Viperin, a Multifunctional Radical SAM Enzyme Biogenesis & Antiviral Mechanisms

Arunkumar Upadhyay

Department of Clinical Microbiology Umeå 2016

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-627-5 ISSN: 0346-6612-1869 Electronic version available at:http://umu.diva-portal.org/ Printed by: Print & Media, Umea University Umeå, Sweden 2016

“Education is an endless journey through knowledge and enlightenment”

A P J Abdul Kalam

To my family

ii Table of contents

Table of contents iii Abstract v Summary in swedish vi List of publications vii Abbreviations viii I Introduction and background 1 1 Interferon: an overview 1 1.1 IFN types 1 1.2 IFN induction 2 1.3 IFN signalling 2 1.4 ISGs 3 2 Viperin 6 2.1 Viperin evolution 6 2.2 Viperin function 7 2.2.1 Antiviral action 7 2.2.2 Virus counter measures 7 2.2.3 Viperin in signalling and immune response 8 2.3 Human viperin and its functional domains 9 2.3.1 N-terminal domain 9 2.3.2 C-terminal domain 10 2.3.3 Central or radical SAM domain 10 3 Radical SAM enzymes 12 4 Biosynthesis of cytosolic and nuclear iron- 14 sulphur proteins 4.1 CIA machinery 14 4.1.1 CIA targeting complex 15 5 Tick-borne encephalitis virus 17 5.1 Natural cycle of TBEV 17 5.2 Pathogenesis of TBEV 18 5.3 Clinical manifestation of TBEV infection 18 5.4 Structure and genomic organization of TBEV 19 5.5 Life cycle of TBEV 20 5.5.1 Binding and entry 20 5.5.2 Replication complex formation and genome 20 replication

5.5.3 Assembly, maturation, and release 22

iii 5.6 TBEV and the interferon system 22 II Aims 25 III Result and discussions 27 6 Identification of proteins that interact with viperin 27 6.1 Interaction of viperin with GeNets pathways 30 7 The role of interacting proteins in viperin 31 function 7.1 Binding of CIA1 to viperin is important for 31 SAM-dependent inhibition of TBEV (paper I) 7.1.1 Viperin inhibits late-step genomic replication 32 of TBEV 7.2 Biogenesis of viperin using the CIA targeting 34 complex (paper II) 7.2.1 Two branches of CIA targeting complex 34 interact at opposite ends of viperin 7.2.2 CIA1 plays a key role in transfer of the Fe/S 36 cluster on viperin 7.3 Viperin sequesters GBF1 for an enhanced 38 secretion of TBEV capsid protein through an unconventional secretory pathway (paper III) IV Concluding remarks 43 Acknowledgements 45 References 47

iv Abstract

Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is an interferon-induced antiviral protein. It has three distinct functional domains: the N-terminal domain, the radical SAM (S-adenosylmethionine) domain for binding iron-sulphur cluster, and the C-terminus domain. Viperin has a broad antiviral effect, and is also involved in the immune response signalling. However, the function and antiviral mechanism of viperin are still not well characterized. Thus the overall aim of the thesis was to investigate and better understand the function of viperin and its antiviral mechanism by identifying the cellular network of interaction partners. Affinity purification and mass spectrometry analysis were used to identify cellular proteins that interact with viperin.

CIA1 (also known as CIAO1), a factor involved in loading of iron-sulphur (Fe/S) cluster was identified and confirmed to interact at the C-terminus of viperin. It was also seen that the C-terminal domain and the functional SAM domain of viperin was essential for loading the Fe/S cluster onto viperin. On a closer look at the biogenesis of viperin, we identified and confirmed viperin interaction with CIA2A, CIA2B, (also known as FAM96A and FAM96B respectively) and MMS19, which are other factors involved in the transfer of Fe/S clusters onto cytosolic Fe/S apo-proteins. Surprisingly, MMS19, which has been shown to act as an adapter protein for other Fe/S proteins, only interacted indirectly and was not required for transferring the Fe/S cluster. Similarly, the interaction of viperin with both the isoforms of CIA2 was interesting, but the role they play in viperin biogenesis and antiviral function is still not clear and requires further investigation.

Study of the antiviral action of viperin against tick-borne encephalitis virus (TBEV) showed that the activity of the SAM domain is essential for the strong inhibition of genome replication of TBEV. Furthermore, viperin also affects the assembly and release of TBEV. Viperin interacts with GBF1 and downregulates its activity, thus preferentially inducing the secretion of viral capsid protein from the cell, and therefore disrupting the formation of infectious virus particles. The N-terminal domain of viperin is important for its effect on assembly and release.

In summary, this work contributes to our general understanding of viperin biogenesis in the cell regarding the loading of Fe/S cluster onto

v viperin. It also addresses the importance of the different domains for its antiviral function against TBEV. Finally, mass spectrometry and viperin interactome analysis implicate many other interesting cellular pathways or processes that might give us a better understanding of viperin’s function and antiviral mechanism.

vi Populärvetenskaplig sammanfattning på svenska (Summary in swedish)

Virus är parasiter och använder cellernas byggstenar för att föröka och sprida sig till andra celler. Cellerna har utvecklat strategier för att förhindra virusväxt. En viktig del i detta försvar mot virusangrepp är typ I interferoner (IFN α/β). När en cell blir infekterad av virus så utsöndras IFN som en alarmsignal till närliggande celler som i sin tur börjar producera skyddande proteiner. Vi har i denna avhandling identifierat att ett av dessa IFN- stimulerade cellulära proteiner är mycket aktivt mot fästing buren encefalit virus (TBEV). Detta protein heter viperin, och målet med denna avhandling var att studera detta antivirala protein i detalj, både vad gäller hur proteinet aktiveras men även vilka delar av TBE virusets livscykel som påverkas. I dessa studier har jag använt TBEV som modell virus. TBEV är det vanligast förekommande flaviviruset i Europa och Asien och kan orsaka encefalit (TBE). Viruset överförs till människan via bett från fästingar eller i vissa fall genom opastöriserade mejeriprodukter. Varje år insjuknar ca 13 000 människor i TBE i Europa trots att ett fungerande vaccin finns tillgängligt. Om man blir sjuk så kan man idag bara erbjudas symptomlindrande behandling. I denna avhandling så har jag identifierat cellulära proteiner som binder till viperin. Jag har gjort det genom att använda viperin som bete och fiska ut proteiner från cellen som viperin binder till. Dessa proteiner har sedan identifierats med en metod som kallas mass spektrometri. Detta har lett till att vi kunnat identifiera ett antal cellulära proteiner, däribland ett komplex av 4 proteiner även kallat CIA komplexet och GBF1 som interagerar med viperin. Vi har därefter karaktäriserat betydelsen av dessa proteiner för viperins antivirala verkan och kunnat visa att CIA komplext är absolut nödvändig för att mogna viperin till ett aktivt protein. GBF1 å andra sidan är ett protein som har en viktig roll i cellen för transport av proteiner och här har vi kunnat koppla viperin-GBF1 interaktionen till en förändrad transport av ett specifikt virusprotein, vilket i sin tur leder till att färre nya viruspartiklar bildas. Sammanfattningsvis så har jag i denna avhandling identifierat det antivirala proteinet viperin samt ett antal cellulära proteiner viktiga för dess funktion. Jag har även gett en detaljerad bild av hur aktiveringen av proteinet sker, vilka andra proteiner som behövs samt hur viperin påverkar olika steg i TBEVs livscykel. Resultaten i denna avhandling visar tydligt att viperin är

vii ett multifunktionellt protein som hämmar TBEV på flera olika sätt och denna funktion gör viperin till ett viktigt cellulärt protein i kroppens försvar mot TBEV-infektion.

viii List of publications

I Viperin is an iron-sulphur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity

Upadhyay A.S*, Vonderstein K*, Pichlmair A, Stehling O, Bennett K.L, Dobler G, Guo J.T, Superti-Furga G, Lill R, Överby A.K* and Weber F*

Cellular Microbiology (2014), 16: 834–848. doi:10.1111/cmi.12241

* Contributed equally II Unusual iron-sulphur cluster maturation of the antiviral radical SAM protein, viperin

Upadhyay A.S, Stehling O, Hubel P, Panayiotou C, Edlund K, Pichlmair A, Rösser R, Lill R and Överby A.K

Manuscript III Viperin induces secretion of tick-borne encephalitis virus capsid particles by interacting with GBF1

Vonderstein K, Nilsson E, Hubel P, Nygård Skalman L, Upadhyay A.S, Pasto J, Pichlmair A, Lundmark R and Överby A.K

Manuscript

Article reprinted with permission of the publisher

ix Abbreviation

ABCE1 ABC transporter E family member 1 AP-1 Activator protein 1 ABCE1 adaptor inducing interferon β ARF1 ADP-ribosylation factor 1 BFA Brefeldin A CHIKV Chikungunya virus JNK c-Jun N-terminal kinase COPI Coat protein I COPII Coat protein II Co-ip Co-immunoprecipitation CIAPIN1 Cytokine-induced apoptosis inhibitor cig5 Cytomegalovirus-inducible gene 5 CFD1 Cytosolic [Fe-S] cluster deficient 1 CIA Cytosolic iron-sulphur protein assembly CIA1 Cytosolic iron-sulphur protein assembly protein 1 CIA2A Cytosolic iron-sulphur protein assembly protein 2A CIA2B Cytosolic iron-sulphur protein assembly protein 2B DENV Dengue virus DPYD Dihydropyrimidine Dehydrogenase POLD1 DNA polymerase 1 ER Endoplasmic reticulum E Envelope protein of tick-borne encephalitis virus elF2 Eukaryotic translation initiation factor 2 XPO1 Exportin 1 FPPS Farnesyl pyrophosphate synthase GATA-3 GATA-binding protein 3 GO Gene Ontology GOBP Gene Ontology Biological process GOCC Gene Ontology Cellular component GPAT Glycerol-3-phosphate acyltransferase GBF1 Golgi-specific brefeldin A resistance guanine nucleotide exchange factor 1 HBV Hepatitis B virus

x HCV Hepatitis C virus HCMV Human cytomegalovirus HIV Human immunodeficiency virus FLUA Influenzavirus A IFN Interferon IRF Interferon regulatory factors ISGF3 Interferon -stimulated gene factor 3 ISGs Interferon -stimulated genes ISRE Interferon -stimulated regulatory elements IFITM Interferon-inducible transmembrane IFNAR Interferon-α receptor IFNGR Interferon-γ receptor IFNLR1 Interferon-λ receptor 1 IRAK1 Interleukin 1 receptor associated kinase 1 IOP1 Iron only hydrogenase-like protein 1 IRP1 Iron regulatory protein 1 ISC Iron-sulphur cluster assembly JAK Janus kinases JEV Japanese encephalitis virus KPNB1 Karyopherin subunit beta 1 LFQ Label-free quantification LDs Lipid droplets LC- MS/MS Liquid chromatography - mass spectrometry M Membrane protein of tick-borne encephalitis virus MMS19 Methyl methanesulfonate protein 19 ABCB7 Mitochondrial ABC transporter 7 MAVS Mitochondrial antiviral signaling protein MyD88 Myeloid differentiation primary response 88 MxA Myxovirus resistance gene A NDOR1 NADPH Dependent Diflavin Oxidoreductase 1 NS Non-structural proteins NF-κB Nuclear factor-kappaB NOD Nucleotide binding oligomerization domain NBP35 Nucleotide binding protein 35 ORF Open reading frame

xi MAPK p38 mitogen-activated protein kinase PAMPs Pathogen-associated molecular patterns PRRs Pattern-recognition receptors pDCs Plasmacytoid dendritic cells PRRSV Porcine reproductive and respiratory syndrome virus PKR Protein kinase R RIG-I Retinoic acid-inducible gene I protein RNaseL RNase latent SAM S-adenosylmethionine SARS Severe acute respiratory syndrome STAT Signal transducer and activators of transcription STING Stimulator of interferon genes protein SMN Survival motor neuron Th1 T helper cell 1 Th2 T helper cell 2 TAP Tandem affinity purification TBE Tick-borne encephalitis TBEV Tick-borne encephalitis virus TRIF Toll interlukin-1 receptor domain-containing TLRs Toll-like receptors TRIM Tripartite motif TN50 Truncation at N terminus 50 amino acids TRAF6 Tumour necrosis factor receptor associated factor 6 X-S Unknown sulphur-containing component UTRs Untranslated regions VAP-A Vesicle-associated membrane protein-associated protein A vMIA Viral mitochondria-localized inhibitor of apoptosis Viperin Virus-inhibitory protein, endoplasmic reticulum- associated, interferon-inducible VLPs Virus like particles WNV West Nile virus XPD Xeroderma pigmentosum D ortholog OAS 2’,5’-oligoadenylate synthetase 2’-5’A 2’-5’ oligoadenylate 5’-Ado. 5´-deoxiadenosyl radical

xii

Introduction and Background I

1. Interferon: an overview

The innate immune system is the first line of defence against any infection. The interferon (IFN) system is a very important part of the innate immune system, that plays a crucial role in early defence and clearance of invading pathogens. It was more than 60 years ago, that the antiviral effect of IFN was first described. In 1957, Issacs & Lindenmann coined the term “interferon” to describe a substance, probably produced by cells, that interfered with influenza virus infection [2]. In line with this, for decades and until very recently PEGylated IFN and ribavirin were jointly used as an antiviral for the treatment of hepatitis C virus (HCV) infection [3]. Over the years, great strides have been made in understanding the mechanisms governing IFN production, IFN gene regulation, and IFN-mediated signalling [4, 5].

1.1. IFN types

IFN belongs to a distinct family of proteins that fall into three classes based on the receptor complex used for IFN signalling. Type-I IFNs are the largest class of IFNs. All IFNs of this class signal through the type-I IFN heterodimeric receptor complex comprising IFN-α receptor 1 (IFNAR1) and IFNAR2 [6]. Almost all the cells are capable of producing IFN-α/β; however, specialized immune cells produce the vast majority of IFN-α [7]. The type-II IFN has IFN-γ as the sole member. It signals through the IFN-γ receptor (IFNGR) complex [8]. IFN-γ production is mostly restricted to the immune cells; however, the IFNGR is widely expressed, so almost all cell types are capable of responding to IFN-γ [9]. Type-III is the most recently discovered member of the IFN family. Type-III IFNs use IFN-λ receptor 1 (IFNLR1) for their signalling [10]. The members of the different classes of IFN are listed in Table 1.

IFN Members Receptor Type- I IFN-α, IFN-β, IFN-ε, IFNAR1/2 receptor IFN-τ, IFN-ζ, IFN-κ, and complex IFN-ω Type- II IFN-γ IFNGR Type- III IFN-λ1, IFN-λ2, IFN-λ3, IL-10R2 and IFNLR1 and IFN-λ4 Table 1: Overview of different classes of IFNs and their members.

1.2. IFN induction

Activation of the IFN system relies on the ability of the host to use specific pattern-recognition receptors (PRRs) (e.g. Toll-like receptors, RIG-I-like receptor, NOD-like receptor) to detect the invading pathogen [11-13]. The principle of innate immune recognition largely falls in two categories: (1) recognition of conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) (e.g. lipopolysaccharides, peptidoglycan, flagellin, genetic material, and intermediate genetic material of pathogen) that are essential for the life cycle of pathogens, and (2) detection of pathogen-inflicted damage or stress [14-16].

Upon recognition of a PAMP, a PRR activates a signalling cascade through an adapter molecule (e.g. MAVS, TRIF, MyD88, or STING) [17-20]. The signalling cascade leads to activation of kinases to phosphorylate the transcription factors, interferon regulatory factors-3 (IRF3) and IRF7 [21, 22]. After phosphorylation, these two IRFs homodimerize and translocate to the nucleus to form a complex with other transcription factors and upregulate type-I IFN genes (Figure 1) [23].

Figure 1: Pictorial representation of IFN induction. Virus infection leads to PRRs such as RIG-I to recognize the invading pathogen by identifying PAMPs such as dsRNA intermediate of virus replication. PRRs then signal through the adaptor protein such as MAVS resulting in phosphorylation of transcription factors such as IRF3 and IRF7. IRF3 and IRF7 then translocate into the nucleus to bind and activate specific promoters for IFN expression. The figure is adapted and reprinted with the permission from the publisher [2].

1.3. IFN signalling

Newly synthesized IFNs are secreted by the cell. IFN binds to its receptor on the cell surface and orchestrates signalling through the JAK-STAT (Janus kinases - signal transducer and activators of transcription) pathway [24]. There are four JAKs, three of which are

ubiquitously expressed and function in IFN signalling. They bind to the receptor chain on the inner side of the membrane [25]. In the absence of any stimulus, IFN receptor is bound to a JAK protein in an inactive conformation. Once activated, JAKs phosphorylate IFN receptor chains, which leads to the binding of STAT proteins [26]. There are seven STAT proteins, of which STAT1 and STAT2 are the most important [27]. Type-I IFN signalling leads to phosphorylation of both STAT1 and STAT2, which leads to heterodimerization and interaction with IRF9, forming the IFN-stimulated gene factor 3 (ISGF3) complex [28-30]. ISGF3 then translocate to the nucleus and binds to IFN- stimulated regulatory elements (ISREs) in the DNA upstream of IFN- stimulated genes (ISGs), resulting in the transcription of hundreds of ISGs (Figure 2).

Figure 2: IFN-1 signalling cascade. IFN α/β binds to the heterodimer receptor complex of IFNAR1 and IFNAR2, resulting in phosphorylation of JAK1 and TYK2 (tyrosine kinase 2). This leads to recruitment and phosphorylation of STAT1 and STAT2. STAT1 and STAT2 then form heterodimer and recruit IRF9 to form IFN-stimulated gene factor 3 (ISGF3). ISGF3 then translocate to the nucleus to induce genes upregulated by IFN-stimulated response elements (ISRE) resulting in expression of antiviral genes. The figure is adapted and reprinted with the permission from the publisher [2].

1.4. ISGs

ISGs were first discovered more than 35 years ago [31]. Since their discovery, more than 350 of ISGs have been identified [32]. Some of the ISGs such as RIG-I, IRF9, and STAT1/2 which act as PRRs, IRFs and signal transducing proteins respectively have a basal level of expression and this is greatly enhanced by IFN, thus increasing the responsiveness of the cell. Other ISGs express effector proteins that have direct antiviral activity and can limit the infection, many of which are still not fully characterized [33]. Since hundreds of ISGs are

upregulated, all the steps of the virus infection cycle can be targeted. Some of the ISGs that are relatively well understood are:

MxA (myxovirus resistance gene A): MxA protein is closely related to dynamin and dynamin-like GTPases [34]. Mx1 is a mice homolog of human MxA, and is one of the earliest identified ISGs known to have antiviral function. MxA protein has antiviral activity against many viruses such as Hepatitis B virus (HBV), poxviruses, Crimean-Congo haemorrhagic fever virus, human parainfluenza virus, influenza virus A, B and C etc. [35]. However, their activity against the virus family Orthomyxoviridae has been well characterized. MxA protein interacts with the newly formed nucleoprotein complex and prevent its import into the nucleus, thus preventing the secondary transcription and genome amplification of the virus [36, 37].

OAS-RNaseL (2’,5’-oligoadenylate synthetase - RNase latent): OAS- RNaseL system is one of the earliest identified interferon pathways and its activity against many viruses has been well characterized. Expression of OAS leads to the synthesis of a secondary messenger, 2’- 5’A (2’-5’ oligoadenylate). 2’-5’A then activates the latent RNaseL, which cleaves viral and cellular single-stranded RNA, thereby limiting virus replication and inducing apoptosis of cell [38-40].

PKR (Protein kinase R): PKR is a protein kinase and was one of the first PRRs to be discovered. PKR is activated by the double-stranded RNAs produced during viral replication. Upon activation, PKR phosphorylates the eukaryotic translation initiation factor 2 (elF2) and thus inhibits the viral and cellular protein synthesis [41]. The role of PKR as a regulator of translation is well established. Recent studies have shown that PKR is important in many other signal transduction pathways, such as activation of JNK (c-Jun N-terminal kinase), MAPK (p38 mitogen-activated protein kinase), STAT1 and 3, and the tumor suppressor p53 [42]. Moreover, PKR has also been shown to be crucial for regulation of the IFN-β transcripts and indirectly play a role in IFNα/β production in response to viral infection [43, 44]. Thus, PKR, a multi-functional ISG, and is believed to be crucial for an optimal antiviral response against many viruses [45].

TRIM (tripartite motif) proteins: The TRIM family is composed of more than 70 members in humans [46]. The main role of TRIM proteins has been shown to be their functioning as E3 ubiquitin ligase. There is growing evidence from recent studies that this protein is multi-functional [46, 47]. TRIM5α is one of the best characterized

members of the TRIM family. It has been shown to inhibit Human immunodeficiency virus-1 (HIV-1) during the early steps of the virus life cycle by directly interacting with the capsid protein and disrupting the capsid shell, thereby prematurely exposing the nucleoprotein [48]. However, the exact mechanism of how this leads to inhibition of HIV-1 is not well understood. Another member of the TRIM family, TRIM79α has been shown to specifically restrict tick-borne encephalitis virus (TBEV) by mediating lysosomal-dependent degradation of the non- structural protein 5 (NS5), an RNA-dependent RNA polymerase that is crucial for virus replication.

IFITM (IFN-inducible transmembrane) proteins: These proteins are ISGs that are present at basal level, but their expression is greatly induced by IFN. To date, five IFITMs have been identified in humans, of which IFITM3 is well characterized. IFITM proteins have been shown to have a broad antiviral effect against many enveloped RNA viruses such as Dengue virus (DENV), West Nile virus (WNV), Marburg virus, Ebola virus, SARS (Severe acute respiratory syndrome) virus, and HIV [49]. IFITM inhibits the early steps in the virus life cycle. They have been shown to inhibit the fusion of virus with the endosomal membrane, and thus prevent the virus from crossing the endosomal or lysosomal membranes and entering the cytosol [50].

Tetherin: This is one of the ISGs whose antiviral activity has been identified only recently. Tetherin is increasingly being understood to be multi-functional. Its main mode of action against envelope viruses has been to inhibit budding of virus from the infected cell [51].

2. Viperin

Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is one of the most strongly upregulated ISGs during viral infection. It was first identified while analysing the IFN-γ response of human macrophages [52]. Later, it was shown to be identical to cytomegalovirus-inducible gene 5 (cig5) isolated during infection of human cytomegalovirus (HCMV) of primary fibroblast cells [53]. Over the last 15 years, viperin has been shown to limit a broad range of RNA and DNA viruses from different families [52, 54- 67].

2.1. Viperin evolution

To establish a productive infection, viruses must overcome many cellular defences. In contrast, cellular factors such as ISGs must be able to inhibit productive viral infection. The opposing goals of the viral and the host factors puts them in an evolutionary arms race to counteract each other. Based on the ratio of nucleotide mutation that alters the amino acid sequence of a protein to the nucleotide mutation that does not alter the amino acid sequence, a selection is considered positive if the ratio is more than one [68].

Viperin is evolutionarily highly conserved in vertebrates from fish to mammals [69]. The protein can be divided into three main domains; the N-terminal domain, the central/radical SAM (s- adenosylmethionine) domain, and the C-terminal domain [70]. Using complete nucleotide-coding sequence, the evolution of viperin in fishes and non-human primates has been studied [71, 72]. Codon substitution analysis has revealed that, while viperin is highly conserved, the three functional domains have experienced different selection pressures in different vertebrates. On the one hand fish viperin seems to have evolved under positive selection mostly in the N-terminal and to some extent at the C-terminal domain [71]. Non-human primate viperin has undergone positive selection mainly in the central and the C-terminal domain [72]. Overall, it could be concluded that, the pressure from different viral infection during the course of evolution may have led to positive selection of viperin to escape viral antagonism and to have a broad antiviral effect.

2.2. Viperin function

2.2.1. Antiviral action

Viperin has an antiviral effect against a broad range of viruses. However, the antiviral mechanism of viperin has only been characterised for a few viruses. During the course of the past 15 years, studies have indicated that the antiviral mechanism of viperin is virus- specific. Viperin has been shown to interact with many viral and host factors, and inhibit different steps of the virus life cycle for different viruses. It has been shown to disrupt lipid rafts by interacting and impairing the host enzyme farnesyl pyrophosphate synthase (FPPS), which is involved in lipid metabolism. This mechanism appears to be specific for influenza virus A (FLUA), HIV and possibly rabies virus [56, 60, 65]. This disruption of lipid rafts traps the virus on the plasma membrane and inhibit its release. Viperin has also been shown to inhibit viral replication by interacting with proteins involved in the formation of replication complex of HCV and DENV-2. It interacts with HCV non-structural protein 5A (NS5A) and host VAP-A (vesicle- associated membrane protein-associated protein A). VAP-A has been shown to bind to NS5A and mediate the formation of replication complex for HCV on the lipid droplets (LDs) [73, 74]. However, in the presence of viperin this interaction is outcompeted, thus disrupting the formation of replication complex and inhibiting HCV replication [57, 66]. Viperin has also been shown to interact with the replication complex of DENV through viral NS3 [55]. Furthermore, pre-expression of viperin is shown to inhibit HCMV infection by reducing the viral structural proteins that are crucial for virus maturation and release [52]. Finally, viperin has also been shown to inhibit an early step in the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in African green monkey Marc-145 cells. Over-expressed monkey viperin inhibited virus at entry, genome replication and translation (Figure 3) [59].

2.2.2. Virus counter measures During the course of evolution, due to constant pressure on ISGs and viruses, both are forced to evolve. While the ISG can undergo positive selection as described above, viruses have developed strategies to counteract the effect of ISGs [75, 76]. For example, Japanese encephalitis virus (JEV) infection induces viperin expression but simultaneously causes proteosomal degradation of viperin [77].

Similarly, HCMV infection induces viperin, but unlike JEV, HCMV redistributes viperin from the endoplasmic reticulum (ER) to mitochondria by a viral protein, vMIA (viral mitochondria-localized inhibitor of apoptosis). Viperin then interacts with and inhibits host protein involved in fatty-acid β oxidation to generate ATP. This leads to reduced amount of cellular ATP, resulting in disruption of the actin cytoskeleton, which enhances the infection of HCMV (Figure 3) [78].

Figure 3: Pictorial representation of viperin’s anti- and pro-viral function in the cell. (1) Viperin interacts and inhibits FPPS activity, disrupting lipid raft formation on the plasma membrane and thus preventing the egress of influenza virus and HIV. (2) Viperin interacts with the replication complex of HCV and DENV through viral NS protein and VAP-A (only for HCV), and inhibits virus replication. (3) HCMV protein vMIA redistributes viperin to the mitochondria, where viperin interacts with and inhibits a host enzyme, leading to disruption of the actin cytoskeleton and thereby enhancing viral infection. The figure is modified and reprinted with the permission from the publisher [70].

2.2.3. Viperin in signalling and immune response Viperin has antiviral and pro-viral function, but it also appears to be involved in immune signalling. Viperin has been shown to be crucial for Toll-like receptor-7 (TLR7) and TLR9 mediated production of IFN in plasmacytoid dendritic cells (pDCs) [79]. It interacts with signalling mediators IRAK1 (interleukin 1 receptor associated kinase 1) and TRAF6 [TNF (tumour necrosis factor) receptor associated factor 6] to recruit them to the lipid droplets (LDs). This leads to ubiquitination of IRAK1 by TRAF6, which in turn leads to translocation of IRF7 to the nucleus and induction of type-I IFN [79]. Furthermore, viperin is also important for T cell activation. Studies on viperin knockout mice have

shown that viperin facilitates T cell-mediated GATA-3 (an important regulator of Th1/Th2 differentiation) activation and optimal Th2 cytokine production by modulating NF-κB (nuclear factor-kappaB) and AP-1 (activator protein 1) activities [80].

2.3. Human viperin and its functional domains

Human viperin has 361 amino acids and a molecular mass of 42 kDa. The protein can be broadly divided into three domains (Figure 4) [70, 81, 82].

Figure 4: Schematic representation of viperin protein with the three functional domains. The N-terminal domain, the C-terminal domain, and the central/radical SAM domain. The four-iron-four-sulphur ([4Fe-4S]) cluster is bound at motif 1 of the radical SAM domain and SAM is bound at motifs 2-4.

2.3.1. N-terminal domain

The N-terminal domain (covering residues 1-42) is highly variable in length and sequence among different species [69]. The N-terminal domain contains an amphipathic α-helix with a leucine zipper. Amphipathic helices are known to bind to membranes and induce membrane curvature [83]. Similarly, the N-terminal domain of viperin has been shown to be important for association of viperin with the endoplasmic reticulum (ER) and lipid droplets (LDs) [84, 85]. The localization and oligomerization of viperin on the cytosolic surface of ER brings the ER leaflets closer to form crystalloid ER. It has been shown that the N-terminal domain by itself cannot oligomerize and form crystalloid ER, but it can inhibit the secretion of soluble proteins, suggesting that the formation of crystalloid ER is not the cause of reduced protein secretion [85]. Truncation of N-terminal domain results in a change in the distribution of viperin from ER and LDs to throughout the cell [85].

This domain has been shown to be crucial for viperin’s antiviral activity against Chikungunya virus (CHIKV). In fact, the N-terminal domain of viperin is sufficient to inhibit the CHIKV infection [54]. This domain has also been shown to be partially responsible for viperin antiviral effect against WNV [63] and HCV [57]. Similarly it has been shown that the N-terminal domain of monkey viperin is important for inhibition of inhibits PRRSV [59].

2.3.2. C-terminal domain

The C-terminal domain (covering residues 218-361) is a highly conserved domain in different species [69]. The function of this domain is unknown, but it is considered to be the major site for protein-protein interactions and/or the site for substrate recognition for the enzymatic activity of viperin [39]. The C-terminal domain is important for viperin dimerization, and along with the N-terminal domain, it rearranges the ER to form crystalloid structures [85].

The C-terminal domain has been shown to be important for antiviral activity of viperin against many viruses [55, 57, 63]. Some of the viral and host factors have been shown to interact with viperin though this C-terminal domain. For example, this domain of viperin is essential for interaction with host factor VAP-A [57], with HCV NS5A and core protein [66], and with the NS3 protein of DENV-2 [55].

Apart from being the major site for protein-protein interaction, the C- terminal domain has been predicted to be important for proper folding of viperin. The last amino acid of viperin is an aromatic one (tryptophan). Any mutation, or removal of this amino acid, or addition of a protein tag at the C-terminus, results in loss of the antiviral activity of viperin against HCV [86].

2.3.3. Central or radical SAM domain

The central/radical SAM domain (covering residues 71-182) is homologous to the MoaA sub-family of radical SAM enzymes [87]. Proteins belonging to this family usually perform chemically difficult reactions such as C-C bond cleavage, C-S bond formation or alkylation [86, 88-90]. The domain has four motifs (1-4) that are conserved throughout all radical SAM enzymes. Motif 1 covers the conserved cysteine segment CxxxCxxC that is responsible for binding to the four- iron-four-sulphur ([4Fe-4S]) cluster [88], while the remaining three motifs bind to the co-factor SAM [91]. In fact, viperin has been shown

to bind to the [4Fe-4S] cluster and also cleave SAM to 5´- deoxiadenosyl radical (5’-Ado.) and methionine, further validating viperin as a radical SAM enzyme [86, 87, 92].

The SAM domain has been shown to be important for the antiviral activity of viperin against many viruses. Mutations in conserved cysteine residues of motif 1 abrogate viperin’s antiviral action against HCV [62], WNV, and DENV [63, 93]. The SAM domain is also important for activity against HIV [65] and Bunyamwera virus [67], and the pro-viral action of viperin during HCMV infection [78, 94]. Furthermore, apart from the significance of SAM domain for the enzymatic activity of viperin, it has been shown to be important for its structural stability. Any mutation that prevents the binding of the [4Fe-4S] cluster to viperin results in an unstable protein [95].

Despite the central importance of the radical SAM domain of viperin, little is known about its maturation, in particular the loading of the [4Fe-4S] cluster [93]. Finally, the target and/or substrate for the radical reaction of viperin is not known. Viperin is the only ISG till date to be identified as a radical SAM enzyme.

3. Radical SAM enzymes

Radical SAM enzymes are considered to be of ancient origin and to be one of the earliest biological catalysts to function by radical mechanism. The radical SAM superfamily, discovered in 2001, is ever- increasing family of metallo-proteins and it currently comprises of more than 2800 proteins. Proteins belonging to this superfamily are involved in catalysis of more than 40 distinct biological transformations for numerous important biological pathways [91, 96]. Although it is a huge family, only a handful of its member have been purified and characterized. A common feature of these proteins is the conserved motif “CxxxCxxC”, which has a [4Fe-4S] cluster as a ligand. Three irons of the cluster are ligated by a single cysteine residue of the CxxxCxxC motif, and the fourth iron is chelated to the α-amino and α- carboxyl group of SAM [88, 90, 96]. The [4Fe-4S] cluster abstracts an electron from the bound SAM. This leads to the cleavage of SAM, generating 5-deoxyadenosyl radical (5’-Ado.) and methionine (Figure 5) [97]. The 5’-Ado. generated then abstracts a hydrogen atom from the relevant substrate to initiate a radical mechanism, or a methyl group is added onto a target substrate for a radical-independent mechanism [98]. Examples of radical SAM enzymes include various enzymes involved in tRNA modification, lipid metabolism, metallo-protein cluster formation, post-transcriptional and post-translational modifications, enzyme activation, and peptide formations (Table 2) [91].

Figure 5: Mechanism of substrate activation by radical-SAM enzymes. S-H: substrate, Ado : 5-deoxyadenosyl radical; S : radical substrate; P: product; Met: methionine; [4Fe– 4S]-SAM: [4Fe–4S] cluster in complex with SAM; SAM shown in red is a ligand of the fourth iron in red. The cysteine residues of the CxxxCxxC motif are shown in green. The figure is modified and reprinted with the permission from the publisher [97].

Protein Function LAM Lysine 2,3-aminomutase Eam Glutamate 2,3-aminomutase Glycerol dehydratase Glycyl radicalization BioB Biotin synthase LipA Lipoyl synthase MoaA Molybdopterin biosynthesis MiaB Methylthiolation of tRNA Phe TYW1 Wybusine biosynthesis in tRNA ThiH Biogenesis of thiazole in thiamine ORF2 Cofactor maturation/amine CofG/CofH Coenzyme F(420) biosynthesis

Table 2: Radical SAM enzymes and their associated biological functions [91].

4. Biosynthesis of cytosolic and nuclear iron- sulphur proteins

Iron-sulphur (Fe/S) clusters are small inorganic protein cofactors. Fe/S proteins perform numerous important functions essential for the cell, such as mitochondrial energy production, protein translation, tRNA modification, transcription, and genome stability [99-104]. Fe/S proteins can be found in the mitochondria, cytosol, and the nucleus. Till date, the insertion of Fe/S clusters in Fe/S apo-proteins requires some 30 known protein components [105, 106].

The maturation of all cellular Fe/S proteins is initiated by the conserved mitochondrial iron-sulphur cluster (ISC) assembly machinery. The cytosolic iron-sulphur protein assembly (CIA) machinery requires the cooperation from the ISC assembly and export machinery for the biogenesis of cytosolic and nuclear Fe/S proteins [107, 108]. Overall Fe/S biogenesis is broadly divided into two main steps. Firstly, an Fe/S cluster is assembled de novo on a scaffold protein, where it is bound in a transient fashion. Secondly, the loosely bound Fe/S cluster is transferred from the scaffold protein to a dedicated targeting factor that finally facilitates the insertion of the Fe/S cluster to its target Fe/S apo-protein [106].

4.1. CIA machinery

The first step in the maturation of cytosolic and nuclear Fe/S proteins by the CIA assembly machinery is dependent on the ISC core assembly machinery. The ABCB7 (mitochondrial ABC transporter 7) facilitates the transport of an unknown sulphur-containing component (X-S) into the cytosol [109]. X-S serves as a sulphur donor for the extra- mitochondrial Fe/S protein maturation [110]. The first step in the CIA machinery involves the scaffold protein complex of NBP35 (nucleotide binding protein 35) and CFD1 (cytosolic Fe/S cluster deficient 1), on which the [4Fe-4S] cluster is assembled. The two proteins form a heterotetrameric complex that binds two different kinds of [4Fe-4S] cluster, a loosely bound [4Fe-4S] cluster at the c-terminus of both proteins and a tightly bound [4Fe-4S] cluster at the N-terminus of NBP35 [111-114]. This reaction requires an electron transfer chain comprised of NDOR1 (NADPH dependent diflavin oxidoreductase 1) and CIAPIN1 (cytokine-induced apoptosis inhibitor 1) [115, 116]. The second step requires the function of IOP1 (iron only hydrogenase-like protein 1) which acts as bridge between the early acting CIA components and the late CIA targeting complex [117-119]. Scaffold

protein complex and IOP1 form the core component of the CIA machinery. Depletion of NBP35 results in a severe defect in the assembly of cytosolic and nuclear Fe/S proteins. The affected Fe/S proteins are the general housekeeping Fe/S proteins and also those involved in iron homeostasis [120]. In the last step, components of the CIA targeting complex may directly interact with the target Fe/S apo- proteins and insert the Fe/S cluster [121].

4.1.1. CIA targeting complex

CIA targeting complex is composed of CIA1, CIA2A, CIA2B, (also called as CIAO1, FAM96A, and FAM96B respectively) and MMS19 (methyl methane sulfonate) [121-124]. CIA1 is a WD40-repeat protein and one of the earliest CIA targeting component identified to take part in the transfer of Fe/S clusters onto Fe/S apo-proteins [122]. This doughnut- shaped protein is believed to be the docking site for the other CIA targeting components and the target Fe/S apo-protein [125]. MMS19 was earlier reported to be important for genome stability. However, the mechanism was not known [126]. Recent studies have shown that MMS19 is part of the CIA targeting complex. In line with the results of the previous studies, MMS19 has been shown to act as an adaptor between the early CIA machinery and many target Fe/S proteins involved in maintaining genomic stability [123, 124, 127]. CIA2A and CIA2B are the two isoforms of CIA2 protein. Interestingly, CIA2B was earlier reported to function in chromosome segregation and was found to be associated with a large complex including MMS19 [126]. However, with recent findings, CIA2B is known to form a complex with CIA1 and MMS19, and enables the maturation of most of the cytosolic and nuclear Fe/S proteins involved in the general housekeeping of the cell [121, 128]. CIA2A is the most recently identified component of the CIA targeting complex. However, unlike CIA2B and MMS19, it interacts with only CIA1 in the CIA targeting complex [121]. Furthermore, CIA2A has been shown to be important for the maturation and stabilization of proteins involved in iron homeostasis, such as IRP1 (iron regulatory protein 1) and IRP2 [121]. Based on the target Fe/S proteins and the components of the CIA targeting complex that are important for maturation, the CIA machinery is divided into two main branches. One branch is where CIA2A is involved in stabilization and maturation of proteins involved in iron homeostasis. The second branch is where CIA2B (along with CIA1 and MMS19) enables the maturation of most of the cytosolic and nuclear Fe/S proteins involved in general housekeeping of the cells. It has been observed that CIA1, CIA2B, and MMS19 form various binary and

ternary sub-complexes in yeast and mammalian cells. Based on the proteomics and functional data analysis of Fe/S proteins, the binary and ternary complexes show target-specific preferences. The ternary CIA targeting complex (CIA1-CIA2B-MMS19) appears to have the largest substrate spectrum and mediates the maturation of many Fe/S proteins such as DPYD (dihydropyrimidine dehydrogenase), ABCE1 (ABC transporter E family member 1), and XPD (xeroderma pigmentosum D ortholog). MMS19 plays a minor role in GPAT (glycerol-3-phosphate acyltransferase) maturation, while CIA2B is only partially important for POLD1 (DNA polymerase 1). All these observations show the versatile and dynamic nature of the CIA targeting complex. Based on their importance, the CIA machinery are further divided into sub-branches (Figure 6).

Figure 6: Schematic representation of Fe/S cluster transfer by CIA machinery. The CIA machinery depends on the ISC machinery for the transfer of the unknown sulphur compound (X-S). The compound is then used for the assembly of Fe/S cluster on NBP35-CFD1 complex. The Fe/S cluster is then transferred to IOP1. IOP1 then with the help of CIA targeting complex transfer the Fe/S cluster on the target Fe/S proteins. The maturation of cytosolic Fe/S proteins is split into two main branches. CIA2A takes part in the maturation of protein involved in maintaining the iron homeostasis of the cell. CIA1, CIA2B, and MMS19 takes part in the maturation of general Fe/S proteins required for the housekeeping of the cell. This branch is further devided into sub branches such as; CIA2B branch that requires CIA1 and CIA2B for maturation of GPAT and CIA2B- MMS19 branch that requires CIA1, CIA2B, and MMS19 for maturation of DPYD and POLD1. The figure is adapted and reprinted with the permission from the publisher [121].

5. Tick-borne encephalitis virus

TBEV is a neurotropic flavivirus that causes TBE (tick-borne encephalitis). TBEV belongs to the family Flaviviridae (with the genera flavivirus, hepacivirus, pestivirus and pegivirus). There are more than 70 viruses in the genus flavivirus, among them several neurotropic and hepatotropic viruses are transmitted by arthropods (DENV, JEV, WNV, yellow fever virus, Zika virus and TBEV) [129-133]. TBEV is the primary cause of viral encephalitis in central and eastern European countries and in Russia, with about 13000 estimated human cases every year [134]. In fact, over the last decade there has been an approximately 300% increase in the number of TBE cases in Europe [135]. This increase is thought to be due to growth in the population and spread of TBEV vectors, which is promoted by factors including climate change, social and political change and changes in the land use [136, 137].

5.1. Natural cycle of TBEV

TBE is a zoonotic disease. In nature, TBEV is maintained in a complex cycle involving ticks as vector and reservoir of the virus and small rodents as hosts for ticks [138]. The virus replicates in ticks and is maintained through mainly trans-stadial transmission and less efficiently by trans-ovarial transmission. Uninfected ticks can become infected by feeding on a viremic host or by co-feeding with the infected ticks on the same host (Figure 7) [139, 140].

Figure 7: Transmission cycle of TBEV. Dashed lines = different developmental stages of tick in cycle. At each stage tick needs blood meal to develop into next stage. Additionally, females need blood meal to produce eggs. Solid lines= presence and transmission of TBEV in ticks and host animal (once infected with the virus tick remains infected throughout its lifespan and occasionally transmit virus through trans-ovarial route). Thickness of arrows indicate the prevalence and efficient transmission of TBEV. The figure is modified and reprinted with the permission from the publisher [141].

Based on phylogenetic analysis and the geographical distribution, TBEV is classified into three different subtypes: European, Siberian, and Far Eastern, with 5-6% divergence at the amino acid level between the different subtypes [142]. Depending on the subtype of TBEV, different tick species may act as the principal vector. While Ixodes ricinus acts as the main vector for European subtype. Far Eastern and Siberian subtypes are primarily transmitted by Ixodes persulcatus [135, 143]. Humans are not part of the natural transmission cycle of TBEV. They are the incidental host and become infected when they are exposed to a bite from an infected tick or when they consume raw milk from an infected animal [144].

5.2. Pathogenesis of TBEV

After an infected tick bite, TBEV replication occurs locally. Dendritic skin cells are assumed to be the first cells for virus replication and transport the virus to the draining lymph nodes, where it replicates in the monocyte lineage cells resulting in primary viremia and subsequently infecting the peripheral tissues, where further multiplication maintains the viremia for several days. After peripheral amplification, the virus enters the circulation, crosses the blood brain barrier and enters the central nervous system (CNS) [145, 146]. Although the exact mechanism by which TBEV breaches the blood brain barrier is not known, entering the CNS is a key factor for the pathogenesis of TBEV, resulting in severe clinical outcome.

5.3. Clinical manifestation of TBEV infection

A large majority of TBEV infections are asymptomatic [147]. The incubation period of TBE may vary between 2 and 28 days with clinical symptoms which are often biphasic [148, 149]. The initial phase correlates with viremia and usually presents with non-specific symptoms of febrile illness [141]. Two-thirds of infected people clear the virus during this phase. However, in one-third of patients a second phase of the disease occurs. During this stage, the virus spreads to the central nervous system and causes fever, headache, vomiting, visual disturbances, paresis, paralysis, encephalitis or even coma. About 40 to 50% of patients with severe disease may also show long term nerve- related sequelae such as depression, lack of concentration or paresis. The mortality rate with TBEV varies from 1 and 20%, depending on the subtype [146, 150-152]. Although TBE can be prevented by vaccination, thousands of people fall ill from this disease every year. Currently, there is no effective antiviral against TBEV, and based on the severity

of the symptoms, the patient might require hospitalization and supportive care. Anti-inflammatory drugs, are considered under specific circumstances for symptomatic relief [153, 154].

5.4. Structure and genomic organization of TBEV

TBEV is an enveloped virus around 50 nm in diameter. The envelope carries two surface proteins, the E (envelope) protein and the M (membrane) protein. The later is derived from a precursor protein, prM. Inside the viral envelope there is a nucleocapsid (NC) consisting of multiple copies of capsid (C) protein and the viral genome. The TBEV genome is a single stranded, positive-sense RNA of approximately 11000 nucleotides. It has a 5’-cap with a single open reading frame (ORF). The ORF is flanked by 5’ and 3’ UTRs (untranslated regions). The viral protein is encoded by the ORF as a single polyprotein, which is co- and post-translationally cleaved by cellular and viral proteases into individual viral proteins (three structural and seven non-structural). The polyprotein is arranged in the order 5’-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3’ (Figure 8) [155, 156].

Figure 8: TBEV genome organization and polyprotein processing. At the top is the viral genome with one open reading frame flanked by untranslated regions, the 5’- and 3’- UTRs. Below the genome is the polyprotein generated from the viral RNA, followed by proteolytic processing of the polyprotein by host and viral proteases into individual viral proteins.

5.5. Life cycle of TBEV (Figure 9)

5.5.1. Binding and entry

In the first step, virus E protein binds to the cellular receptor. The cellular receptor has not been identified, but attachment to heparan sulphate and glycosaminoglycan, which are present in abundance on many cell types of both vertebrates and ticks, is predicted to play a role during binding [157]. After binding, receptor-mediated endocytosis by clatherin-coated pits transports the virion to the endocytic compartment of the cell [158]. The acidic environment of these vesicles leads to reorganization and conformational change of the viral E protein. In the course of these changes fusion of the viral and endosomal membranes is induced, releasing the NC into the cytoplasm [159]. Figure 9: Schematic representation of flavivirus life cycle. In the first step, virus binds to the cell surface and enters the host cell by receptor mediated endocytosis. Low pH in the endosomes then trigger the conformational changes in the virion that leads to fusion of viral and endosomal membrane releasing the genome in the cytoplasm. The viral genomic RNA is then translated into a single polyprotein that is processed by viral and host proteases. The genomic replication occurs in the vesicular compartments formed on the ER. Virus assembly occurs on the surface of the ER when the newly synthesized viral RNA buds into the lumen of the ER. The resultant immature virion is then transported through the trans-Golgi network. During the transport the immature virion is cleaved by a host protease furin resulting in mature, infectious particles. The virions are then subsequently released by exocytosis. The figure is reprinted with the permission from the publisher [160].

5.5.2. Replication complex formation and genome replication

The viral RNA (vRNA) also functions as an mRNA, associating with ribosomes to produce the polyprotein. The transmembrane domain of the viral protein is recognised as a signalling peptide and recruits the vRNA/ribosomes/nacent polypeptide complex to the ER membrane [161, 162]. The nascent polypeptide is then processed by the cellular

and the viral proteases into structural and non-structural (NS) viral proteins. Some of the viral proteins such as NS2B, NS4A and NS4B integrate and alter the membrane of the ER to form a membrane vesicular structure with a small pore connecting the interior of the vesicle to the cytoplasm [161, 163-168]. These vesicles are the site for the formation of replication complexes (RC) and RNA replication [169]. In the first step of RNA replication, NS5-which is an RNA dependent RNA polymerase (RdRp) uses the genomic plus-sense RNA [(+) RNA] as a template for the synthesis of (-) RNA. The (-) RNA remains base-paired with genomic (+) RNA to form dsRNA intermediate. The (-) RNA in the dsRNA intermediate then serves as a template for more (+) RNA. This step requires the helicase activity of NS3 to unwind the dsRNA, and NS5 for RNA replication. The nascent (+) RNA displaces and releases the pre-existing (+) RNA and a dsRNA as a product. The new dsRNA is then recycled to generate more (+) RNA. The genome replication of flaviviruses in general is asymmetric with 10- to 100- times more (+) RNA generated than (-) RNA, the only known function of which is to act as a template for (+) RNA. The (+) RNA generated is methylated and capped by NS3 and NS5. It can function as mRNA in protein translation or it can act as a genomic (+) RNA for virion assembly and release [161, 170-172].

Figure 10: Schematic representation of TBEV RNA synthesis by NS3 and NS5. (+) strand genomic RNA serves as template to produce (-) strand RNA. The (-) strand RNA exist as dsRNA intermediate (replicative form). The (-) strand RNA within the dsRNA is then used as template for (+) strand RNA synthesis. The dsRNA product is then released and recycled for additional (+) strand RNA synthesis. The (+) strand RNA is then capped and methylated to form (+) strand genomic RNA. (+) strand RNA and (-) strand RNA are represented by violet and orange colour respectively.

5.5.3. Assembly, maturation, and release.

Following RNA replication, genomic (+) RNA exits the vesicle through the vesicular pore and is packaged by C proteins into the NC on the cytoplasmic side of the ER membrane [165]. The exact mechanism of NC formation is not understood. Some studies have predicted that since the genomic (+) RNA is negatively charged and the C protein is positively charged, NC is formed in a non-specific electrostatic manner [173, 174]. However, viral genomic RNA is specifically packaged into virions. It has been suggested that to achieve specificity, the organization of genome replication and virion assembly is closely intertwined. Spatial coordination of the two processes has been observed for DENV, where budding of virions was seen just opposite to the pore/opening of the vesicular bodies in which RNA replication takes place [175]. During the process of budding of NC from the cytoplasm in the ER, it acquires the lipid envelope along with E and prM proteins, which are associated with the ER membrane. The mechanism that ensures efficient incorporation of NC into the ER membrane with the E and prM protein is not well understood. However, sequential cleavage of C protein on the cytoplasmic side (release mature capsid) before cleavage of the C-prM junction on ER side has been postulated as one of the mechanism [176, 177].

Following budding, the immature virion clusters in the lumen of the ER and are then transported through the cellular secretory pathways to the extracellular medium. The immature virion has heterodimers of E and prM that completely covers the lipid bilayer to form a smooth protenaceous coat. During the transport through the Golgi to the cell membrane, E and prM are glycosylated. This transport also coincides with a massive conformational change of the viral glycoproteins due to low-pH [178, 179]. The conformation change from the smooth coat to trimeric spikes allows the cleavage of prM to M and pr fragment by a host protease, furin. The pr fragment dissociates after the release of the virion from the cell resulting in a mature virus [180, 181].

5.6. TBEV and the IFN system

For a successful viral infection, viruses have evolved different strategies to overcome the rapid innate immune response of the host [182, 183]. On similar lines, TBEV has been shown to hide the viral dsRNA intermediate in intracellular membrane vesicles [184]. This strategy prevents the cellular PRRs such as RIG-I and/or MDA5 to access and recognize the RNA of TBEV, thus delaying the activation of

the IFN system and giving sufficient time for a successful virus multiplication. Although late, IFN is produced by the infected cells, but it fails to hamper the spread of TBEV, indicating the importance of an early IFN response [184, 185]. In fact, the importance of IFN response was confirmed in vivo in our group. It was found that IFN protects the mice from fatal neurotropic infection against Langat virus, a naturally attenuated member of the TBEV serogroup [186]. This was also found to be true against TBEV (Figure 11).

3 Figure 11: TBEV is sensitive to IFN-α treatment. VeroB4 cells 2 were treated with increasing concentrations of IFN-α for 16h (log10) 1 before infection with TBEV. 24h post-infection, TBEV genomic 0 RNA copy number determined by quantitative PCR.

Fold inhibition of TBEV RNA -1 0 1 5 10 100 1000 IFN (U/ml)

It is well established that the antiviral function of type-I IFN is mediated by hundreds of ISGs that are upregulated by the IFN [2]. To identify the ISG that might be responsible for the sensitivity of TBEV for type-I IFN, a screening of TBEV sensitivity was performed in tetracycline-inducible cells expressing different ISGs that are known to have antiviral activity against different flaviviruses. This screening identified viperin as a potent anti-TBEV protein (Figure 12).

TBEV RNA Control 7 Tetracyclin Figure 12: Viperin was found to inhibit TBEV more than 6 1000 fold upon expression. Tetracycline-inducible 293T cells expressing different ISGs 5 were infected with TBEV. 24h post infection, TBEV genomic 4 RNA copy numbers were

Log10 RNA (a.u.) determined by quantitative 3 PCR. CAT-expressing cells with or without IFN treatment were 2 used as negative and positive T  n R 0 1 1 L A N ri 2 -M 56 R S n controls respectively. C IF PK R-M 0 G C A AS eri ipe K ISG 2 IS S O O h T V P G L t A IS P e C T

Aims II

Viperin is one the most highly upregulated ISGs during viral infection. It has been shown to be antivirally active against many different viruses [70]. It also plays a key role in immune signalling during T cell activation and type-I IFN production in pDCs [79]. Since it has such a diverse range of function, it must interact with several host and viral factors. Although some of these factors have been identified, they cannot explain all the many functions and antiviral mechanisms against many pathogens. The mode of action of viperin is largely uncharacterized. So our hypothesis was that identification of the protein interactome of viperin would help us to understand some of the functions and antiviral mechanism of viperin.

The specific aims were:

I. To identify the cellular protein that interact with viperin. II. To evaluate the cellular interaction partners and determine their role in the biogenesis and antiviral mechanisms of viperin.

Results and discussion III

6. Identification of proteins that interact with viperin (papers I and III)

To identify the host factors that interact with viperin, we first used an inducible cell line (FLP-IN T Rex cells) expressing a viperin with a tandem affinity purification (TAP) tag. After expression of TAP-tagged viperin, tandem affinity purification and mass spectrometry analysis was performed according to previous protocols [187, 188]. Mass spectrometry analysis resulted in identification of several hits. One of these hits was CIA1 (CIAO1), a protein involved in the loading of Fe/S cluster onto cytosolic apo-proteins.

Although we got several hits besides CIA1, this method had a disadvantage. The long TAP-tag made viperin protein antivirally inactive. Therefore, in order to identify host factors that interact with a physiologically active antiviral protein, we performed another mass spectrometry analysis. This time, we used inducible cells (FLP-IN T Rex) that express viperin with a short flag-tag; this protein is antivirally active. Cells inducibly expressing flag-tagged GFP or flag- tagged N-terminally truncated viperin (TN50) was used as a control for the experiment. The expression and affinity purification of viperin and the control proteins was done under type-I IFN treatment or LGTV infection to mimic the physiological condition under which viperin is expressed. Protein enrichment was analysed by LC-MS/MS and proteins significantly enriched with viperin compared to the control GFP were identified. The number or type of hits for viperin, TN5o, and GFP under different conditions of expression did not change significantly. Data analysis with MaxQuant identified only a few hits with the TN50 mutant, compared to viperin. Indicating the importance of N-terminal domain for protein interactions. To concentrate the hits with viperin and reduce the possibility of false hits, stringent permutation was applied to generate label-free quantification (LFQ) values. The LFQ values were then used to generate heat maps of protein enrichment for GFP, TN50, and viperin (Figure 13).

Figure 13: Heat map of proteins significantly changed, against the background (all samples annotated as LFQ intensity GFP, N = 8). The LFQ intensities were determined (prenormalised by LFQ algorithm implemented in MaxQuant) using stringent permutation cut off (FDR of 0.001 and S0 = 1) and displayed as heatmap and clustered by Euclidean distance in the columns and rows. CIA1, CIA2B, CIA2A, (also called as CIAO1, FAM96B, and FAM96A repectively) MMS19, and GBF1 are indicated by an arrow and will be discussed in details below.

Among the proteins that were significantly enriched for viperin were the components of the CIA targeting complex (CIA1, CIA2B, CIA2A and MMS19) and a protein important for retrograde transport (GBF1) in the cell. Since viperin has an Fe/S cluster and has been shown to inhibit protein secretion [85], CIA1, CIA2B, CIA2A, MMS19 and GBF1 were interesting candidate proteins when investigating the function of viperin.

As viperin has many different functions and a broad spectrum of antiviral activity, it most likely associates with a wide variety of protein networks. To understand the interaction network of viperin, we performed an enrichment analysis using

the Gene Ontology (GO) database on the 73 proteins that interacted with viperin. The network showed the functionally enriched GO terms (GOBP: Biological process, GOCC: Cellular component) of all the significantly interacting proteins. Apart from the expected components of the “iron-sulphur assembly”, we could also isolate components from the “nuclear envelope”, “survival motor neuron (SMN) complex” involved in spliceosome snRNP assembly in cytoplasm and in pre- mRNA splicing in the nucleus, “intrinsic to membrane”, and proteins involved in “DNA catabolic processes” (Figure 14).

Figure 14: GO enrichment analysis: The network shows functional enriched GO terms (GOBP: Biological Process, GOCC: Cellular Component) of all the significant interacting proteins. The GO term enrichment was calculated against the total number of identified protein groups by a Fisher exact test using Benjamin-Hockberg FDR for truncation and a threshold value of 0.05. All annotation fulfilling the statistical criteria were selected by a minimal enrichment factor of > 2 against the background. The corresponding interacting proteins were isolated from the matrix and network was generated using Cytoscape.

Some of the identified cellular proteins from the networks have been reported previously to play key roles in different parts of the virus life cycle. For example, karyopherin subunit beta 1 (KPNB1), acts as a docking site for the importin/substrate complex, to the nuclear pore complex. KPNB1 is important for the nuclear import and localization of proteins of HIV, FLUA, DENV for virus replication [189-193]. Conversely, exportin 1 (XPO1) mediates nuclear export to the cytoplasm; this protein is important in the replication of HIV, FLUA, Herpes simplex virus-1 (HSV-1), DENV-2, and HCV [194-201]. Many viruses of different families require nuclear transport during their replication cycle. With viperin showing broad antiviral effects, its interaction with proteins of the nuclear transport is very interesting.

This could possibly be a new, unidentified function of viperin and might perhaps reflect a common mode of action against many viruses. Similarly, the host spliceosomal machinery is important for HIV and FLUA replication [202, 203]. The NS5 protein of DENV-2 has been shown to modulate the host spliceosomal machinery to counteract the host antiviral response [204]. Thus, the SMN complex also appears to be an interesting network to investigate for its involvement in the antiviral mechanism of viperin. Finally, it would also be interesting to look at some of the proteins that are intrinsic to membranes and involved in DNA catabolic processes to improve our understanding of the function and antiviral mechanisms of viperin.

6.1. Interaction of viperin with GeNets pathways

Although enrichment of proteins from several GO networks was identified, with stringent analysis, we might have lost many weak or transient interaction partners of viperin. We therefore took the first 25o strongly enriched proteins and analysed them with reactome prediction software available online (www.reactome.org). Overall, 94 GeNets pathways were predicted to be significantly enriched. Some of the interesting pathways that came up are shown in Table 3.

Name Bonferroni p-value Significant

Influenza viral RNA 3.20E-14 Yes transcription and replication

Host interaction of HIV 6.40E-14 Yes factors

NEP NS2 interacts with the 1.90E-07 Yes cellular export machinery

Late phase of HIV life cycle 1.20E-06 Yes

Table 3: List of the interesting GeNets pathway with viperin

As expected, we identified more networks that interact with viperin, indicating that the protein is multi-functional in its antiviral action. Viperin specifically interacted with FLUA and HIV networks. This could be due to the amount of information available on these viruses, compared to any other virus. Overall, study of the interaction of viperin with the identified factors and networks would lead to a much better understanding of its function and its broad spectrum of antiviral effects.

7. The role of interacting proteins in viperin function

7.1. Binding of CIA1 to viperin is important for SAM- dependent inhibition of TBEV (paper I)

Viperin has a conserved central domain that carries a motif for binding the [4Fe-4S] cluster, and the co-factor SAM [87, 92]. Mass spectrometry analysis of TAP-tagged viperin identified CIA1 (CIAO1) as one of the strongest hits. CIA1 is a component of the CIA machinery, which is involved in the late-step transfer of Fe/S clusters onto apo- proteins.

Interaction of viperin with CIA1 was confirmed by co- immunoprecipitation (co-ip) assay. Viperin has three distinct domains, and by screening viperin mutants, I could show that the C-terminal domain of viperin was the only domain that was important for this interaction. To correlate the viperin-CIA1 interaction with the known function of CIA1, loading of the Fe/S cluster onto viperin was studied in the presence of 55Fe. Affinity purification and scintillation counting of viperin and its mutants showed that mutations at the SAM domain or C-terminal domain completely abolished the loading of Fe/S cluster onto viperin. This indicated the importance of the C-terminal domain for viperin-CIA1 interaction and of a functional site in the SAM domain for loading of the Fe/S cluster. Moreover, a functional SAM domain and C-terminal domain was absolutely necessary for the viperin antiviral activity of viperin against TBEV. The results suggested that, when induced, viperin will interact with CIA1 through its C-terminal domain. The interaction will result in the transfer of Fe/S cluster onto the SAM domain. The SAM domain might then be involved in some enzymatic activity important for viperin function.

Studies have shown that the SAM domain is crucial for antiviral action of viperin against HIV, HCV, DENV1, WNV, and Bunyamwera virus [62, 63, 65, 67]. Conversely a functional SAM domain is also required for the pro-viral action of viperin during HCMV infection [94]. All these studies investigating the importance of the SAM domain have used a viperin mutant (CxxxCxxC to AxxxAxxA) that cannot bind an Fe/S cluster. However, it has been reported that the Fe/S cluster is important for the general stability of viperin [95]. So, the findings with the SAM domain mutants raise many questions regarding the activity and stability of this mutant protein. To validate our findings for SAM domain, we manipulated the levels of SAM in the cell by over-

expression of a bacteriophage T3 SAMase, without any mutational alterations to viperin. Depletion of SAM resulted in a complete loss of antiviral activity of viperin against TBEV, confirming the importance of the enzymatic activity of the SAM domain for its function.

Figure 15: Schematic representation of viperin and its mutants in the study.

Interestingly, many proteins in the group of radical SAM enzymes bind the substrate at the C-terminus of the enzyme. In line with this, the C- terminal domain of viperin has been shown to interact with viral proteins such as DENV-2 NS3 [55], HCV NS5A [57], and some host factor pro-viral for HCV, hVAP-A [57]. These interactions are crucial for the action of viperin against HCV and DENV-2. Similarly, the C- terminal domain is very important for viperin dimerization [85]. All these observations suggest that the C-terminal domain is important for both the maturation of viperin and its binding to the target protein. However, there appears to be virus-specific differences as the N- terminal domain of viperin is important for its antiviral activity against CHIKV [54]. When we analysed the interactome of viperin, many interaction partners were lost in the absence of the N terminal domain. This might be because the intracellular localization is important, or because the N-terminal domain is important for binding and function. The N-terminal domain is also the most variable part of viperin, and may have been subjected to positive selection, whereas the C-terminal domain is highly conserved across in different species [70-72].

7.1.1. Viperin inhibits late-step genomic replication of TBEV

Viperin is antivirally active against many viruses. However, only a handful of studies have identified the steps in the virus life cycle that are affected by viperin. It has been shown that viperin interacts and inhibits FPPS, resulting in depletion of lipid rafts (LDs) on the cell

membrane [60]. LDs are required by many viruses for entry and release [205]. Viperin has therefore been shown to prevent the budding of FLUA and HIV from the cell [60, 65]. FPPS is involved in the synthesis of cholesterol [206]. TBEV requires cholesterol for its entry [207], so we looked at the binding and entry of TBEV. Viperin expression did not affect the level of cholesterol in the cell; nor did it affect the binding or entry of TBEV. Moreover, over-expression of FPPS did not relieve the antiviral function of viperin, suggesting a different mode of action against TBEV.

Figure 16: Schematic representation of viperin’s mode of action against TBEV. The steps involved in the antiviral action of viperin can broadly be divided into six steps as follows. Step 1, CIA1 binds at the C-terminus of viperin. Step 2, CIA1 transfers the Fe/S cluster onto the motif (Cx3Cx2C) in the SAM domain. Step 3, the Fe/S cluster extracts an electron from the co-factor, SAM, bound to the SAM domain. Step 4, SAM is cleaved into 5’ deoxyadenosyl radical (5’ Ado.) and methionine. Step 5, 5’ Ado. abstracts a hydrogen atom from an unknown substrate (X) which could be a cellular or a viral protein and this reaction generates a product Y. Step 6, the product Y can then directly or indirectly inhibit the synthesis of plus-sense RNA of TBEV.

Interestingly, we did not identify FPPS as a binding partner for viperin in our interactome analysis, but this may have been due to the different cell types used in the studies. Viperin has been shown to have an effect on the replication of many viruses. During HCV infection, viperin interferes with the interaction between host factor (VAP-A) and viral protein NS5A, therefore disturbing the formation of viral replication complex (RC) [57]. Similarly, viperin inhibits DENV2 replication by interacting with a viral protein, NS3, that is important for the viral RC [55]. In line with its effect on other members of the family Flaviviridae, viperin affected the replication of TBEV. Expression of viperin reduced the amount of TBEV RNA in the host cell already 5 hours after infection. To investigate the replication step that was being affected, we differentiated positive-strand RNA from negative-strand RNA during detection. Viperin was found to selectively inhibit the production of positive-stranded RNA and the effect was comparable to that of the cyclohexamide control, a compound that inhibits any de novo synthesis of protein. As the negative-strand RNA synthesis was not affected by viperin we could conclude that protein translation is not a target of viperin, and that viperin selectively inhibits the synthesis of positive-stranded RNA. Based on the results up to this point, a model for the viperin antiviral action of viperin against TBEV is presented in the Figure 16.

7.2. Biogenesis of viperin using the CIA targeting complex (paper II)

Over the last few years, our knowledge of the maturation of Fe/S proteins has grown exponentially [105, 106]. This is particularly due to the recent identification of components involved in the maturation of Fe/S apo-proteins, such as CIA2A, CIA2B and MMS19 [121, 123, 124, 128]. However, along with our increased understanding of the loading of Fe/S clusters onto apo-proteins, the maturation pathways have become more and more complex [106-108, 208]. The importance of individual components involved in the transfer of Fe/S cluster onto apo-proteins appears to be target-specific [106, 121]. So, to understand viperin maturation, we set out to investigate the newly identified CIA targeting factors (CIA2A, CIA2B, and MMS19) in viperin biogenesis.

7.2.1. Two branches of the CIA targeting complex interact at opposite ends of viperin.

Viperin interacts with CIA1, so it was used as a positive control. The co- ip of viperin with individually over-expressed candidate proteins and

endogenous proteins confirmed viperin interaction with all the components of the CIA targerting complex. Using full length viperin and its mutant, we could also identify the domain in viperin important for these interactions. Interestingly, the two distinct branches of the CIA targeting complex required opposite ends of the amino acid sequence of viperin for their interaction. The general CIA targeting complex (CIA1, CIA2B, and MMS19) bound at the C-terminal domain of viperin and the N-terminal domain was important for its interaction with CIA2A (Figure 17).

Figure 17: Schematic representation of the interaction of viperin with the two branches of CIA targeting complex at opposite ends of the primary sequence: the general CIA targeting complex (CIA1, CIA2B, and MMS19) at C-terminal domain and CIA2A at the N-terminal domain.

Although cytosolic Fe/S proteins have been shown to interact with the CIA targeting complex, one protein interacting with both CIA2A and CIA2B is very rare [121]. CIA2A and CIA2B are involved in the maturation of distinct set of cytosolic Fe/S proteins. On the one hand, CIA2B enables the maturation of almost all cytosolic Fe/S proteins involved in the general housekeeping of the cell. On the other hand, CIA2A enables the maturation of cytosolic Fe/S proteins involved in iron homeostasis of the cell [121]. Iron is a redox-active metal that is required in multiple metallo-proteins for different life processes. Some viruses such as HCV [209], HIV [210] and HCMV [211] have been shown to alter the iron homeostasis of the cell to promote the infection and severity of disease [212-216]. Thus, interaction of viperin with CIA2A was very interesting.

The CIA targeting complex is very dynamic. Functional analysis of the individual components has suggested a unique combination of individual components of the complex to assist in maturation for different Fe/S apo-proteins. We therefore wanted to investigate the interaction of individual component of the CIA targeting complex with viperin, and how far the binding of one component to viperin would influence the interaction of other components. This was done by over- expression and depletion of specific components followed by co-ip. Our results indicate that CIA1 is the major interaction partner of viperin because its depletion decreases viperin association with both CIA2B

and MMS19. Conversely, CIA2B or MMS19 depletion has no effect on viperin-CIA1 binding. However, CIA2B depletion decreases viperin- MMS19 binding, indicating that the C-terminal domain of viperin might directly interact with CIA1 which recruits CIA2B, and this complex then facilitates the binding of MMS19. The interaction of CIA1-CIA2B-MMS19 complex at the C-terminal domain appears to be independent of CIA2A because depletion of the latter has no effect on viperin association with the complex. In contrast, viperin-CIA2A interation appears to be improved when CIA1, CIA2B, or MMS19 is depleted. This effect may be due to the fact that CIA2A i) is stabilised by viperin and/or CIA1 binding, and ii) may compete with CIA2B for complex formation with CIA1. The presence of either of the CIA2 isoform is crucial to stabilize CIA1 [121] and thus appears to be important in maintaining viperin-CIA1 interaction. Overall, the data suggests that the N-terminus of viperin binds CIA2A independently of other CIA factors, but its cellular amount is regulated by association with CIA1, which shuttles between the two CIA2 isoforms (Figure 18).

7.2.2. CIA1 plays the key role in transfer of the Fe/S cluster on viperin

To determine the functional significance of the individual components in viperin maturation, one or more factors of the CIA targeting complex was removed and loading of the Fe/S cluster was determined by measuring the 55Fe on viperin. IOP1, a component of the core CIA machinery, was used as a positive control for viperin maturation. IOP1 and CIA1 depletion led to a significant decrease in the transfer of the Fe/S cluster on viperin. Individual depletion of CIA2A, CIA2B, or MMS19 did not show any effect on the transfer of the Fe/S cluster onto viperin. However, depletion of both CIA2A and CIA2B reduced the transfer of the Fe/S cluster on viperin. This affect may have been because the stability of CIA1 stability was reduced in the absence of CIA2A and CIA2B. Hence from these results it can be concluded that viperin interacts with CIA1, CIA2B, MMS19, and CIA2A, but is solely dependent on CIA1 (among the CIA targeting complex), which is also the major interacting partner for the transfer of the Fe/S cluster onto viperin (Figure 18).

Figure 18: Schematic representation of loading of the Fe/S cluster onto viperin by the CIA targeting complex. Multimeric complexes of CIA targeting components are present in the cell. The general CIA targeting complex interacts with viperin at the C-terminal domain and CIA2A at N-terminal domain. CIA1 bound at the C-terminus recruits CIA2B, this complex then binds MMS19. The CIA1 bound at the C terminal domain then transfer the Fe/S cluster onto the SAM domain of viperin.

CIA1 belongs to a large family of WD40 proteins, and is important for all the cytosolic and nuclear Fe/S proteins tested [121]. Structurally CIA1 is doughnut-shaped [125]. Proteins of the WD40 family have been shown to serve as docking sites and mediate molecular recognition of partner proteins [217, 218]. CIA1 serving as the docking site has been predicted based on its interaction with IOP1 and other members of the CIA targeting complex such as MMS19, CIA2B, and CIA2A [121, 122]. Moreover, CIA1 (along with CIA2B and MMS19) binds to the majority of cytosolic and nuclear Fe/S proteins. However, the functional significance of these interactions is not always clear. On the one hand MMS19 has been shown to be less important for GPAT maturation [121]. While on other hand CIA2B has been shown to be partially important for POLD1 assembly [121]. Therefore, it seems that individual constituents of the CIA targeting complex deliver the Fe/S clusters to dedicated client Fe/S apo-proteins. Interestingly, viperin’s dependence on only one component of the CIA targeting complex for the transfer of Fe/S cluster is different from the maturation of other reported Fe/S proteins, where at least two components of the CIA targeting complex is required for maturation. Similarly, it is very surprising that CIA2 isoform play a role in CIA1 stability and improved interaction of viperin with CIA1, but is functionally not required for loading of the Fe/S cluster on viperin. One possible hypothesis could be that since CIA1 can interact with either of the CIA2 isoforms, depletion of one isoform might be compensated for by use of the other isoform. However, since the two CIA2 isoforms bind at opposite ends

of the primary sequence of viperin, it could be that loading of the Fe/S cluster can be mediated from either terminal domain of viperin. But, this seems unlikely because any mutation at the C-terminal domain abolishes the loading of Fe/S cluster on viperin [93]. Additional studies are required to investigate and define the role of CIA2 isoforms regarding the stability and transfer of the Fe/S cluster onto viperin. Another hypothesis could be that since viperin is an ISG, this protein (in comparison to other Fe/S proteins) might have experienced more selection pressure due to viral infections. Thus for its robust maturation and antiviral activity, viperin may have evolved is such a way as to only require one component of the CIA targeting complex i.e. CIA1 for loading of the Fe/S cluster. Although all these scenarios appear possible, additional studies will be required to confirm our hypothesis. Based on the results to so far, viperin can be placed in a unique branch of the CIA machinery, where loading of the Fe/S cluster is dependent on the early CIA machinery and only one component of the CIA targeting complex: CIA1 (Figure 19).

Figure 19: Schematic representation of viperin maturation pathway by the CIA machinery. CIA targeting complex is split into two main branches. CIA1, CIA2B, and MMS19 take part in the maturation of general Fe/S proteins that are required for the housekeeping of the cell. This branch is further divided into CIA2B branch and CIA2B- MMS19 branch. CIA2A-CIA1 (CIA2A branch) take part in the maturation of proteins involved in maintaining the iron homeostasis of the cell. Since viperin requires only CIA1 for maturation, it is placed in a new unique branch, CIA1 branch. The picture is adopted and reprinted with the permission from the publisher [121].

7.3. Viperin sequesters GBF1 for an enhanced secretion of TBEV capsid protein through an unconventional secretory pathway (paper III)

Viperin has a strong effect on the genome replication of TBEV (paper I). However, its effect on the infectious virus particle is even stronger, by tenfold (Figure 20).

TBEV titer in FLPin cells 10 Control Figure 20: Tenfold greater reduction of TBEV titre relative to genomic RNA copies 8 Tetracyclin by viperin. FLP-IN T Rex cells that 10 6 inducibly expressed viperin upon addition Log of tetracycline were infected by TBEV. 4 twenty-four hours post infection, the titre of TBEV was determined by plaque assay. 2 Titer (Pfu/ml) RNA (au)

This indicated that viperin might also affect the life cycle of TBEV after genome amplification. Assembly and release of virions is highly dependent on genome replication in life cycle of the virus [131]. However, owing to a very strong inhibition effect of viperin on genome replication, its effect on assembly and release would be masked. Thus, to study the effect of viperin on assembly and release, we transiently expressed only the structural proteins of the virus (C, E and prM) to produce virus-like particles (VLPs). Viperin expression specifically enhanced the secretion of C protein. This effect was independent of any other structural proteins of TBEV (E and prM). Furthermore, secretion of C particles was found to be induced by viperin in a dose-dependent manner, and was dependent on the N-terminal domain of viperin.

Flavivirus virions are formed by budding into the ER, and virus release is via the conventional secretory pathway (Figure 21).

Figure 21: In the conventional secretory pathway, protein exit the ER via COPII-coated vesicles and arrive at the Golgi. In the Golgi, COPI coated- vesicles are responsible for the intra-Golgi transport and the retrograde transport between the Golgi and ER. Vesicles exiting the trans-Golgi are clathrine coated. The figure is reprinted with the permission from the publisher [1] .

The preferential increase in C particle release caused by viperin was different from its effect on soluble cellular protein or membrane-bound vesicular stomatitis virus glycoprotein [85]. Taken together, our observations and results indicated that viperin has an as yet undescribed cellular function. We therefore looked at the secretory pathway to understand the effect of viperin on C particle release. Inhibition of COPII-vesicles was achieved by expression of a mutant Sar1 protein. Sar1 is involved in the transport of COPII-vesicles from ER to Golgi [219]. Expression of the Sar1 mutant inhibited secretion of all the structural protein of TBEV (E, prM, and C) independently of viperin expression. The results indicated that egress of both prME VLPs and C particles of TBEV was COPII-mediated and that viperin did not affect this step. Next, COPI and clathrine-coated vesicle formation was inhibited by brefeldin A (BFA). This resulted in inhibition of prME release, but secretion of C particles was enhanced and was similar to the effect observed with viperin. This indicated that the increased release of C particles was due to inhibition of COPI and the later steps in the secretory pathway. Overall, it could be concluded that C particles are released in a COPII-dependent but COPI- independent manner.

COPI complex formation and vesicular transport is much more complex than earlier proposed. Studies over the years have identified many factors that contribute to our current understanding of COPI- mediated transport [220]. So in order to investigate the effect of viperin on COPI-mediated transport, we performed a pull-down experiment followed by mass spectrometry analysis of viperin. To identify novel interaction partners that could explain the phenotype, I compared the wild-type viperin with the TN-50 mutant that did not affect the C particle secretion. Among the several significant hits identified, one of them was Golgi-specific brefeldin A resistance guanine nucleotide exchange factor 1 (GBF1). This protein came up with the wild-type viperin but not with TN-50. The main function of GBF1 is to activate a GTPase, ADP-ribosylation factor 1 (ARF1) [1, 221, 222]. ARF1 is involved in assembly of COPI-coated vesicles [223]. Interaction of viperin with GBF1 was confirmed and the N-terminal domain of viperin was found to be important for this interaction. Live cell microscopy showed vesicular structures in which GBF1 was surrounded by viperin. Furthermore, over-expression of GBF1 abrogated the induced secretion of C particles by viperin, suggesting that viperin may sequester GBF1 and inhibit its function, leading to

excess release of C protein in the form of C particles and therefore disturb the virion assembly.

Release of naked C particles has already been reported for HCV [224]. However, this is the first time that the release of C particle has been shown for any flavivirus. It is also the first time when an ISG antiviral mechanism is to preferentially induce the secretion of a viral structural protein (C particles) so that the formation of infectious virion is inhibited. Although these findings are very interesting more studies should be performed in vivo to validate the results under physiological condition. GBF1 was first identified in the context of enterovirus infection, where it was shown that the virus inhibits GBF1 as a counter measure to limit the host cell response against infection [225, 226]. However, in many studies GBF1 has been shown to be pro-viral for many different viruses [227-232]. Many viruses form vesicle-like structures where viral RNA replication occurs. GBF1 has been shown to be part of these vesicles. In fact GBF1 is crucial for the formation of such vesicles and therefore for the replication of these viruses [230, 232-234]. Although GBF1 is important for COPI-coated vesicle formation and in protein transport, the pro-viral function of GBF1 has mostly been through the formation of vesicular structure for replication of viral RNA. GBF1 was recently shown to be important in the transport of DENV C protein on LDs. The accumulation of C on the LDs is important for the genome encapsidation during the formation of infectious virions. The use of this non-canonical function of the COPI system by DENV for the accumulation of C on the LDs has not been seen before [227]. On the one hand it seems that apart from being involved in the secretory pathway, GBF1 is also important at different steps of the virus life cycle of different viruses. On the other hand, viperin inhibits many viruses at different steps of virus life cycle. Thus, it would be interesting to study the interaction and function of viperin and GBF1 regarding other viruses.

Concluding remarks IV

Viperin is an important IFN inducible cellular protein that has a radical SAM enzyme activity. It is one of the strongest upregulated ISGs, which shows a broad antiviral effect, however, the antiviral mechanism appears to be virus specific. Additionally, viperin has also been shown to be important in immune signalling. With increasing evidence that viperin plays a multifunctional role, the work in this thesis was aimed to investigate and understand some of the functions of viperin. Viperin has been shown to interact with both host and viral factors, however, they cannot fully explain the antiviral mechanism. Therefore, we decided to look at the protein interactome of viperin. A number of proteins involved in different functional networks were identified to be interacting with viperin. Protein network (known as CIA targeting complex) involved in the late step transfer of Fe/S cluster on cytosolic Fe/S apo-protein were identified as the strongest interacting partners and since viperin has been shown to have the Fe/S cluster, CIA targeting complex was very interesting candidate proteins to study further. With co-ip, we could confirm the interaction of viperin with all the factors of CIA targeting complex (CIA1, CIA2A, CIA2B, and MMS19). We could also identify that CIA1, CIA2B and MMS19 (involved in maturation of Fe/S protein involved in housekeeping of cell) bound at the C-terminal domain, and CIA2A (involved in stability and maturation of protein involved in maintaining iron homeostasis of cell) required N-terminal domain of viperin. Although viperin bound to all the CIA targeting factors, only one factor (CIA1) was essential to transfer Fe/S cluster onto the SAM domain of viperin. These findings are very interesting, as most of the radical SAM enzyme investigated require two or three components (CIA1, CIA2B, with or without MMS19) for their maturation, indicating that viperin maturation is different than other radical SAM enzyme. We also found that viperin is a potent antiviral protein against TBEV infection, and showed the importance of SAM domain (Fe/S cluster binding site) and C-terminal domain (CIA1 binding site) for its antiviral action. Thus we can conclude that CIA1 is essential for the antiviral activity of viperin against many viruses, were the antiviral effect of viperin require the radical SAM domain activity. The physiological significance of viperin interaction with CIA2A, CIA2B and MMS19 is not clear yet and further studies must be done to elucidate their role in viperin function.

In this thesis, we show that viperin is a very strong inhibitor of TBEV by targeting the virus in multiple ways. Interestingly, viperin specifically targets positive stranded RNA synthesis, as well as, interfering with particle assembly, by inducing secretion of C particle. Different domain of viperin play significant role in these targets. The SAM domain and C-terminal domain is essential to inhibit the genome replication, while, N-terminal domain is responsible for the effect on virus assembly.

Although, we have identified viral positive RNA synthesis as a target, we do not know the exact mechanism, if it’s a viral factor, or cellular factor important for the TBEV genome replication. Similarly, we show that viperin interacts with GBF1 to preferentially induce the secretion of C particle and thus disrupt virus assembly, but we still do not know the pathway by which C particles are released. Therefore, further studies are required to elucidate these gaps. The protein interactome of viperin will be a great asset for these studies, and to investigate the antiviral mechanism of viperin against other viruses. The work in the thesis helps to broaden our current understanding of viperin in general. Hopefully, future work on the viperin interactome that has many interesting networks yet to be studied will help us to further improve our understanding of viperin function and mode of action.

Acknowledgement

As I look back on my life, I realize that every time I thought I was being rejected from something good, I was actually being re-directed to something better. I am happy that I took this journey which had many ups and downs, but in the end it has helped me to grow both professionally and personally. This journey would not have been possible without the support of many people and even though it’s extremely difficult to acknowledge them in words, I would like to express my gratitude.

First I would like to thank my supervisor Anna for giving me the opportunity to pursue research under her supervision. Thank you for all the encouragement and support. I am extremely grateful to you for showing lots of patience and resilience in educating me and guiding me. Your critical thinking and dedication to research were an inspiration on every step of my research journey. I would also like to thank my co- supervisors Niklas and Richard for the discussions and suggestions on my projects. Niklas, thank you for being around and guiding me whenever I needed your help.

This work would not have been possible without the help from our collaborators. I am very lucky to have visited some of them. I am grateful to Roland and Oliver for all the support both professionally and personally during my stay in Marburg and also thereafter. Roland, thank you for your advice and being there for me whenever I needed any help. Oliver, thank you for teaching me all the techniques required for my project and also showing me the city. I am thankful to you and all the group members in Marburg for their hospitality, especially Som to help me quickly adjust to Marburg. I would also like to thank Andreas and Philipp for being a very nice host during my stay in Munich. Thank you Philipp for performing the mass spec and its analysis and thereafter being very patient in answering my questions.

I would like to thank all the lab members of our group. It’s difficult to imagine this journey without you all. Kirstin, thank you for all the help and support throughout my stay in Umea, thank you for making my life easy in the lab. Richard, it has been a real pleasure knowing you. I am extremely grateful for your help in almost everything. Thank you for all those wonderful dinners. You are one person who is irreplaceable in our group. Chaitanya, thank you for all those weekend parties and extending my life beyond the boundaries of the lab. Emma, thank you being there for me and helping me in the lab. Jenny, thank you for all the experiments you did for me. Karin, thank you for all the help in the lab. Elvira, thank you for all those discussions and suggestions when I needed them. Christos and Yi Ping, coffee breaks without you guys are

not the same anymore. Raquel, thank you for all those discussions about life. Naveed, thank you for all the dinners and fun time.

I would also like to thank all the people at the unit of virology for making a very pleasant working environment. Katrine, Magnus, Fredrik, Ya- Fang, Jan, Kickan, Annasara, Naresh, Anandi, Koushik, Maria, Mårten and Göran, thank you for all the kind support during my Phd. Lars, Stefan, and Rickard, thank you for all the discussion and suggestions for my project.

Life outside the lab could be dull without friends and family. I have been very fortunate to have good friend’s. Anubhav (Chacha), Naresh, Chaitanya, Anandi, Chinmay and Richard thank you for being my family in Umea. Thank you all for being there for me through all the highs and lows of my life in Umea. Mridula, Radha, Steffi, and Mahesh thank you for all the funtime in Umea.

I would also like to thank my friends, Gaurav, Sandip, and Vishal for their support and word of encouragement during stressful times. I am grateful to my brother, Sandeep and sister, Neetu for their love and support during my Phd. I also wish to thank my fiancé, Amita for her love and support during my stay in Umea.

Finally, I dedicate this thesis to my parents, Mr Surendranath Upadhyay and Mrs Sita Upadhyay, whose love and devotion has always motivated me.

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