbat frag 7 for GCTCTGCATCACTTAAATTCAGAG rbat frag 8 for GGGAGTTTCTTGGAATCCATTCCGGAAATCTG rbat frag 10 rev CGATTGTGATGATTTAGTATCTCACAAGGTCATC rbat frag 12 for CAACATTCCACAGGATGGAGCCATCAAAATTATTC rbat frag 14 rev GTATCTCATTTAGGCCTGCC rbat frag 15 for GACTATCACATGAATGTAATTGATATTCAGGAT rbat frag 15 rev TAAGGTATGTTAAGGCTTTATGCTC rbat frag 16 for GACTGGAGAGTTAACAGATCTG rbat frag 16 rev AGGGGCATTACCATGTAA rbat frag 17 for TATGTGTGCCCCGAAAGTGTTAG rbat frag 17 rev CATGATTAGCCAATAATGACTCTGC rbat frag 18 for TGTGCATCATATTCGCTAAAAGAGAAAG rbat frag 18 rev ATATATCTTCATTAAGGGCTGTATC rbat frag 19 for CGAATGGATCCACCTTAGGTTAATGAG rbat frag 19 rev CAACTCCATTCTCAGTG rbat frag 20 for GGGTAGGATCCTAACACAAG rbat frag 20 rev ACCCGAGGCATAACTACCTCTCGATC rbat frag 21 for TACACATTAATGGAACGAAGTGTC
79 Appendix rbat frag 21 rev CAAATGCCCAGATGTATACACCTGCTAAC rbat frag 22 for TAGAGTGCCCTACATCGGATCAAAGACAG rbat frag 22 rev CTAATCCAATTGCTAGTATAATCAG rbat frag 23 for TTACTTGAGAAAATACCATTATTGGC rbat frag 23 rev CGGATTGCATTGAGGAGCTTTCTTGATAA rbat frag 24 for CTGCTTCAGACTGTTGAATATG rbat frag 24 rev CTTGTGTATGATGTACTCAACACAAGCC rbat frag 25 for GAAAGTGTCCCCTATAGATTAATTC rbat frag 25 rev CGACAATTAATCTGATTATAGTG rbat frag 26 for CAGAATTGGCTAGATTGATTACAATCC
Primers for sequencing pCG1 for CCTGGGCAACGTGCTGGT pCG1 rev GTCAGATGCTCAAGGGGCTTCA
80 Appendix
8.3 Sequences of F peptides used in the FRET assay
Name Sequence hMuV P8-L Dabcyl – LGSRRHKRFE – Edans hMuV P8-P Dabcyl – PGSRRHKRFE – Edans hMuV P8-S Dabcyl – SGSRRHKRFE – Edans hMuV P8-T Dabcyl – TGSRRHKRFE – Edans
batMuV P8-L Dabcyl – LSSRRRKRFE – Edans batMuV P8-S Dabcyl – SSSRRRKRFE – Edans batMuV P8-P Dabcyl – PSSRRRKRFE – Edans batMuV P8-T Dabcyl – TSSRRRKRFE – Edans hMuV full-length Dabcyl – NIASPSPGSRRHKRFAGE – Edans batMuV full-length Dabcyl – SIAPPSSSSRRRKRFAGE – Edans amino acid residue at P8 (P95 of MuV F)
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