ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2019

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1583

Human adenovirus – host cellinterplay

The role of the cellular zinc finger proteins andmitochondrial DNA

KWANGCHOL MUN

ISSN 1651-6206ISBN 978-91-513-0696-4urn:nbn:se:uu:diva-389805

Dissertation presented at Uppsala University to be publicly examined in C8:305, BiomedicalCentrum (BMC), Husargatan 3, Uppsala, Thursday, 5 September 2019 at 09:00 for thedegree of Doctor of Philosophy (Faculty of Medicine). The examination will be conductedin English. Faculty examiner: PhD Stefan Schwartz (Department of Laboratory Medicine,Lund University).

AbstractMun, K. 2019. Human adenovirus – host cell interplay. The role of the cellular zincfinger proteins and mitochondrial DNA. Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine 1583. 61 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0696-4.

Human adenovirus (HAdV) is an abundant DNA virus with significant clinical relevance sinceit cauces a variety of respiratory, ocular, and gastrointestinal diseases. It is also intensively usedas a therapeutic tool to treat cancers and to boost immune responses. In order to achieve a bettercontrol over the HAdV epidemiology and improved utilization for clinical applications, it iscrucial to understand the molecular interaction between the host cell and HAdV.

The aim of the current thesis is to delineate the molecular interactions between HAdV type5 (HAdV-C5) protein VII (pVII), two cellular zinc finger proteins (MKRN1, ZNF622) andmitochondrial DNA (mtDNA). In paper I, we have identified MKRN1 as one of the novel pVII-interacting proteins. Surprisingly, endogenous MKRN1 protein is down-regulated in the HAdV-infected cells due to its proteasomal degradation. Further, the pVII(wt) promoted MKRN1 self-ubiquitination, which may explain the overall instability of the MKRN1 protein in the infectedcells. In addition, we show that the MKRN1 protein is also down-regulated in measles virus- andvesicular stomatitis virus-infected cells. In paper II, we report that the cellular ZNF622 proteininteracts with the pVII protein. Intriguingly, ZNF622 expression was enhanced in HAdV-C5-infected cells, implying its anti-viral role. Surprisingly, lack of the ZNF622 protein significantlyenhanced formation of the infectious HAdV-C5 virions. Finally, we propose a model howthe ZNF622/NPM1/pVII protein complex regulates the pVII protein binding to viral DNA.In paper III, we report that HAdV-C5 infection enhanced mtDNA release into cytosol. Theenhanced mtDNA release can be partially explained by accumulation of the pVII protein sinceits down-regulation diminished mtDNA release into cyotosol. We also report pVII-regulatedgene expression profile and show that cellular cytokine IL-32 mRNA accumulates in responseto the pVII protein expression.

Collectively, in this thesis we provide molecular characterization how two cellular zinc fingerproteins (MKRN1 and ZNF622) and mtDNA behave in the context of lytic HAdV-C5 infection.The ZNF622 may act as a bona fide anti-viral factor blocking infectious virion formation viatargeting the essential viral core protein pVII. The MKRN1 protein is efficiently eliminatedin the infected cells, highlighting the essence of HAdV-C5-controlled proteasome. Finally,dynamical change of mtDNA induced by HAdV-C5 infection, might initiate a novel signalingpathway beneficial for the cells or the viruses.

Keywords: Adenovirus, Protein VII, Ubiquitination, Chromatin remodeling, Mitochondria

Kwangchol Mun, Department of Medical Biochemistry and Microbiology, Box 582, UppsalaUniversity, SE-75123 Uppsala, Sweden.

© Kwangchol Mun 2019

ISSN 1651-6206ISBN 978-91-513-0696-4urn:nbn:se:uu:diva-389805 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-389805)

Dedicated to my parents

Main supervisor Tanel Punga, Associate Professor Department of Medical Biochemistry and Microbiology Uppsala University Sweden Associate supervisor Göran Akusärvi, Professor Department of Medical Biochemistry and Microbiology Uppsala University Sweden Opponent Stefan Schwartz, Professor Department of Laboratory Medicine Lund University Sweden Committee members Aristidis Moustakas, Professor Department of Medical Biochemistry and Microbiology Uppsala University Sweden Mikael Berg, Professor Department of Biomedical Sciences and Veterinary Public Health Swedish University of Agricultural Sciences Sweden Sara Lind, Associate Professor Department of Chemistry Uppsala University Sweden

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Raviteja Inturi, Kwangchol Mun, Katrin Singethan, Sabrina Schrei-ner and Tanel Punga (2018). "Human adenovirus infection causes the cellular MKRN1 E3 ubiquitin ligase degradation involving the viral core protein pVII." J. Virol., 92(3), 01154--01117. doi: 10.1128/jvi.01154-17.

II Kwangchol Mun, Tanel Punga (2019). "Cellular Zinc Finger Pro-tein 622 Hinders Human Adenovirus Lytic Growth and Limits Bind-ing of the Viral pVII Protein to Virus DNA." J Virol, 93(3). doi: 10.1128/JVI.01628-18.

III Kwangchol Mun, Raviteja Inturi, Tanel Punga (2019). “Human ad-enovirus core protein pVII influences mitochondrial DNA dynamics and expression of the pro-inflammatory cytokine IL-32.” Manu-script.

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11Adenoviruses ............................................................................................ 12

Adenovirus taxonomy .......................................................................... 12HAdV structure .................................................................................... 13HAdV life cycle ................................................................................... 14The HAdV-C5 pVII protein ................................................................. 16

Structure of protein VII ................................................................... 16Function of the pVII protein ............................................................ 17

Zinc finger proteins .................................................................................. 20Makorin ring finger protein 1 (MKRN1) ............................................. 20Zinc finger protein 622 (ZNF622) ....................................................... 21

Mitochondria ............................................................................................ 22Structure of mitochondria .................................................................... 22Function of mitochondria ..................................................................... 23Mitochondrial DNA (mtDNA) ............................................................ 23Mitochondria and viruses ..................................................................... 24

Mitochondria and cellular anti-viral innate immunity .................... 24MtDNA and viruses ........................................................................ 24

Current investigation ..................................................................................... 26Paper I. Human adenovirus infection causes the cellular MKRN1 E3 ubiquitin ligase degradation involving the viral core protein pVII .......... 26

Identification of novel HAdV-C5 interacting proteins ........................ 27The MKRN1 protein undergoes proteasomal degradation in HAdV-C5-infected cells .................................................................................. 27The precursor pVII protein enhances MKRN1 protein self-ubiquitination ....................................................................................... 28

Paper II. Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA .................................................................................................................. 30

ZNF622 specifically interacts with the HAdV-C5 pVII protein ......... 31The ZNF622 protein accumulates during the HAdV-C5 infection time course ................................................................................................... 31Lack of the ZNF622 protein increases the extracellular pVII accumulation. ....................................................................................... 31

Lack of the ZNF622 protein enhances cell lysis and production of infectious HAdV-C5 particles .............................................................. 32Elimination of ZNF622 expression enhances the pVII protein binding to viral DNA ........................................................................................ 33Reintroduction of ZNF622 affects virus growth .................................. 34Reintroduction of ZNF622 reduces pVII binding to viral DNA .......... 34ZNF622 forms a complex with the NPM1 protein in HAdV-C5-infected cells ........................................................................................ 35ZNF622 associates with chromatin in adenovirus infected cells ......... 35

Paper III. Human adenovirus core protein pVII influences mitochondrial DNA dynamics and expression of the pro-inflammatory cytokine IL-32 36

HAdV-C5 infection induces mtDNA release to cytosol ...................... 36The pVII protein is partially involved in the mtDNA release ............. 37The pVII protein is not sufficient to release mtDNA into cytosol ....... 38Protein VII protein expression alters host gene expression ................. 39

Discussion and future perspectives ............................................................... 41Paper I ....................................................................................................... 41Paper II ..................................................................................................... 42Paper III .................................................................................................... 44

Conclusions ................................................................................................... 47Acknowledgements ....................................................................................... 48References ..................................................................................................... 50

Abbreviations

Ad ASK1 Avp CHD3 cGAS DBP DDR DENV HAdV HAdV-C5 HCMV HMGB1 HTLV HSV-1 hpi IFN kbp MeV MHC MKRN1 MLP MLTU MPK38 MYBL2 NLS nt TRAIL VSV ZFP ZNF622

Adenovirus Apoptosis signal-regulating kinase 1 Adenovirus protease Chromodomain helicase DNA binding protein 3 Cyclic GMP-AMP synthase DNA binding protein DNA damage response Dengue virus Human adenovirus Human adenovirus type 5 Human cytomegalovirus High mobility group box 1 Human T-lymphotropic virus Herpes simplex virus 1 hours post infection interferon kilo base pair Measles virus Major histocompatibility complex Makorin ring finger protein1 Major late promoter Major late transcription unit Murine protein serine/threonine kinase 38 MYB proto-oncogene like 2 Nuclear localization signal nucleotide TNF-related apoptosis-inducing ligand Vesicular stomatitis virus Zinc finger protein Zinc finger protein 622

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Introduction

We are living in a world where the news about virus disease outbreaks is reaching us more than once a week. The emergence of new viruses and re-emergence of known viruses are causing devastating diseases around the globe. This prompts us to wonder what we could do to control various virus infections. My answer to this question is straightforward - we need to gain more knowledge on basic virology. Clearly, we need to advance our in-depth understanding on the molecular interactions between the viruses and host cells. The molecular interaction between the viruses and host cells has a direct relevance to the survival of viruses and host cells. Therefore, better understanding of virus-host cell interactions can benefit us in several ways including efficient prevention of viral diseases, improved diagnostics and new therapeutic strategies. Moreover, it can bring us the opportunity to up-date our knowledge on fundamental cell biology.

Throughout the years, the virologists have accumulated extensive knowledge on the virus-host interactions. Further, with the development of new methodologies and innovative techniques, researchers have identified multiple viral factors responsible for evading host immunity as well as cellu-lar factors responsible for limiting or promoting viral infection.

However, we have to admit that our knowledge on the virus-host interac-tion is still limited. The complicated nature of virus-host cell molecular in-teractions is causing numerous challenges. Since plentiful virus and host factors (e.g., DNA, RNA, proteins) interfere at different stage of virus life cycle, it is often difficult to scrutinize the relevance of individual molecular alterations. Therefore, we still lack complete functional repertoire of virus and host factors. The viruses and host cells are still evolving diverse and sophisticated strategies to maintain their survival from the threats imposed by each other. The investigation of these “molecular arms race” between the viruses and the host cells is certainly challenging, but it is attainable by ex-tensive research and development of new methodologies.

In an attempt to investigate one of these challenges, I have studied how two cellular zinc finger proteins (MKRN1 and ZNF622) and mitochondrial DNA interfere with human adenovirus infection.

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Adenoviruses Adenoviruses (Ad) are double-stranded DNA (dsDNA) viruses discovered in human adenoids by Rowe and colleagues in 1953 [1]. In general, adenovi-ruses have a linear dsDNA genome ranging from 30 to 46 kbp. This virus has a broad host range including fish, reptile, birds and mammals. Human adenoviruses (HAdVs) are well-known pathogens, causing different types of respiratory, ocular, gastrointestinal and liver diseases [2, 3]. Emerging HAdVs with the mutation and recombination events can cause severe illness in infected patients. For example, recombinant HAdVs have caused the out-breaks of pneumonia and acute respiratory distress syndrome (ARDS) [4-6]. HAdVs can induce lytic (acute) and persistent infections in humans. Alt-hough HAdV lytic infections have been studied extensively, much less is known about HAdV persistent infections. Based on available data, certain HAdV types (e.g., HAdV-C5, HAdV-C2) can persist long-term in B- and T-lymphocytes [7]. Further, it has been shown that HAdV modulates cellular interferon (IFN) signaling to establish persistent infection [8]. It has also been proposed that reactivation of persistent HAdVs in immunocompro-mised patients can lead to high-rate of mortality among this particular group of patients [9]. HAdVs have been also suspected as potential tumorigenic agents in human cancer since the discovery that HAdV-A12 can induce tu-mors in hamsters [10]. However, at present there is not enough experimental evidence confirming that HAdVs can indeed induce human cancer.

HAdV is one of the most popular viral vectors for both therapeutic appli-

cations and basic research. Numerous studies have been done to develop oncolytic HAdVs, which can specifically target and destroy the tumor cells. This approach is very often called as a oncolytic virotherapy [11]. HAdV has also been used as a vaccination platform to express foreign antigens [12]. Moreover, HAdV has been widely used as an experimental model system to study the fundamental questions of molecular biology. For example, research on HAdVs as a model system has elucidated how mature mRNAs are spliced from pre-mRNAs [13, 14].

Adenovirus taxonomy There are 5 genera of adenoviruses: Mastadenovirus, Aviadenovirus, Atade-novirus, Siadenovirus and Ichtadenovirus. HAdVs belong to the genus Mas-tadenovirus. There are 7 HAdV species (A, B, C, D, E, F, G) and more than 70 types of HAdVs have been identified [15, 16]. This classification is based on HAdV serological features, oncogenicity in rodents and genome sequence variations. Official HAdV naming takes into consideration both virus species and type [17]. For example, HAdV-C5 indicates that it is a type 5 virus, which belongs to species C. Majority of the HAdV studies have been done

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using highly similar HAdV-C2 and HAdV-C5. Therefore, the following descriptions of HAdV structure and life cycle are based on HAdV-C2 and HAdV-C5 studies.

Fig. 1. The structure of HAdV.

HAdV structure HAdV is a non-enveloped virus consisting of the external capsid and internal core structures. HAdV capsid has a diameter of 70-90 nm and consists of 13 structural proteins [18]. Major components of the capsid are: hexon, penton and fiber proteins (Fig. 1). There are 240 copies of the hexon trimers in the HAdV capsid and it accounts for almost 63% of the capsid mass [19]. The assembly of hexon is the most important factor for the accurate shaping and stability of the virion. The penton complex at the vertex consists of pen-

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tameric penton and trimeric fiber. Penton proteins bind to cell surface integ-rins for the internalization of virus particles. In contrast, the fiber proteins consist of a tail, a shaft and a knob structure, which has binding sites for the cell surface receptors [20]. Minor components of the capsid are the protein IIIa, protein VI, protein VIII, protein IX. Monomeric protein IIIa, trimeric protein VI and protein VIII locate inner capsid, holding adjacent capsid components together. Trimeric protein IX exists on the outer surface of cap-sid and acts like a cement protein, which stabilizes hexon-hexon interface [21-23].

The HAdV core structure contains three basic proteins: protein VII, pro-tein V and protein Mu (µ). Protein VII is a histone-like protein condensing virus DNA together with protein µ inside virus capsid. Detailed structure and function of protein VII will be described in the following section. The pro-tein V (pV) is believed to mediate the binding between the viral core and capsid proteins [24].

In addition, the core contains terminal protein (TP), protein IVa2 and ad-enovirus protease (Avp). The TP is a monomeric protein covalently linked to both 5′ ends of HAdV genome and it plays an essential role in the initiation of virus DNA replication [25]. Protein IVa2 is a sequence-specific DNA-binding protein and is involved in HAdV genome packaging and transcrip-tional activation of the major late promoter (MLP) [26, 27]. Avp specifically cleaves precursors of minor capsid and core proteins including proteins IIIa, VI, VII, VIII, µ, TP and L1 52,55k [28-30]. This particular feature of Avp is needed for HAdV virion maturation [31]. In the literature, the precursor pro-teins include prefix “p” in their names (e.g., pVII), whereas the mature, Avp cleaved, proteins do not have it (e.g., VII).

HAdV life cycle HAdVs have a broad tropism, extending from ocular to gastrointestinal and respiratory tissues [9]. The initial HAdV contact with the host cells is initiat-ed by the attachment of virus surface proteins to the cellular receptors. The interaction of fiber protein with coxsackie and adenovirus receptor (CAR) is followed by the interaction between penton proteins and integrins (αvβ3, αvβ5). In addition to CAR, alternative receptors including heparin sulfate glycosaminoglycans, CD46, CD80, CD86 and sialic acid are also utilized by different HAdV types for cellular attachment [32]. These interactions result in the uptake of virus into clathrin-coated pits. Sequential entry of HAdV occurs following the steps of endosomal escape, transport through microtu-bule and uncoating of capsid proteins from viral genome. Finally, the viral DNA associated with protein VII is imported into the nucleus where virus genome replication and transcription occurs [33-35].

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HAdV life cycle can be divided into two phases: early and late phase. The turning point from early to late phase is defined by the onset of virus DNA replication [36]. There are five early genes (E1A, E1B, E2, E3 and E4), which are expressed during the early phase. At the onset of early phase, tran-scription of the E1A gene is initiated by cellular RNA polymerase II. Alter-native splicing of E1A pre-mRNA results in five different transcripts; 13S, 12S, 11S, 10S, 9S. The main role of the major E1A proteins (encoded by the 13S and 12S mRNAs) is to activate other virus early gene expression and therefore to stimulate virus DNA replication. These main E1A proteins also bind to retinoblastoma protein (Rb) to suppress its cell cycle regulatory func-tion. Suppression of Rb perturbs the finely orchestrated control over the re-lease of transcriptional activator E2F, which in turn has a positive effect on virus DNA replication [37]. The E1B proteins inhibit E1A-induced apoptosis either by directly interacting with the mitochondrial pro-apoptotic proteins (i.e., E1B-19K) or by inhibiting tumor suppressor protein p53 (i.e., E1B-55K) [38]. E2 gene encodes proteins necessary for virus DNA replication including adenovirus polymerase (Ad-Pol), virus DNA-binding protein (DBP) and terminal protein (TP) [39]. E3 gene encodes proteins required for the evasion of host immune responses. The E3 proteins are involved in the prevention of MHC I-restricted antigen presentation and degradation of the Fas ligand and TRAIL receptors [40]. The E4 region encodes at least seven proteins crucial for efficient virus production [41]. In association with E1B-55K, E4 proteins (E4orf3, E4orf6) inactivate Mre11-Rad50-Nbs1 (MRN) DNA repair complex and p53 [42, 43]. In addition, the E4orf6/E1B-55K protein complex is known to mediate selective export of viral mRNAs into the cytoplasm while inhibiting the export of cellular mRNAs [44].

Virus late genes are expressed from a common major late transcriptional

unit (MLTU) via sequential RNA processing including alternative polyad-enylation and splicing. MLTU produces five different families of late pre-mRNAs (known as L1, L2, L3, L4 and L5) by alternative polyadenylation. These pre-mRNAs are further processed into at least 20 different mRNAs by alternative splicing [45, 46]. All MLTU mRNAs contain so-called tripartite leader, a 200 nt leader sequence at their 5′ end. The tripartite leader enables MLTU mRNAs to be efficiently translated in the absence of functional host cell translation initiation factor eIF4F. Efficient translation of viral late mRNAs also require expression of the L4-100K protein and virus-associated RNA I (VA RNAI) [47]. The latter is an abundant non-coding RNA, which blocks the function of anti-viral protein kinase R (PKR) [48]. Further, VA RNAs are substrates for the cellular enzyme Dicer, which generates viral microRNA-like molecules, so-called mivaRNAs [49, 50].

The L1 unit encodes capsid structural proteins IIIa and L1 52,55k, both

involved in the virion assembly [51]. Also the L2 unit expresses capsid

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structural proteins: protein III, protein V, protein µ and protein VII [52]. The L3 unit encodes hexon, protease and protein VI [53]. The L4 unit encodes proteins involved in the regulation of virus DNA transcription, viral pre-mRNA splicing, viral mRNA translation (L4-100K, L4-33K, L4-22K) and in structure organization (pVIII) [54, 55]. The L5 unit encodes the fiber pro-tein, which is essential for virus particle attachment to the recipient cell [53].

HAdV life cycle completes through the assembly of virions in the cell nu-

cleus and the release of assembled virus particles by cell lysis. The signaling of capsid assembly is derived from cis-acting components (left terminus of HAdV genome) and possibly from trans-acting components [56]. Assembly of the HAdV capsids is finalized through the cleavage of precursor proteins (i.e., pIIIa, pVI, pVII, pVIII, pre-µ, pTP and L1 52/55k) by viral protease Avp [30]. Final release of the virions is facilitated by the host cells lysis mediated by the adenoviral death protein (ADP) encoded from the E3 unit [57].

The HAdV-C5 pVII protein

Structure of protein VII HAdV-C5 protein VII is a multifunctional protein essential for virus growth [58]. Due to its high basic amino acid content (23% of arginine and lysine), the protein VII shows a limited protein sequence homology with cellular histone 3 (H3) and protamine, a protein replacing histones during spermato-genesis [59]. Analogously to the cellular histones, the protein VII is a DNA-binding protein, able to bind non-specifically to virus DNA and non-virus DNA [60-63]. Because of these biochemical characteristics, the protein VII is regarded as viral histone-like protein [64].

Protein VII is expressed as a precursor protein and is eventually processed into a mature protein during the late phase of virus infection [65]. In HAdV-C5, the precursor protein is synthesized as a 198 amino acid long polypep-tide. During the final maturation steps the N-terminal 24 amino acids pro-peptide is cleaved off by the viral protease Avp, which leads to accumulation of the mature protein VII (Fig. 2) [64]. Based on available data, the mature protein VII is one of the most abundant viral core protein [19, 24]. It is im-portant to mention that research groups are using different abbreviations for the precursor protein VII (e.g., pVII, pVII(wt), pre-pVII, ppVII) and for the mature protein VII (e.g., VII, pVII(Δ24)). In the present thesis we use abbre-viations pVII(wt) for the precursor protein VII and pVII(Δ24) for the mature protein VII. If the precursor or mature form is not specified we use abbrevia-tion pVII.

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Biochemical studies have revealed that pVII contains several nuclear lo-calization signals, which target the protein into cell nucleus [66]. The pVII protein is extensively modified. The protein is post-translationally phosphor-ylated and acetylated, however the exact role of these modifications in HAdV-infected cells has remained enigmatic (Fig. 2) [67, 68].

Function of the pVII protein Since the pVII is a DNA-binding protein, its function has been investigated in connection with viral DNA and host chromatin. The known functions of the pVII protein can be summarized into two categories: i) viral DNA re-modeling, and ii) evasion of host defense system by targeting host chroma-tin. Most of pVII functional information is available about the virus DNA remodeling by the pVII(Δ24) protein. Since pVII(Δ24) is condensed with virus DNA inside the virus capsid, it has been believed that the pVII(Δ24) protein is involved in organizing HAdV DNA within the capsid [69]. How-ever, an elegant study by Ostapchuk and colleagues has challenged this view [58]. In their study it was shown that the pVII(Δ24) protein is not essential in virion assembly, but instead it plays an essential role during the early stages of lytic virus infection [58].

Fig. 2. Post-translational modification of precursor and mature protein VII. Individ-ual acetylation (Ac) and phosphorylation (P) sites are indicated. Also nuclear locali-zation signal (NLS) locations are indicated.

The pVII(Δ24) protein remains associated with virus DNA after the entry into nucleus, protecting virus genome from DNA damage response at the early phase of infection [70, 71]. There are controversial reports about the fate of pVII(Δ24) after the nuclear entry. Some reports show that pVII(Δ24) remains tightly associated with virus DNA throughout the early phase of infection [61, 72], while other reports show that pVII(Δ24) is removed se-

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quentially from virus DNA [63, 73]. In vitro studies report limited DNA replication and transcription in case virus DNA is condensed only with pVII(Δ24), implying that the interaction of the protein with virus DNA should be remodeled to increase the efficiency of replication and transcrip-tion [74, 75]. This view is further supported by the fact that several host cell chromatin-remodeling proteins (e.g., TAF-I/SET, TAF-III/NPM1) have been identified as the pVII-interacting partners [74, 76-78].

In addition to remodeling virus DNA, the pVII(Δ24) also counteracts the function of the host cell immune-related proteins to evade host immune re-sponse. It interacts with the immune danger signals, such as high mobility group box (HMGB) family proteins, and sequesters two of them (HMGB1, HMGB2) to host chromatin to inhibit their extracellular signaling function [67]. Table 1. Cellular proteins identified as pVII interacting partners

Gene name Known cellular function Reference

ARMCX2 Tissue integrity Tumorigenesis

Paper I

ANP32A/pp32 Cell proliferation Cell differentiation Apoptosis

[72]

BAZ1A Chromatin remodeling Paper I

C1QBP/p32 Ribosome biogenesis Transcriptional regulation Inflammation

Paper I

CHD3 Chromatin remodeling Paper I CTPS1 Synthesis of nucleic acids Paper I CUL3 Ubiquitination [79] HMGB1 Inflammation [67] HMGB2 Chromatin regulation Paper I, [67] HMGB3 Chromatin regulation Paper I MKRN1 Ubiquitination Paper I

TAF-III/NPM1 Protein chaperone Centrosome duplication Cell proliferation

[77]

PTGES3L-AARSD1 Unknown Paper I RACK1 Translational repression Paper I PHF13/SPOC1 Chromatin regulation [80]

TAF-I/SET Chromatin regulation Apoptosis

Paper I, [62, 72]

SETSIP Transcriptional activation Paper I

ZNF622 Transcription Ribosome biogenesis

Paper I, Paper II

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The updated list of protein VII interacting proteins indicates that pVII

may play multiple functions through the interaction with several cellular proteins (Table 1). However, the functional significance of most of these interactions remains obscure. The pVII protein also interacts with several HAdV proteins including IVa2, L1 52/55k, E1A and pV [81, 82]. The pVII protein interaction with the IVa2 and L1 52/55k proteins is suggested to influence virus DNA packaging [81]. The pVII protein is also reported to regulate virus DNA transcription by interacting with the E1A protein [82]. Further, the pVII protein associates with viral pV protein inside the virus core to stabilize the core structure [83].

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Zinc finger proteins Zinc finger proteins (ZFP) are a big family of proteins that have particular zinc finger (ZNF) motif present in the protein sequence. Typically, ZNF is a short amino acid sequence, which can coordinate one or more zinc ions (Zn2+) to stabilize protein folding. The requirement of Zn2+ for the function of gene regulatory proteins was first reported in 1983 [84]. The ZFPs are classified into 30 types according to their ZFN motif structure [85]. Typical example of ZFN motif is the C2H2-motif where 2 cysteins (C) and 2 histi-dines (H) are coordinated by Zn2+. Due to high abundance of this particular ZFN motif, it is often regarded as the classical ZNF type. The non-classical ZFN motifs have slightly different cysteine and histidine combinations, for example C3H1, C2C2, C3HC4. Since several ZFN motifs are often found close to each other, they can form so-called zinc finger domains (ZFD). The most abundant ZFD are the C2H2, RING finger (really interesting new gene), PHD (plant homeodomain) [86].

ZFPs are very diverse and are essential for a variety of biological func-tions. Those functions include chromatin remodeling, transcription activa-tion, regulation of apoptosis, folding and assembly of proteins, stress re-sponse, cell proliferation and differentiation. These functional effects are accomplished via ZFP interactions with various DNA, RNA, protein and lipid molecules [87-91]. In the present thesis, the structure and function of two ZFPs: MKRN1 and ZNF622, will be discussed in connection with the HAdV-C5 pVII protein.

Makorin ring finger protein 1 (MKRN1) Makorin ring finger protein 1 (MKRN1) is a ZFP with multiple biological functions. MKRN1 was first identified as an ancestral gene of the MKRN protein family. There are three MKRN proteins (MKRN1, MKRN2 and MKRN3) identified within the human MKRN protein family. The MKRN1 protein consists of four C3H1-motifs and one C3HC4-type RING finger do-main (Fig. 3).

Fig. 3. Structure of the MKRN1 protein. Image is derived from [78].

The MKRN1 protein is known as an E3 ubiquitin ligase since it has a func-tional RING finger domain, one of the fundamental features of the E3 ubiq-uitin ligases [92]. MKRN1 mediates ubiquitination of several substrate pro-teins including Fas-associated protein with death domain (FADD), human

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telomerase reverse transcriptase (hTERT), p14ARF, p21, p53, peroxisome-proliferator-activated receptor γ (PPARγ), AMP-activated protein kinase (AMPK) [92-98]. Since MKRN1 induces p53, p21 and p14ARF degradation it is thought to be an important regulator of the cell cycle and apoptosis [93, 95]. Recent study also showed that the MKRN1 can control glucose con-sumption and lipid accumulation by targeting the AMPK. Hence, MKRN1-dependent AMPK degradation might be a novel therapeutic target for meta-bolic diseases [98]. Interestingly, MKRN1 has emerged as a potential anti-viral protein. The first evidence for that came from a study showing that MKRN1 can specifically induce proteasomal degradation of the West Nile virus capsid protein [99]. Also, porcine MKRN1 (pMKRN1) has been shown to modulate porcine circovirus type 2 (PCV2) replication. Here, the pMKRN1 protein can specif-ically induce ubiquitination and proteasomal degradation of the PCV2 capsid protein to reduce virus progeny production [100]. In line with these studies, my study (see Paper I) also shows that HAdV-C5 infection interferes with the MKRN1 protein [78].

In addition to the E3 ubiquitin ligase activity, the MKRN1 protein is also

known as transcriptional regulator [101]. It has also been reported that MRKN1 protein is an RNA-binding protein involved in the transport of RNA in response to stress treatments [102].

Zinc finger protein 622 (ZNF622) Zinc finger protein 622 (ZNF622, also known as ZPR9) is a C2H2-type of ZFP. Available experimental data connects ZNF622 to apoptosis regulation, cell signaling and maturation of 60S ribosome [103-105]. It was first identi-fied as a substrate protein of murine protein serine/threonine kinase 38 (MPK38) [103]. Three potential ZNF motifs were identified through compu-tational analysis of ZNF622 protein sequence (Fig. 4) [106]. Protein-protein interaction studies have shown that ZNF622 interacts with several proteins including MPK38, MYB proto-oncogene like 2 (MYBL2) and apoptosis signal-regulating kinase 1 (ASK1) [103, 107, 108]. Among these interac-tions, most research has been focused on the interaction between MPK38 and ZNF622. Here, the MPK38 phosphorylated ZNF622 positively regulates redox-sensitive ASK1, TGF-β and p53 signaling pathways, which in turn can lead to enhanced apoptotic cell death [104].The ZNF622 protein co-purifies with proteins involved in the biogenesis of 60S ribosomal subunit in mammalian cells [105, 109]. Since Rei1, a ZNF622 homolog in Saccharo-myces cerevisiae, is involved in 60S ribosomal subunit maturation [110], it is

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possible that ZNF622 might be involved in 60S ribosomal subunit matura-tion process in the mammalian cells [105, 109, 111].

Fig. 4. Structure of the human ZNF622 protein. ZNF indicates the annotated zinc finger motif (ZNF).

Mitochondria

Structure of mitochondria Mitochondria are double membrane-bound cellular organelles, which func-tion as cellular powerhouse in eukaryotic cells. The size and number of mi-tochondria varies depending on the species, cell type and physiological con-ditions [112]. Structurally, the mitochondria consist of an outer membrane, inner membrane, inter-membrane space, matrix and cristae (Fig. 5).

Fig. 5. The simplified structure of a mitochondrion.

The outer and inner mitochondrial membranes are composed of proteins and phospholipids. The outer membrane encloses the entire mitochondria and

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has transporting channels, also known as porins, for different molecules in-cluding nucleotides and metabolites [113, 114]. The outer membrane also contains the Bax and Bak proteins involved in the formation of mitochondri-al permeability transition pore [115]. The inner mitochondrial membrane contains proteins involved in oxidative phosphorylation, ATP synthesis, metabolite transport, protein import and mitochondrial fusion and fission [116]. In contrast to the outer membrane, the inner membrane is highly im-permeable to all molecules [117]. The mitochondrial matrix is the space enclosed by the mitochondrial inner membrane. The matrix contains numer-ous molecules including mitochondrial DNA (mtDNA) genome, enzymes involved in ATP synthesis, mitochondrial ribosomes and tRNA [118]. The mitochondrial cristae are compartmentalized structure of the mitochondrial inner membrane and the shape of cristae is organized by the association of mitochondrial-shaping proteins [119].

Function of mitochondria Mitochondria are involved in diverse cellular functions including energy conversion, calcium signaling, apoptosis, cellular proliferation, cellular me-tabolism, hormonal signaling and innate immune response [120, 121]. Oxi-dative phosphorylation in mitochondria converts chemical energy from glu-cose, pyruvate and NADH into ATP [122]. Mitochondrial uptake of Ca2+ is known to regulate important cellular functions including cell metabolism and cell survival by buffering the level of Ca2+ [123]. Mitochondria are important mediators in the activation of apoptosis where the release of the proteins from mitochondrial intermembrane space prompts activation of the effector caspases [124]. Upon microbial infection or cellular damage, mitochondria can function as signaling platform for the activation of the innate immunity [125]. Since mitochondria play several important roles in cell, the impair-ment of mitochondrial function often causes multiple disorders [126].

Mitochondrial DNA (mtDNA) Human mitochondrial genome is a circular, double-stranded 16 kbp DNA molecule. MtDNA encodes 37 genes (13 genes for subunits of oxidative phosphorylation system, 22 genes for mitochondrial tRNA and 2 rRNA genes) [127]. Each mitochondrion may have several copies of mtDNA alt-hough it varies between different cell types [128].

MtDNA is packaged with mitochondrial transcription factor A (TFAM), forming a condensed nucleoid structure [129]. In addition to mtDNA pack-aging, TFAM plays an essential role in the transcription of mtDNA [130]. In contrast to nuclear DNA, mtDNA does not contain histones. This feature makes mtDNA prone to stress-induced damages [131].

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MtDNA mostly lacks introns and has one non-coding region, also known as control region, which contains the promoter for the mtDNA transcription. The control region also serves as a regulatory sequence for mtDNA replica-tion [129]. The initial mtDNA transcription produces a polycistronic tran-script, which covers nearly the whole genome and terminates near the con-trol region. The initial transcripts are co-transcriptionally processed to gen-erate individual RNA molecules [129]. The mtDNA replication requires proteins, which are different from the nuclear DNA replication machinery. The polymerase involved in mtDNA replication is DNA polymerase-γ (POLγ): a complex of one catalytic subunit (POLγA) and two accessory subunits (POLγB) [132]. Further, POLγ is involved in mtDNA repair and is the main source of mutations found in mtDNA [133].

Mitochondria and viruses

Mitochondria and cellular anti-viral innate immunity Mitochondrial homeostasis is an important part of the host cell well-being since mitochondria play essential cellular functions [134, 135]. Therefore, any perturbation in cellular homeostasis is likely to be reflected to or pro-cessed by mitochondria. Upon virus infection, mitochondria function as a signaling platform for innate immune signaling triggered by the pathogen-associated pattern recognition receptors [136, 137]. Recent studies have demonstrated that mitochondrial anti-viral signaling (MAVS) protein regu-lates the activation of the NF-κB and IRF3 proteins as a response to Sendai virus infection, suggesting the role of mitochondria in innate immune signal-ing [138, 139]. Since mitochondria produce energy and are involved in anti-viral response, they are often targeted and modulated by viruses including HSV, HCMV, HIV, HTLV and EBV [140]. Manipulating the mitochondria can enable the viruses to take the control over the cellular immunity and metabolism [141].

MtDNA and viruses Extracellular stresses (e.g., cellular damage, virus infection) can induce re-lease of mtDNA into cytosol [142]. The exact mechanism how mtDNA is released from the mitochondria is unclear, but it was suggested that the per-meabilisation of the mitochondrial inner membrane induces mtDNA release during apoptosis [143]. MtDNA release into cytosol is recognized as one of the damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR) [144]. Typical examples of DNA-sensing PRRs are the cGAS and TLR9 proteins [145]. The recognition of DAMPs by PRRs can sequentially activate the pro-inflammatory immune responses [144].

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The important role of mtDNA in anti-viral innate immune response has been suggested by recent studies [142, 146]. Moderate mtDNA stress in-duced by the loss of TFAM, can cause mtDNA release into cytosol. The cytoplasmic mtDNA seems to activate cGAS-STING-IRF3 signaling path-way followed by enhanced expression of interferon-stimulated genes (ISGs) and the augmentation of type I interferon response [146]. This signaling event, initiated by mtDNA release, can block herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) infections. Also a dengue virus (DENV) infection can interfere with mtDNA dynamics. Here, DENV infec-tion can trigger cGAS-dependent anti-viral response through the release of mtDNA into the cytosol in the infected cells [142]. In addition, release of the cyto-mtDNAin DENV-infected cells can activate TLR9 signaling in dendrit-ic cells followed by production of the IFNs [147].

However, viruses have evolved countermeasures to inhibit anti-viral in-nate immunity mediated by mtDNA [148]. One strategy utilized by the vi-ruses is the degradation of key signaling proteins throughout the interaction between the viral proteins and signaling proteins [148, 149]. For example, as an active survival strategy against mtDNA signaling, DENV NS2B protein targets and degrades the cGAS protein [148]. Another strategy is used by HSV-1, which can cause mtDNA degradation using viral nuclease UL12.5 [150]. This suggests that at least 2 viruses, HSV-1 and DENV, have evolved strategies to block mtDNA signaling for efficient virus replication.

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Current investigation

Paper I. Human adenovirus infection causes the cellular MKRN1 E3 ubiquitin ligase degradation involving the viral core protein pVII HAdV pVII is a multifunctional viral protein with essential functions during the early phase of virus life cycle [58]. Despite of these facts, relatively little is known how the pVII protein interferes with the cellular proteins and what are the functional consequences of it. To eliminate this gap in our knowledge, we decided to identify cellular proteins specifically interacting with the HAdV-C5 pVII protein. Hence, the paper I reveals some of the pVII interacting proteins and focuses on the cellular MKRN1 protein as a novel pVII interactor. The main conclusion of Paper I is shown below (Fig. 6).

Fig. 6. Protein VII facilitates MKRN1 ubiquitination.

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Identification of novel HAdV-C5 interacting proteins To characterize cellular proteins specifically interacting with the HAdV-C5 pVII protein, we performed a yeast two-hybrid (Y2H) screening experiment using the human lung cancer cell line cDNA library. We identified 13 poten-tial cellular proteins interacting with the pVII(wt) protein (Table 1). The proteins having the highest number of clones were C1QBP, SET, HMGB2, HMGB3. In addition, we recovered rare cDNAs, encoding the SETSIP, ZNF622, CHD3, MKRN1, BAZ1A, CTPS1, RACK1 proteins. The specifici-ty of our Y2H screen was strengthened by the observation that the SET, HMGB2 and HMGB3 proteins are already established pVII interactors [67, 151]. Further, we performed a proximity ligation assay (PLA) in HeLa cells expressing the pVII(wt)-Flag protein after doxycycline treatment to validate the Y2H screening results in mammalian cells. All the tested proteins showed detectable proximity ligation in the pVII(wt)-Flag protein expressing cells (Paper I, Fig. 1). Subsequent quantification showed that interaction of the HMGB2, C1QBP, MKRN1, SET, BAZ1A, CHD3 proteins with pVII(wt)-Flag was above the background signal.

Since our previous study has shown that the pVII(wt) protein stability can be regulated by the ubiquitin-proteasome system [79], we decided to study the identified E3 ubiquitin ligase MKRN1 and its interference with the pVII(wt) protein (Paper I, Fig. 2).

The MKRN1 protein undergoes proteasomal degradation in HAdV-C5-infected cells In general, HAdV infection induces proteasomal degradation of several host proteins, reprograming the cellular machinery to be optimal for virus growth [152-154]. In order to investigate if MKRN1 protein is also degraded by virus infection, we evaluated the expression of MKRN1 in HAdV-C5-infected cells. H1299 cells were infected with HAdV-C5 and the expression levels of endogenous MKRN1 protein was monitored during HAdV infec-tion time course. Since we were interested also in the pVII protein expres-sion during infection time course, we generated a unique replication-competent virus, HAdV-pVII-Flag. This virus expresses the pVII-Flag fu-sion-protein with the Flag antibody epitope tag merged to the C-terminus of the pVII protein. The expression of MKRN1 decreased steadily as the infec-tion proceeds and it was not detectable from 20 hpi and onwards (Fig. 7). Importantly, the MKRN1 protein decrease coincided with the accumulation of the pVII-Flag protein. To certify if decrease of the MKRN1 protein is caused by its proteasomal degradation, an inhibitor of proteasomal degrada-tion (MG132) was administered to HAdV-C5-infected cells. MG132 treat-ment restored the expression of MKRN1 protein, indicating that the MKRN1 protein undergoes proteasomal degradation in HAdV-C5-infected cells. The

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expression of MKRN1 mRNA was not affected in infected H1299 cells, supporting the hypothesis that MKRN1 decrease is due to proteasomal deg-radation but not due to transcriptional downregulation (Paper I, Fig. 3C).

Fig. 7. MKRN1 is down-regulated as the pVII accumulates in HAdV-C5-infected cells. Image from Paper I, fig. 3D.

The precursor pVII protein enhances MKRN1 protein self-ubiquitination The MKRN1 protein binds to the substrate proteins via the C-terminal RING finger domain to enhance ubiquitination and proteasomal degradation of the target proteins [92, 155]. MKRN1 protein is also self-ubiquitinated and de-graded, implying that activity of MKRN1 has to be also efficiently regulated within a cell [92, 156].

Based on our experiments, the pVII(wt), but not the pVII(Δ24) protein, is able to enhance MKRN1 ubiquitination (Paper I, Fig. 5A). It is known that the mutation of histidine to glutamate at 307 amino acid (H307E) in the RING finger domain deprives MKRN1 to ubiquitinate the substrate proteins [92, 93]. However, the mutated MKRN1 (H307E) protein itself was ubiqui-tinated in our in vivo ubiquitination experiment in H1299 cells, suggesting that MKRN1(H307E) can still serve as a substrate for ubiquitination (Paper I, Fig. 5B). In contrast to HA-MKRN1(wt), ubiquitination of the HA-MKRN1(H307E) protein was not enhanced by the pVII(wt)-Flag protein. The differential effect by pVII(wt) was not due to different affinity of the MKRN1 proteins since HA-MKRN1(wt) and HA-MKRN1(H307E) bound to pVII(wt)-Flag in a similar manner. The observation that MKRN1(H307E) was ubiquitinated urged us to further study the details of this particular mu-tation. In vitro ubiquitination experiments with the purified E1 (His-UbE1), E2 (His-UbcH5a) and E3 (GST-MKRN1) proteins showed that the

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MKRN1(H307E) protein is defective in self-ubiquitination (Paper I, Fig. 5D).

Since the pVII(wt) protein did not enhance MKRN1(H307E) self-ubiquitination, we hypothesized that this mutant protein might be more sta-ble in HAdV-C5 infected cells when compared to wild-type MKRN1. To test this hypothesis, we infected H1299 cells expressing either HA-MKRN1(wt) or HA-MKRN1(H307E) protein along with HAdV-C5 virus and blocked de novo protein synthesis with cycloheximide. Truly, in the presence of cycloheximide, HA-MKRN1(wt) protein showed faster decay than the HA-MKRN1(H307E) protein, suggesting that the latter is more resistant to proteasomal degradation in HAdV-C5-infected H1299 cells (Pa-per I, Fig. 5E).

Collectively, our data indicate that pVII(wt), but not pVII(Δ24), enhances

the MKRN1 protein self-ubiquitination (Fig. 6). Further, our data show that also other viruses, such as measles virus (MeV) and vesicular stomatitis vi-rus (VSV) infections reduce MKRN1 protein levels in the target cells. This may indicate a general strategy of viruses to eliminate the MKRN1 protein expression (see Discussion section).

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Paper II. Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA Interestingly, we identified an obscure cellular zinc finger protein ZNF622 as a novel pVII interactor in the Y2H screen (Paper I, Table 1). Hence, the aim of this paper was to investigate the functional significance of the ZNF622 and pVII interaction in HAdV-C5-infected cells. The graphical summary of the main results of paper II is shown below (Fig. 8): Fig. 8. The proposed model for the ZNF622 protein interplay with the cellular NPM1 and viral pVII proteins in HAdV-C5 infected cells. Image is from paper II, Fig. 10.

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ZNF622 specifically interacts with the HAdV-C5 pVII protein To confirm ZNF622-pVII interaction in mammalian cells, we performed co-immunoprecipitation assay in U2OS cells infected with the replication com-petent HAdV-C5, HAdV-pVII-Flag. The interaction between ZNF622 and pVII was slightly affected by endonuclease (e.g., benzonase) treatment (Pa-per II, Fig. 1A). In order to study which region of the ZNF622 protein is involved in binding to the pVII protein, we constructed plasmids expressing three different ZNF622 N- and C-terminal deletion proteins (Paper II, Fig. 1B). These plasmids were transfected along with pVII-Flag expressing plasmid into H1299 cells followed by immunoprecipitation with an anti-Flag antibody. Two mutant proteins, 1-108 and 1-359, both lacking the C-terminal part of ZNF622, did not bind to the pVII-Flag protein. In contrast, a mutant 109-477, lacking the N-terminal part of ZNF622 was still able to interact with the pVII-Flag protein (Paper II, Fig. 1C).

Taken together, our data indicate that the ZNF622 protein specifically in-teracts with the HAdV-C5 pVII protein and that the C-terminal region (360-477) of ZNF622 is likely involved in this interaction.

The ZNF622 protein accumulates during the HAdV-C5 infection time course Since efficient HAdV infection relies on modulation of the host cell protein expression [157], we tested ZNF622 expression in HAdV-C5-infected cells. The ZNF622 protein expression was elevated in all the tested cell lines (HEK293, HeLa, A549, U2OS) after 24 hours post-infection (hpi) (Paper II, Fig. 1D). Quantitative mRNA analysis further showed that ZNF622 mRNA was elevated during the early stage (6 to 12 hpi) of infection in U2OS cells. The enhanced accumulation of ZNF622 mRNA was not observed during the late stage (18 to 24 hpi) of virus infection (Paper II, Fig. 1E).

Together, our results indicate that the ZNF622 protein accumulation is enhanced in various human cell lines infected with the HAdV-C5.

Lack of the ZNF622 protein increases the extracellular pVII accumulation. Since the ZNF622 protein showed an enhanced accumulation in the infected cells, we tested if the ZNF622 protein expression affects HAdV-C5 growth. We generated U2OS cell line where the ZNF622 protein expression was eliminated due to nucleotide deletion introduced within the first exon of ZNF622 gene using the CRISPR/Cas9 genome editing system. The U2OS cell line was chosen because the genome editing can be carried out with high efficiency in this particular cell line [158]. Both parental U2OS (hereafter as

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U2OS(wt)) and ZNF622 knockout U2OS (hereafter as U2OS(KO)) cell lines were infected with HAdV-C5 and the accumulation of HAdV-C5 late pro-teins (pVII-Flag, pV, and capsids) were monitored. Virus capsid proteins accumulated equally well in both cell lines even though slightly higher level of some HAdV-C5 proteins at some particular time points (e.g., pVII-Flag, at 72 hpi) was observed in the U2OS(KO) cells (Paper II, Fig. 2A).

To test if ZNF622 knockout affects viral DNA replication, we measured the virus DNA replication in U2OS(wt) and U2OS(KO) cells. Quantitative analysis revealed that relative virus DNA accumulation was similar in both cell lines (Paper II, Fig. 2B). To investigate if ZNF622 knockout affects the accumulation of capsid proteins in the extracellular space, we tested the presence of the pVII-Flag protein in cell growth medium. The pVII-Flag protein was detected in the extracellular space of U2OS (KO) cells at 72 hpi and 96 hpi, but was not detected in that of U2OS(wt) cells (Paper II, Fig. 2C).

Together, this indicates that lack of ZNF622 does not have a major impact on late virus protein accumulation inside the cell, but it increases the accu-mulation of the extracellular ZNF622 protein.

Lack of the ZNF622 protein enhances cell lysis and production of infectious HAdV-C5 particles Based on elevated accumulation of the extracellular pVII-Flag protein (Paper II, Fig. 2C), we hypothesized that formation of infectious virus particles may differ between these two U2OS cell lines. This assumption was supported by a visual difference between U2OS(wt) and U2OS(KO) cells infected with the HAdV-pVII-Flag (Paper II, Fig. 3A). The majority of U2OS(KO) cells were rounded and detached at 72 hpi and 96 hpi whereas U2OS(wt) cells did not show similar visual phenotype. This results was confirmed by cell viabil-ity test, showing that infected U2OS(KO) cells had significantly lower via-bility compared to U2OS(wt) counterparts.

Next we compared the virus particle formation in U2OS(wt) and U2OS(KO) cells. For this purpose, the HAdV-pVII-Flag virus particles were purified by CsCl gradient from the infected U2OS(wt) and U2OS(KO) cells. The heavy density band, which contains mature virus particles, was more intense and sharp in the virus sample purified from U2OS(KO) cells when compared to U2OS(wt) cells (Fig. 9). Moreover, the light density band, which corresponds to immature virus particles, was more diffused in the virus sample purified from U2OS(wt) cells compared to U2OS(KO) cells. The ratio between the heavy density band and light density band (H/L) was 0.8 in U2OS(wt) virus sample while it was higher (1.4) in U2OS(KO) sam-ple. This indicates that virus isolated from U2OS(KO) cells has more mature virus particles compared to sample from U2OS(wt) cells. Interestingly, when

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the purified viruses were titrated in 911 cells, we observed a clear difference in infectious virus titers depending on the host cell origin. Although there was 1.9-fold increase in physical titer, the infectious titer showed a 7.1-fold increase in favour of U2OS(KO) cells as the host (Fig. 9).

To analyze the intracellular and extracellular accumulation of infectious virus particles in U2OS(wt) and in U2OS(KO) cells, cell pellets and growth medium from HAdV-C5-infected U2OS(wt) and U2OS(KO) cell lysates were used to re-infect 911 cells. Both intracellular and extracellular virus titer were higher in in U2OS(KO) sample compared to that of U2OS(wt) sample (Paper II, Figs. 3F and 3G).

Collectively, our data implies that lack of the ZNF622 protein has a posi-tive effect on maturation and infectivity of the HAdV-C5 particles.

Fig. 9. Purification (A) and titration (B) of HAdV-C5 from U2OS(wt) and U2OS (KO) cells. Modified image from Paper II, Fig. 3.

Elimination of ZNF622 expression enhances the pVII protein binding to viral DNA Based on the observation that ZNF622 interacts with the pVII protein (Paper II, Fig. 1), we hypothesized that ZNF622 may alter pVII function. In particu-lar, we were interested to investigate if ZNF622 affects pVII function as a DNA-binding protein. Chromatin immunoprecipitation (ChIP) assay showed that the pVII-Flag protein interaction was significantly increased on virus DNA in U2OS(KO) compared to U2OS(wt) cells (Paper II, Fig. 4B). The best pVII-Flag interaction was observed at the E1A promoter region while the lowest interaction was observed at the L2 unit. The pVII-Flag protein binding to DNA was not detectable at the cellular GAPDH promoter.

Together, our data indicate that lack of the ZNF622 protein specifically enhances pVII-Flag protein binding to virus DNA.

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Reintroduction of ZNF622 affects virus growth Since we are aware of the concern that CRISPR/Cas9 genome-editing sys-tem may have off-target effects [159], we decided to confirm the phenotype by reintroducing ZNF622 into U2OS(KO) cells. In order to do that, four different cell clones were generated by stable transfection: 1) U2OS(KO) expressing the ZNF622-HA(wt) (hereafter as KO+ZNF622-HA(wt), 2) U2OS(KO) expressing the ZNF622-HA(1-359) (hereafter as KO+ZNF622-HA(1-359), 3) U2OS(KO) expressing the ZNF622-HA(109-477) (hereafter as KO+ZNF622-HA(109-477), and 4) U2OS(KO) cells transformed with the empty pcDNA3.1 plasmid as a control (hereafter as KO+pcDNA).

Constitutive expression of the proteins in HAdV-pVII-Flag infected cell lines revealed no obvious difference in the viral late protein (i.e., pVII-Flag) accumulation (Paper II, Fig. 5A). Similarly to our previous data (Paper II, Fig. 1C), ZNF622-HA(wt) and ZNF622-HA(109-477) proteins associated with pVII whereas ZNF622-HA(1-359) was unable to associated with pVII in the established U2OS cell lines (Paper II, Fig. 5B).

Reintroduction of ZNF622 reduces pVII binding to viral DNA Since knockout of ZNF622 increased pVII binding to virus DNA, we hy-pothesized that reintroduction of wild-type ZNF622 into U2OS(KO) cells may reduce pVII binding to virus DNA. To test it, we performed ChIP anal-ysis in the aforementioned four U2OS(KO) cell lines expressing ZNF622 mutants. Reintroduction of ZNF622-HA(wt) reduced pVII-Flag binding to viral DNA in all the analyzed viral DNA genomic regions (Paper II, Fig. 7A). This is consistent with the phenotype of U2OS(wt) cells (Paper II, Fig. 4B), confirming that ZNF622 elimination enhances pVII binding to viral DNA. However, the binding of pVII to viral DNA was not reduced in cells expressing ZNF622 mutant proteins such (ZNF622-HA(1-359) and ZNF622-HA(109-477)) (Paper II, Fig. 7A).

Knowing that the formation of infectious viral particles was enhanced in U2OS(KO) cells (Paper II, Fig. 3), we hypothesized that reintroducing the ZNF622(wt) protein into U2OS(KO) cells may affect infectious virus proge-ny formation. Truly, virus titer was significantly reduced in the KO+ZNF622-HA(wt) cells at 72 hpi and 96 hpi compared to other ZNF622 mutant expressing cells (Paper II, Fig. 7B).

Together, the data confirm that full-length ZNF622 reduces pVII binding to viral DNA coinciding with lower infectious virus titer.

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ZNF622 forms a complex with the NPM1 protein in HAdV-C5-infected cells Interestingly, the ZNF622-HA(109-477) protein, which is still able to bind to pVII (Paper II, Fig. 1C), was unable to block pVII binding to virus DNA. Hence, we hypothesized that additional factor(s) might bind to the N termi-nus (amino acids 1-108) of ZNF622 to control pVII binding to virus DNA. Among the candidate proteins, we focused on the cellular histone chaperon nucleophosmin 1 (NPM1) since previous studies suggested that NPM1 in-hibits pVII loading to virus DNA [160]. First, we confirmed that endogenous NPM1 interacts with ZNF622 through co-immunoprecipitation (Paper II, Fig. 8A). In order to identify ZNF622 domains involved in NPM1 interac-tion, we did another co-immunoprecipitation using the aforementioned four ZNF622 mutant cell lines. The ZNF622(109-477)-HA protein showed re-duced biding to NPM1, implying that the N terminus of ZNF622 may be involved in the interaction between ZNF622 and NPM1 (Paper II, Fig. 8B). Next, we investigated how the complex formation between the pVII, ZNF622 and NPM1 proteins. Through co-immunoprecipitation assays, we found that NPM1 binding to pVII was decreased in ZNF622(KO) cells.

ZNF622 associates with chromatin in adenovirus infected cells To further dissect the function of ZNF622, we performed cell nuclei salt extraction to evaluate ZNF622 intranuclear localization. The ZNF622 pro-tein accumulated in the high-salt resistant chromatin fractions in the HAdV-C5-infected cells (Paper II, Fig. 9B). The nuclear ZNF622 protein was main-ly detected in the high salt fraction, overlapping with the pVII-Flag fraction-ation pattern. Considerable localization changes were detected for the cellu-lar HMGB1 and HMGB2 proteins in infected nuclei as reported previously [67, 71]. Next, we investigated if elimination of ZNF622 has impact on pVII-Flag chromatin localization. Knockout of ZNF622 did not alter pVII-Flag chromatin localization (Paper II, Fig. 9C). In addition, HMGB1 and HMGB2 intranuclear localization pattern did not show any change in U2OS(KO) cells. Interestingly, reduced chromatin-association was observed for the NPM1 protein in U2OS(KO) cells (Paper II, Fig. 9C), implying that the ZNF622 protein partly regulates the expression and/or stability of the NPM1 protein in HAdV-C5-infected cells.

Together, our results indicate that the ZNF622 protein localizes to chro-matin in HAdV-C5-infected cells.

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Paper III. Human adenovirus core protein pVII influences mitochondrial DNA dynamics and expression of the pro-inflammatory cytokine IL-32 It is evident that various virus infections have an impact on mitochondria function [140]. Therefore, the aim of Paper III was to study if HAdV-C5 infection influences cellular mtDNA dynamics. The results of the study show that HAdV-C5 infection increases mtDNA accumulation in the cyto-sol, partially controlled by the virus encoded core protein pVII. The pro-posed model how mtDNA interferes with HAdV-C5 is provided below (Fig. 10).

Fig. 10. Suggested model for mtDNA dynamics in HAdV-C5 infected cells.

HAdV-C5 infection induces mtDNA release to cytosol The release of mtDNA from mitochondria to cytosol (cyto-mtDNA) can be regarded as one of the hallmarks of mtDNA dynamics [161]. To investigate if HAdV-C5 infection has an impact on mtDNA dynamics, we analysed cyto-mtDNA in the cytosolic fractions of HeLa cells infected with HAdV5-pVII-Flag. For this purpose, we set-up a qPCR experiment using primers detecting 2 different mtDNA genes: mt-CYB and mt-ND4. The accumula-tion of cyto-mtDNA was observed in virus-infected cells at 16 hpi and 24hpi (Paper III, Fig. 1A). This coincided with the accumulation of the viral late protein pVII-Flag at the same time points post-infection (Paper III, Fig. 1C,

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lanes 8, 9 and 11,12). The striking difference in the cyto-mtDNA release between 16 hpi and 24 hpi prompted us to analyse the virus DNA replica-tions at these specified time points. Virus DNA indicated a clear increase from 16 hpi to 24 hpi, coinciding with the elevated cyto-mtDNA (Paper III, Fig. 1D).

The possible contamination of genomic DNA should be considered when analyzing mtDNA since the transmission of mtDNA to the nuclear genome may occur [162]. In order to exclude the possibility that the detected mtDNA signal is due to the nuclear DNA contamination, we compared the relative amount of nuclear DNA (GAPDH) between different samples. No consider-able difference of nuclear GAPDH DNA was observed between different cytosolic extracts (Paper III, Fig. 1E), confirming that the increase of cyto-mtDNA was not due to nuclear DNA contamination.

Furthermore, to ensure that the increase of cyto-mtDNA was not due to increased cell death, cell viability of the infected cells was tested. No con-siderable difference in cell viability was observed between mock- and virus-infected. Also cell cytotoxicity analysis revealed that the virus-infected cells did not undergo extensive cell lysis ((Paper III, Fig. 2A)

Collectively, our data show that HAdV-C5 enhances cyto-mtDNA accu-mulation during the late phase of infection.

The pVII protein is partially involved in the mtDNA release Since we found that the accumulation of cyto-mtDNA coincides with pVII expression in the late phase of infection (Paper III, Fig. 1), we hypothesized that the viral late proteins, such as pVII, may be involved in the mtDNA release. To test our hypothesis, we had to develop an efficient knockout sys-tem, which can down-regulate the expression pVII. In order to develop such a system, several options were considered including the knockout of pVII from HAdV genome and the knockdown of the pVII mRNA by siRNA. However, these options were not suitable. First, targeting of the pVII tran-script will also affect the expression of other viral late genes since they orig-inate from the same MLTU (see Section “HAdV life cycle”) [163]. Second, since pVII is an essential gene, pVII-knockout-virus will not grow in the cells [58]. Therefore, we decided to use MKRN1 overexpression as an alter-native method to knockdown the pVII protein expression. In paper I, we suggested that HAdV-C5 infection targets endogeneous MKRN1 for degra-dation. We hypothesized that HAdV-C5 infection has to overcome the po-tential anti-viral activity of MKRN1 for an efficient virus growth. In line with that, we hypothesized that artificial overexpression of the MKRN1 pro-tein may affect the pVII protein through the pVII-MKRN1 complex for-mation. To test our hypothesis, we generated U2OS stable cell lines express-ing different MKRN1 mutants including MKRN1(wt)-HA, MKRN1(1-267)-

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HA and MKRN1(H307E)-HA (Paper III, Fig. 3A). Overexpression of the MKRN1(wt) protein reduced pVII expression in U2OS cells infected with HAdV-pVII-Flag virus (Paper III, Fig. 3B). In addition, overexpression of the E3 ligase-deficient MKRN1(H307E) also down-regulated pVII, indicat-ing that the observed pVII degradation was not due to MKRN1 E3 ligase activity (Paper III, Fig. 3B, lanes 2 and 3).

To test if reduction of the pVII protein affects mtDNA release to cytosol,

we quantified cyto-mtDNA in the U2OS cell constitutively expressing MKRN1(H307E)-HA in the presence and absence of HAdV-pVII-Flag in-fection. Infected control cells showed considerable cyto-mtDNA increase compared to mock-infected control cells (Paper III, Fig. 3D, lanes 1 and 2). In contrast, infected MKRN1(H307E)-HA overeexpressing cells did not show similar cyto-mtDNA increase when compared to mock-infected MKRN1(H307E)-HA cells (Paper III, Fig. 3D, lanes 3 and 4). In the absence of HAdV-C5 infection, U2OS cells expressing MKRN1(H307E)-HA showed a minor cyto-mtDNA increase compared to non-infected control cells. Cell lysates from the same fractionation experiment were further ana-lyzed by western blotting. In accordance with Paper III, Fig. 3B, expression of the MKRN1(H307E)-HA reduced pVII nuclear accumulation, whereas the levels of another virus late protein, hexon, was not affected (Paper III, Fig. 3E (lanes 4,10 and lanes 6,12) and Fig. 3F). More careful analysis of the viral late proteins revealed also that the pV protein, was affected by MKRN1(H307E) (Paper III, Fig. 3E, WB:pV).

Taken together, our results show that MKRN1(H307E) overexpression can be used as an efficient strategy to reduce pVII levels in the HAdV-C5-infected cells. Further, our data suggest that down-regulation of the pVII protein levels may affect cyto-mtDNA accumulation in the virus-infected cells.

The pVII protein is not sufficient to release mtDNA into cytosol Since our data (Paper III, Fig. 3D) suggested that down-regulation of the pVII protein can affect cyto-mtDNA accumulation in infected cells, we in-vestigated if overexpression of the pVII protein alone is sufficient to induce cyto-mtDNA accumulation. Cyto-mtDNA in HeLa cells transfected with plasmids expressing the pVII(wt) or pVII(∆24) proteins was analyzed. Over-expression of these proteins (Paper III, Fig. 4B) did not increase cyto-mtDNA, indicating that transient expression of these proteins alone was not sufficient to increase cyto-mtDNA (Paper III, Fig. 4A lanes 1-3). Since dif-ferent epitope tags may alter protein functions, we tested if expression of the pVII(wt) protein with C-terminal tGFP tag (hereafter referred to as pVII-tGFP) can influence mtDNA release. Similarly to the Flag-tagged pVII, ex-pression of the pVII-tGFP protein (data not shown) did not alter the cyto-

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mtDNA levels (Paper III, Fig. 4A, lanes 4 to 5). These results indicate that additional HAdV proteins might be involved in the machinery influencing cyto-mtDNA accumulation in the infected cells.

To identify additional HAdV factors involved in mtDNA release to cyto-sol, we transiently overexpressed HAdV early and late proteins shown either to target mitochondria functions (E1A and E1B-19K) or known to bind to the pVII protein (L1 52/55k and pV) (See also section “HAdV life cycle”). The early proteins (E1A and E1B-19K) inhibited the release of mtDNA, with the most prominent effect observed in the E1B-19K plasmid transfected cells (Paper III, Fig. 4C). In contrast, transfection of the plasmids expressing the late viral proteins (L1 52/55k and pV) did not inhibit mtDNA release into cytosol. Interestingly, the expression of the pV showed a slight increase in cyto-mtDNA (Paper III, Figure 4C).

Taken together, our data indicate that the pVII protein alone is not suffi-cient to elevate cyto-mtDNA levels in HeLa cells.

Protein VII protein expression alters host gene expression According to previous reports, cyto-mtDNA acts a pro-inflammatory signal and induces the expression of immune response-related genes [146, 147, 161]. In connection with that, our results indicating that pVII may influence cyto-mtDNA levels prompted us to investigate the expression of immune response-related genes in the presence of pVII. Further, recent reports have suggested that pVII protein associates with host cell chromatin, implying the possibility that pVII may influence the host gene expression [71].

Therefore, we decided to investigate if the pVII(wt) protein influences

host gene expression. For this purpose, we generated an inducible HeLa cell line, which can express the pVII(wt)-Flag protein after doxycycline(Dox) treatment (Paper III, Fig. 5A). To analyze gene expression profile in the pVII(wt)-Flag expressing cells, we performed AmpliSeq RNA sequencing analysis. The data analysis showed that in the pVII(wt)-Flag protein express-ing cells, 8761 genes were up-regulated, 6327 genes were down-regulated and expression of 5724 genes did not change (Paper III, Fig. 5B). Since the release of mtDNA is connected with the expression of inflammation-related genes [146, 147, 161], we focused our analysis on these particular genes. Notably, we identified several inflammation-related gene transcripts influ-enced by the pVII protein expression (Paper III, Fig. 5C). The pVII protein up-regulated genes included complement 3 (C3), caspase recruitment do-main family member 10 (CARD10), interleukin 32 (IL-32), myc proto-oncogen (MYC) and protein kinase C zeta (PRKCZ). The pVII protein down-regulated genes included caspase 3 (CASP3), caspase 9(CASP9), dual specificity phosphatase 1 and 2 (DUSP1, DUSP2), signal transducer and activator of transcription 5A (STAT5A) and tachykinin receptor 1 (TACR1).

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We were particularly interested in the IL-32, a pro-inflammatory cyto-kine, which has recently being recognized as a novel cytokine controlling both bacterial and viral infections [164]. To confirm the up-regulation of IL-32 mRNA by the pVII(wt)-Flag protein, we analyzed IL-32 expression in Dox-treated HeLa stable cell line. In line with the Ampliseq sequencing ex-periment (Paper III, Table 1), IL-32 mRNA was increased in the presence of pVII(wt)-Flag (Paper III, Fig. 5D). The increase of IL-32 mRNA expression was further confirmed in the U2OS cells infected with HAdV-pVII-Flag (Paper III, Fig. 5E).

Collectively, our Ampliseq sequencing approach revealed the host gene

expression profile in the presence of the pVII(wt)-Flag protein. Further, we show that the mRNA expression of pro-inflammatory cytokine IL-32 expres-sion increases in pVII(wt)-Flag expressing HeLa cells as well as in the HAdV infected U2OS cells.

41

Discussion and future perspectives

Paper I Here we identified a novel function of the HAdV-C5 pVII(wt) protein.

Although the pVII has been recognized as an abundant viral DNA-binding protein able to pack viral DNA into chromatin-like structure, our study re-vealed that the pVII(wt) protein exhibits also other functions. This is exem-plified by the experiments showing that the pVII(wt) protein can enhance MKRN1 self-ubiquitination. Hence, we believe that pVII may function as a viral co-factor for MKRN1 to control MKRN1 proteolytic turnover. Even though, we have identified a novel, and unexpected, function for the pVII(wt) protein, we still have several unanswered questions. For example, although the pVII(wt) protein enhances MKRN1 self-ubiquitination, it is not enough to cause MKRN1 proteasomal degradation in the transient plasmid overexpression experiments (data not shown). Since the MKRN1 protein is very efficiently degraded in HAdV-C5-infected cells, we believe that there is another factor, which together with pVII contributes to MKRN1 proteasomal degradation. Hence, the future studies should reveal the factor(s) which to-gether with pVII(wt) assure the MKRN1 degradation in the infected cells.

Our data indicate that transient overexpression of the MKRN1 protein

specifically reduces the pVII protein levels (see also Paper III, Fig. 2). Therefore, we believe that MKRN1 may function as an anti-viral protein to counteract virus infections. This would mean that under low pVII protein concentration, MKRN1 would preferably degrade pVII and thereby will diminish virus growth. However, as the pVII protein expression is drastically enhanced during the infection (see Fig. 7), it will saturate all binding sites on the MKRN1 protein and will target it for self-ubiquitination. Even though, it is an attractive model, we still lack convincing evidence that MKRN1can function as an anti-viral protein. Based on our unpublished experiments, HAdV-C5 replicates with the same efficiency in the MKRN1 knockout (i.e., siRNA-treated) and control cells. This result suggests that MKRN1 does not per se act as an anti-viral protein. However, it is possible that the anti-viral role of MKRN1 can be substituted by other members of the MKRN family (e.g., MKRN2, MKRN3). Therefore, we are eager to understand whether other members of the MKRN protein family exhibit the anti-viral potential and if they are targeted in HAdV-infected cells by the pVII(wt) protein.

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Curiously, the MKRN1 protein is also eliminated in MeV- and VSV-infected cells. This indicates that different viruses target the MKRN1 pro-tein. It remains to be tested if the decreased MKRN1 proteins levels in MeV- and VSV-infected cells are due to reduced MKRN1 gene expression or/and protein degradation.

Paper II We report a novel functional interaction between the HAdV-C5 pVII and ZNF622 proteins. This interaction was detectable in different buffer condi-tions, implying that this mutual interaction is stable. Further, our data also indicate that the mutual binding between these two proteins is probably par-tially mediated by their binding to DNA/RNA, since endonuclease benzo-nase treatment slightly reduced protein-protein binding (Paper II, Fig. 1A). The pVII protein did not bind to ZNF622 mutant lacking C-terminal region. Since the C-terminal region (360-477) does not contain any annotated ZFNs, the interaction between ZNF622 and pVII is independent of these motifs. It is important to mention that although the ZNF622 protein contains annotated ZFNs, there is no experimental proof about the function of these motifs. Further, our salt-fractionation experiments showed that both, ZNF622 and pVII, were found in the high salt-resistant chromatin fractions (Paper II, Fig. 9B) in virus-infected cells. Hence, in our future experiments we plan to map the exact localization of ZNF622 on the host cell chromatin and potential binding of the pVII protein on ZNF622-targeted chromatin loci. Further, we are eager to understand the biochemical requirements needed for ZNF622/pVII binding on host cell chromatin in virus-infected cells.

The elevated ZNF622 protein level in HAdV-C5-infected cells was de-

tected in all the cell lines tested in the study. This implies that ZNF622 in-crement is a general, and not cell type-specific, effect in response to virus infection. The underlying molecular mechanism of the ZNF622 protein in-crement is still enigmatic. Since we did not observe enhanced ZNF622 tran-script accumulation during the late phase of infection, we believe that the observed ZNF622 protein accumulation is a consequence of a post-transcriptional regulation. It is possible that the ZNF622 protein levels are increased due to enhanced protein stability or translation during the late phase of infection. Regarding the translation, it is important to mention that during the late phase of HAdV infection, mainly the viral late transcripts are translated using an ill-defined ribosome shunting mechanism [165]. The preferred translation of the late viral mRNAs is due to the presence of so-called tripartite leader at the 5’ end of the late viral transcripts [166]. Hence, in our future studies we are interested to study the potential mechanisms (e.g., ZNF622 protein stability, preferred translation of ZNF622 mRNA due

43

to tripartite leader-like structure) responsible for the ZNF622 protein accu-mulation during the late phase of infection.

One of the important findings in the Paper II was the observation that ac-

cumulation of the infectious HAdV-C5 virions was clearly increased in ZNF622(KO) cells (Paper II, Fig. 3). The increase of the infectious virions can be due to better virus release from cell (i.e., virus in the growth media) or by enhanced formation of the virions (i.e., virus in the intact cells). Based on our results we favour the latter scenario as higher levels of infectious virus particles were detected both in the intact cells and cell growth media.

Formation of the new HAdV particles depends on a proper assembly of the viral capsid proteins with the virus DNA genome. This process is a very error-prone, since only a small fraction of virus particles can form infectious virus particles and hence infect/replicate in the recipient cells. This phenom-enon can be explained by comparing the physical virus titer (i.e., total num-ber of formed virus particles) and infectious virus titer (i.e., virus particles able to replicate in the recipient cells). In general, only 5-10% of the HAdV-C5 particles are infectious in the total virus pool [167]. Interestingly, our data show that HAdV-C5 purified from ZNF622(KO) cells was 7,1-fold more infectious compared to the virus isolated from the unmodified cells (See Fig. 9). This effect is not due to enhanced formation of the new virus particles, since the virus physical titer did not increase in a similar manner.

The increased virulence of the virus grown in the ZNF622(KO) cells may

have of great importance for virotherapy applications. Even a small im-provement of infectious HAdV titers will reduce the total virus amount needed to administer to the patients. Also the improved virus growth proto-col (e.g., using the ZNF622(KO) cells) can significantly reduce the costs for producing the infectious virus. These are important points, since low infec-tious titer and high purification costs of oncolytic HAdVs can hamper their extensive clinical usage [168].

Interestingly, this observation (i.e., enhanced virus lytic growth in U2OS(KO)) cells correlates well with enhanced pVII-Flag interaction on virus genome. Therefore, it is likely that the improved virus infectious titer is achieved due to a more efficient and/or specific-binding of the pVII protein to virus DNA within the virus particle. Since the enhanced pVII DNA bind-ing was not detected on cellular DNA (i.e., GAPDH promoter), we speculate that the ZNF622 is a potential anti-viral protein inhibiting proper accumula-tion of pVII on virus DNA. In addition, our study show that also the NPM1 protein is involved in pVII DNA binding regulation. Together, this has led to model (see Fig. 8) whereby formation of the trimeric ZNF622/NPM1/pVII protein complex has a negative impact on HAdV-C5 life cycle. Taken, to-gether, our data from Paper I and II can be summarized into the following model (Fig. 11).

44

Fig. 11. Comprehensive model for the interplay of pVII with the ZNF622 and MKRN1 proteins.

Paper III Recent reports have shown that the mtDNA can play an important role in innate immune response [142, 146, 147]. The results showing that both DNA virus (e.g., HSV-1, [143]) and RNA virus (e.g., DENV, [140]) infections can cause mtDNA release and induction of the immune response, clearly signify mtDNA as an important signaling molecule in some virus infections.

In the present paper, we show that mtDNA is accumulated in the cytosol of the cells infected with well-known DNA virus, HAdV-C5. Further, our data indicate that the accumulation of cyto-mtDNA partially depends on the pVII protein expression in the virus infected cells. Previously, the bovine adenovirus type 3 (BAdV-3) pVII protein was shown to directly interfere with the mitochondrial functions [169]. In line with that, our subcellular fractionation experiments showed that a small amount of the HAdV-C5 pVII protein can be found in the mitochondrial fraction in HeLa cells. However, pVII was not detected in mitochondrial fraction in U2OS cells although the same subcellular fractionation method was used. In spite of localization dif-ference between HeLa and U2OS cells, pVII detection in the HeLa mito-chondria fraction was always reproducible. This may indicate that pVII mi-

45

tochondrial localization in virus-infected cells might be cell type (i.e., HeLa) specific phenomena.

The pVII is an exclusively nuclear protein under immunofluorescence ex-

periments [79].This does not support the hypothesis that pVII directly inter-acts with the mitochondria. Further, the observation that transient expression of the pVII protein did not cause mtDNA release, may indicate an indirect role of the pVII protein in cyto-mtDNA release. Notably, the transient ex-pression experiments revealed that the expression of the E1B-19K consider-ably blocked cyto-mtDNA detection (Paper III, Fig. 4C). This was expected since E1B-19K is known to bind to the pro-apoptotic Bax proteins and thereby block the release of the mitochondrial inner components [170]. Hence, it is more likely that the pVII protein in connection with an addition-al viral protein may indirectly influence mtDNA release. In this regard it is interesting to note that also the pV protein levels were reduced in MKRN1(H307E) expressing cells. This is not surprising, since both, pVII and pV are nuclear proteins and form a complex in the infected cells [171]. Hence, it is possible that pVII, together with the pV protein, regulates the expression of one or several mitochondrial inner and outer membrane pro-teins, which regulate mitochondrial membrane permeability. For example, a recent study showed that expression of the pro-apoptotic BAX and BAK proteins can cause mitochondrial inner and outer membrane permeabilisa-tion, which in turn leads to mtDNA release [143]. Our RNA sequencing experiment revealed that the pVII protein expression clearly alters host gene expression. Therefore, it remains to be validated if expression of the mito-chondrial membrane proteins is altered in the HAdV-C5-infected cells. Fur-ther, it will be important to evaluate if pVII acts together with pV on repro-graming the host transcriptiome and what are the exact functions of these individual proteins regarding the mtDNA dynamics.

Another important question is why HAdV-C5-infection induces the re-

lease of mtDNA to cytosol. One possible explanation would be that the re-lease of mtDNA is needed to induce host cell immune response. This view is supported by the DENV-infection studies, which showed that cyto-mtDNA activates TLR9 signaling in dendritic cells followed by production of the IFNs [147]. A different strategy is used in HSV-1 infected cells, where re-leased mtDNA engages cGAS-STING pathway to drive ISG expression [146]. Interestingly, HAdV infection is recognized by the cGAS protein, which in turns leads to induction of the type I IFN anti-viral cascade [172]. An alternative explanation is that released mtDNA is needed for efficient virus growth. For example, cyto-mtDNA might be recognized as the signal to initiate or enhance virus release from the infected cells. Hence, further studies are needed in order to reveal the exact consequences of cyto-mtDNA in HAdV-C5-infected cells.

46

Finally, we show that the pVII protein increases the expression of pro-

inflammatory cytokine, IL-32. At present, we lack the data which can ex-plain the molecular mechanism how pVII regulates IL-32 expression. We speculate that pVII might be involved in the transcriptional control of IL-32 through direct DNA binding. It is important to note that IL-32 is involved in the infection of other viruses including EBV, HIV-1 and influenza virus [173-175]. Hence, one our working hypothesis is that is that IL-32 up-regulation may allow HAdV-C5 to maintain persistent infection based on similar role of IL-32 in maintenance of the EBV latency [175].

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Conclusions

Paper I (Fig. 11)

• Novel pVII interacting cellular proteins were identified • The MKRN1 E3 ubiquitin ligase specifically interacts with the

HAdV5-C5 pVII protein • The MKRN1 protein is degraded during HAdV-C5 infection • The pVII protein may induce MKRN1 degradation by enhanced

MKRN1 protein self-ubiquitination

Paper II (Fig.11)

• The ZNF622 protein accumulates and interacts with the pVII protein in HAdV-C5-infected cells

• The ZNF622 protein affects pVII binding to viral DNA • The ZNF622 protein forms an inhibitory complex with NPM1 and

pVII • Lack of ZNF622 enhances formation of infectious HAdV-C5 virions

Paper III (Fig. 10)

• mtDNA accumulates in the cytosol in HAdV-C5-infected cells • The pVII protein is partially involved in the cyto-mtDNA accumula-

tion • The pVII protein alone is not sufficient to induce mtDNA release • The pVII protein regulates expression of several immune response-

related genes

48

Acknowledgements

All the work was done in the Department of Medical Biochemistry and Mi-crobiology, Faculty of Medicine, Uppsala University. The current work was supported by Erasmus Mundus Lotus+ project and Swedish Cancer Society.

Here, I want to express my sincere gratitude to all the people who sup-ported and helped me to finish my doctoral study.

First of all, I want to express my gratitude to my main supervisor, Dr. Tanel Punga. I feel the extreme shortage of my wordings to fully express my appreciation towards your help, patience and inspiration. Throughout my 4 years’ journey to achieve being a Doctor of Philosophy (PhD), you were always there to help, encourage and inspire me. I think I would not have been able to accomplish anything if you were not there for me. Your opti-mistic attitude towards all the failure taught me an invaluable wisdom in my life. Thank you for being such an inspirational supervisor in the face of diffi-culties and frustration.

I want to thank my co-supervisor, Prof. Göran Akusjärvi for his uncon-ditional support and inspiration. Your critical comments on Friday seminars always have been precious inspiration to advance the projects.

I want to thank Prof. Catharina Svensson for all the support and invalu-able comments in Friday seminars.

I think I can never thank enough for the help of Dr. Anette Carlsson. Thank you for always being the right person whenever I needed help.

I want to express my gratitude to the mentor of my master studies, Dr. Daniel Öberg who firstly introduced me the exciting field of virus research. Thank you for being my first mentor in virus research and designing the recombinant adenovirus, which became an indispensible tool for my doctoral studies.

I want to thank Dr. Raviteja Inturi for helping me with all the tips and tricks in the lab. I am also thankful for the happiness, encouragement and gossip you brought to the lab whenever I was depressed by the failed exper-iments.

I want to thank Dr. Mahmoud Darweesh for sharing ample knowledge on immunology and providing inspiration for my project.

I am grateful for the constructive criticism and inspiration from Prof. Göran Magnusson, Dr. Anders Bergqvist, Dr. Wael Kamel, Priya Devi, Dr. Mahesh Anagandula, Amanda Westergren Jakobsson, Jianxiang (Gegen) Wang and Benjamin Danish on Friday seminars.

49

I want to thank Alexis Fuentes for all the support with my PhD studies. I also want to thank IMBIM administrative staff Veronica Hammar, Rehné Åkerblom and Malin Strömbom for all the support and help.

I want to thank the former adenovirus group members Dr. Ming Hu, Dr.Chengjun Wu, Dr. Susan Lan and Dr. Farzaneh Assadin for sharing a joyful memory in the lab.

I want to thank Aida Paivandy, Viktor Ek, Carl-Fredrik Johnzon, Christopher von Beek, Xinran Zhao, Fabio Rabelo Melo, Mirjana Gru-jic, Lu Zhang, Sultan Alanazi, Ann-Marie Gustafson, Sebastin San-thosh, Marco Maccarana, Eva Skovajsova and Abdul Alim for all the help and encouragement in the B9:3 corridor.

I want to thank Ulrica Ouline and the staff at the international office of Uppsala University for all the support.

I want to thank Yanjun Zan, Yanyu Zhang, Kehuan Wang, Tilman Rönneburg, Niki Sarri, Deepesh Kumar Gupta, Lei Chen, Yunzhou Yang, Shreya Singh Pundir, Liangwen Liu, Zaiqiu Cao, Melanie Herre, Wangshu Jiang, Zhirong Fu, Anahita Hamidi, Jinfen Wang, Weihua Ye, Husam Alsarraf, Elin Karlsson and Emma Åberg for being helpful and cheerful colleagues outside the lab.

I want to express my special thanks to Chandra Sekhar Mandava (Chandu) and Geeta Mandava for your kindness.

Last, I want to thank my best Korean friend Chang-il Kim for sharing all the ups and downs of our lives in Sweden.

50

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