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
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
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
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
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
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
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
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
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
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.
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
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.
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.
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;
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
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.
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).
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
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).
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).
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).
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
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).
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
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)
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
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
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).
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
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.
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.
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, nadine.krueger@tiho-hannover.de
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%
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.
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, nkrueger@dpz.eu
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%
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.
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, nkrueger@dpz.eu
State of publication: submitted
Authors contribution (SH):
Scientific design: 50%
Investigation, data collection: 75%
Evaluation, formal analysis: 75%
Scientific writing: 75%
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.
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
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.
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
