The role of SAMHD1 in restriction and immune sensing of and retroelements

Die Rolle von SAMHD1 in der Restriktion und

Immunerkennung von Retroviren und Retroelementen

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Alexandra Herrmann

aus Biberach an der Riß

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 31.07.2018

Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer

Gutachter: Prof. Dr. Lars Nitschke

Prof. Dr. Manfred Marschall

Table of content

Table of content

I. Summary ...... 1

I. Zusammenfassung ...... 3

II. Introduction ...... 5

1. The human immunodeficiency virus ...... 5 2. Transposable elements ...... 7 3. Host restriction factors ...... 10 4. The restriction factor SAMHD1 ...... 11 5. Involvement of SAMHD1 in autoimmunity and antiviral responses ...... 14 6. The cGAS/STING pathway of cytosolic DNA sensing ...... 15 III. Objectives ...... 19

1. The role of SAMHD1 in retroviral infection ...... 19 2. The role of SAMHD1 in the inhibition of endogenous retroelements ...... 19 IV. Material and Methods ...... 21

1. Materials ...... 21 1.1. Chemicals...... 21 1.2. Buffers, solutions, and media ...... 21 1.2.1. ...... 21 1.2.2. Bacterial culture ...... 21 1.2.3. Standard buffers and solutions ...... 21 1.2.4. methods ...... 22 1.2.5. Virus preparation ...... 22 1.2.6. LEAP assay ...... 22 1.3. Vectors ...... 23 1.3.1. Empty vectors ...... 23 1.3.2. SAMHD1 expression vectors ...... 23 1.3.3. Expression vectors for retroelements ...... 25 1.3.4. Vectors for virus preparation ...... 25 1.3.5. Others ...... 26 1.4. Oligonucleotides ...... 26 1.4.1. Cloning of SAMHD1 mutants ...... 26 1.4.2. Cloning of LINE-1 expression vectors ...... 27 1.4.3. LEAP assay ...... 28 1.4.4. cDNA synthesis and qRT-PCR ...... 28 1.4.5. Genotyping...... 28 1.5. Antibodies...... 29 1.5.1. Primary antibodies ...... 29 Table of content

1.5.2. Secondary antibodies ...... 30 1.5.3. Direct antibody ...... 30 1.6. Enzymes...... 30 1.7. Biological materials ...... 31 1.7.1. Cell lines ...... 31 1.7.2. Bacterial strains ...... 31 1.7.3. Mouse strains ...... 32 2. Methods...... 32 2.1. Cell culture ...... 32 2.1.1. Bone marrow preparation and BMDC isolation ...... 32 2.2. methods and transfections ...... 33 2.2.1. Cloning and plasmid preparation ...... 33 2.2.2. RNA preparation ...... 33 2.2.3. cDNA synthesis and qRT-PCR ...... 33 2.2.4. Transfections ...... 34 2.3. Virus preparations ...... 35 2.3.1. HIV-GFP ...... 35 2.3.2. HIV-Luc ...... 35 2.3.3. shRNA-encoding lentiviral particles: ...... 35 2.4. Infection assays ...... 36 2.4.1. Infection of THP-1 cells with HIV-GFP ...... 36 2.4.2. Infection of THP-1 cells with HIV-Luc ...... 36 2.4.3. Infection of mBMDCs with HIV-GFP ...... 36 2.4.4. Infection of mBMDCs for RNA isolation ...... 36 2.5. Transcriptome analysis of murine BMDCs ...... 37 2.6. Retrotransposition assays ...... 37 2.6.1. GFP-based retrotransposition assay ...... 37 2.6.2. Neomycin-based retrotransposition assays ...... 37 2.7. LINE-1 assay ...... 38 2.8. LEAP assay ...... 38 2.9. ...... 39 2.9.1. Preparation of cell lysates ...... 39 2.9.2. Co-immunoprecipitation ...... 39 2.9.3. SDS-Polyacrylamide (SDS-PAGE) and western blot ...... 40 2.10. Indirect immunofluorescence staining ...... 40 2.11. Intracellular dNTP quantifications ...... 41 2.12. Genotyping ...... 41 V. Results ...... 43

1. The role of SAMHD1 in retroviral restriction and sensing ...... 43 1.1. SAMHD1 and the cGAS/STING pathway restrict HIV-GFP infection in non-cycling human THP-1 cells ...... 43 Table of content

1.2. SAMHD1, signaling through the IFN-I receptor, and the cGAS/STING pathway impair HIV-GFP infection in primary murine BMDCs ...... 45 1.3. HIV-GFP infection induces the expression of different in wt and SAMHD1 KO BMDCs...... 47 1.4. Spontaneously elevated transcripts are further upregulated upon HIV-GFP infection in the absence of SAMHD1 ...... 50 1.5. Analysis of highly upregulated genes in SAMHD1 KO BMDCs upon HIV-GFP infection ..... 51 1.6. SAMHD1- and IFNAR-dependent regulation in HIV-GFP infected BMDCs ...... 54 1.7. Analysis of genes which are directly induced upon HIV-GFP infection in murine BMDCs ...... 56 1.8. Validation of transcriptome analysis results by quantitative RT-PCR ...... 58 2. The role of SAMHD1 in the inhibition of endogenous retroelements ...... 59 2.1. Inhibition of LINE-1, but not HIV-1, is regulated by phosphorylation of SAMHD1 at T592 in cycling cells ...... 59 2.2. SAMHD1 inhibits various transposable elements in cycling cells ...... 66 2.3. Restriction of LINE-1 depends on an enzymatically active SAMHD1 and an intact allosteric binding site ...... 70 2.4. Murine SAMHD1 potently restricts retrotransposition of human LINE-1 ...... 73 2.5. Nuclear localization of SAMHD1 is not required for LINE-1 inhibition ...... 75 2.6. The dNTPase activity of SAMHD1 is not sufficient for LINE-1 restriction ...... 76 2.7. SAMHD1 does not affect LINE-1 expression ...... 79 2.8. SAMHD1 does not impair LINE-1 reverse in vitro ...... 81 2.9. SAMHD1 directly interacts with ORF2p in functional LINE-1 RNPs ...... 82 2.10. SAMHD1 does not promote the accumulation of LINE-1 RNPs in cytoplasmic stress granules...... 85 VI. Discussion ...... 90

1. The role of SAMHD1-dependent restriction and sensing in mice ...... 90 2. The role of SAMHD1 in the inhibition of endogenous retroelements ...... 96 VII. Abbreviations ...... 104

VIII. References ...... 106

IX. Appendix ...... 120

1. Supplementary information ...... 120 2. Curriculum vitae ...... Fehler! Textmarke nicht definiert. 3. Publications ...... 145 4. Contributions to national and international conferences ...... 145 4.1. Oral presentations ...... 145 4.2. Poster presentations ...... 145 5. Acknowledgement ...... 147

Summary

I. SUMMARY

The dNTPase SAMHD1 reduces the intracellular dNTP pool and thereby most likely restricts HIV•1 infection in non-cycling cells. Furthermore, mutations in samhd1 are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS), which is characterized by elevated IFN•I levels in the absence of viral infection. In AGS, loss of SAMHD1 function is thought to cause accumulation of nucleic acids derived from replicating retroelements, which are subsequently detected by intracellular immune sensors like the cytosolic DNA receptor cGAS. Within the present study, the endogenous as well as the HIV-1-induced immune responses in the absence of SAMHD1 were analyzed by infecting BMDCs isolated from wt, SAMHD1, IFNAR, and IFNAR/SAMHD1 KO mice with VSV-G-pseudotyped HIV-GFP reporter virus. An enhanced infectivity in double KO cells compared to SAMHD1 or IFNAR single KO cells was found, thereby demonstrating an IFN-dependent antiviral block in the absence of SAMHD1. Moreover, the transcriptome of HIV-GFP infected wt, SAMHD1, IFNAR, and IFNAR/SAMHD1 double KO cells was compared to that of uninfected cells. Without viral challenge, enhanced ISG transcript levels in cells lacking SAMHD1 were found, of which the majority was also further upregulated upon infection. Analysis of IFNAR/SAMHD1 double KO cells confirmed that this upregulation is IFN- dependent. These findings strongly suggest that most of the upregulated genes play a role in the autoimmune and the antiviral response and indicate that the same intracellular sensor is responsible for both immune reactions. To analyze the involvement of cGAS and the downstream signaling partner STING in sensing of HIV-1, infection studies of SAMHD1-competent or deficient BMDCs lacking cGAS or STING were performed. An enhanced infectivity of cGAS/SAMHD1 and STING/SAMHD1 double KO cells was found, suggesting that SAMHD1 prevents the induction of an anti-HIV immune response through the cGAS/STING pathway and highlights the involvement of cGAS as a sensor for HIV-1 infection. Furthermore, SAMHD1 restricts the retrotransposition of endogenous LINE-1 () retroelements. The replication of L1 in the absence of SAMHD1 has been suggested as a trigger for AGS. In contrast to HIV-1, SAMHD1 is active against L1 in cycling cells. The present study revealed that in agreement with its antiviral activity, the anti-L1 activity of SAMHD1 is also regulated by phosphorylation at threonine 592 (T592) and depends on the enzymatically active site of SAMHD1 as well as on the allosteric dGTP-binding site. Quantification of intracellular dNTP level revealed that the dNTPase activity is unaffected by phosphorylation at T592, thus indicating that the dNTP depletion is necessary but not sufficient for L1 restriction. Interestingly, co-immunoprecipitation experiments revealed that SAMHD1 directly interacts with functional L1 RNPs and that this interaction seems to be regulated by T592- phosphorylation. These findings suggest that SAMHD1 restricts L1 through locally-restricted dNTP degradation in close proximity to the L1 RT. Conclusively, the data provided within this thesis highlight the role of SAMHD1 in antiviral and autoimmune responses and also demonstrate that SAMHD1 contributes to genome stability by restricting endogenous retroelements like L1.

1

2 Zusammenfassung

I. ZUSAMMENFASSUNG

Die dNTPase SAMHD1 verhindert die retrovirale Infektion von ruhenden Zellen höchstwahrscheinlich durch Abbau intrazellulärer dNTPs. Darüber hinaus stehen Mutationen in samhd1 mit der Autoimmunerkrankung Aicardi-Goutières-Syndrom (AGS) in Verbindung, für die erhöhte IFN-Level in Abwesenheit einer viralen Infektion charakteristisch sind. Es wird vermutet, dass die Abwesenheit von funktionellem SAMHD1 im AGS zu einer Akkumulation von endogenen Nukleinsäuren führt, welche durch intrazelluläre Immunsensoren wie den zytosolischen DNA-Rezeptor cGAS erkannt werden. Innerhalb dieser Arbeit wurde die endogene sowie die virusinduzierte Immunantwort durch Infektion von BMDCs aus wt, SAMHD1, IFNAR und IFNAR/SAMHD1 KO-Mäusen mit einem VSV-G-pseudotypisierten HIV-GFP-Reportervirus untersucht. Es wurde eine erhöhte Infektiösität von IFNAR/SAMHD1 Doppel-KO- im Vergleich zu SAMHD1 oder IFNAR KO-Zellen gefunden, was einen IFN-abhängigen antiviralen Block in Abwesenheit von SAMHD1 beweist. Zudem wurde das Transkriptom von infizierten wt, SAMHD1, IFNAR und IFNAR/SAMHD1 Doppel-KO-Zellen mit dem von nicht infizierten Zellen verglichen. In Abwesenheit einer viralen Infektion wurden in Zellen ohne SAMHD1 hochregulierte ISG- Transkriptlevel nachgewiesen, von denen die Mehrheit durch Infektion noch weiter erhöht wurde. Die Analyse von IFNAR/SAMHD1 Doppel-KO-Zellen zeigte, dass diese Hochregulation IFN- abhängig ist. Zusammenfassend lassen diese Ergebnisse darauf schließen, dass zum Großteil dieselben Gene in der endogenen und der virusinduzierten Immunantwort beteiligt sind. Um die Beteiligung von cGAS sowie des Adapterproteins STING zu untersuchen wurden Infektionsversuche in SAMHD1-kompetenten oder -defizienten Zellen durchgeführt, denen cGAS bzw. STING fehlt. Es wurde eine erhöhte Infektiösität von cGAS/SAMHD1 und STING/SAMHD1 Doppel-KO-Zellen festgestellt, was darauf schließen lässt, dass SAMHD1 das Auslösen einer Immunantwort durch den cGAS/STING-Signalweg verhindert. Zusätzlich zu HIV-1 restringiert SAMHD1 außerdem die Retrotransposition von endogenen LINE-1 (L1) Retroelementen. Die Replikation von L1 in Abwesenheit von SAMHD1 wird als Auslöser für AGS diskutiert. Diese Arbeit zeigt, dass L1 im Gegensatz zu HIV-1 in sich teilenden Zellen gehemmt wird. Übereinstimmend mit der antiviralen Aktivität wird die anti-L1-Aktivität von SAMHD1 über Phosphorylierung an Threonin 592 (T592) reguliert und benötigt ein intaktes aktives Zentrum sowie intakte allosterischen dGTP-Bindungsstellen. Eine Quantifizierung intrazellulärer dNTP- Spiegel zeigte, dass die dNTPase-Aktivität nicht durch Phosphorylierung an T592 beeinflusst wird. Dies deutet darauf hin, dass dNTP-Abbau nötig, aber nicht ausreichend für die L1- Restriktion ist. Interessanterweise zeigten Ko-Immunopräzipitationsexperimente, dass SAMHD1 mit funktionalen L1-RNPs interagiert und dass diese Interaktion sehr wahrscheinlich über Phosphorylierung reguliert wird. Diese Ergebnisse deuten darauf hin, dass SAMHD1 L1 inhibiert, indem es lokal begrenzt die dNTPs in der unmittelbaren Umgebung der L1-RT abbaut. Schlussfolgernd heben die in dieser Arbeit dargestellten Daten die Rolle von SAMHD1 in der antiviralen und Autoimmunantwort hervor und machen deutlich, dass SAMHD1 zum Erhalt der Genomstabilität beiträgt, indem es endogene Retroelemente wie L1 inhibiert. 3

4 Introduction

II. INTRODUCTION

1. The human immunodeficiency virus

The acquired immunodeficiency syndrome (AIDS) was first described in 1981 (Gottlieb et al., 1981; Masur et al., 1981; Siegal et al., 1981) and defines an impairment of the immune system, which allows opportunistic infections to become -threatening. Later, the human immunodeficiency virus (HIV) was identified as the causative agent of AIDS (Barre-Sinoussi et al., 1983; Popovic et al., 1984). HIV is part of the family Retroviridae and belongs to the genus Lentivirus. HIV can be divided into two groups, HIV-1 and HIV-2 (reviewed in Nyamweya et al., 2013). HIV-1 is more infectious, more virulent, and the cause of the vast majority of HIV infections globally. In contrast, HIV-2 is less pathogenic, less infectious, and largely confined to West Africa. Until today, HIV-1 infection is a global health burden. According to the World Health Organization (WHO), approximately 37 million people worldwide were living with HIV-1 in 2016. Since the start of the epidemic, almost 76 million people have become infected particularly in low and middle socio-economic countries (http://www.who.int/hiv/data/en/). After infection, HIV can be spread sexually or via blood products. Moreover, transmission from an infected mother to her child during pregnancy and childbirth as well as by breast feeding can occur.

HIV virions are membrane-enveloped, approximately 100 nm in diameter, and contain two copies of positive-sensed single-stranded (ss) genomic RNA. The HIV RNA is about 9.2 kb in length and encodes the canonical open reading frames (ORFs) gag (group-specific antigen), pol (polymerase), and env (envelope), as well as regulatory and accessory . The regulatory proteins Tat (transactivator of transcription) and Rev (regulator of expression of viral proteins) are essential for viral replication. The accessory proteins Vif (viral infectivity factor), Vpu (viral protein U), Vpr (viral protein R), and Nef (negative factor) are dispensable for replication in many in vitro systems but are highly advantageous for viral replication and pathogenesis in vivo (reviewed in Malim and Emerman, 2008). The Gag polyprotein consists of the structural proteins Matrix (MA, p17), Capsid (CA, p24), and Nucleocapsid (NC, p7), as well as the p6 protein. The Gag and Gag-Pol precursors are cleaved during virus maturation by the viral protease. MA proteins mediate targeting of Gag polyproteins to the plasma membrane and form an inner shell beneath the viral membrane after cleavage. CA proteins form the conical capsid, which is characteristic for mature, infectious virions and surrounds the nucleocapsid. NC proteins regulate packaging and condensation of viral RNA into viral particles. The p6 protein is important for facilitating virus release. Additionally, in case of HIV•1, p6 mediates incorporation of Vpr into virions (Kondo and Gottlinger, 1996). In contrast, HIV-2 incorporates the viral accessory protein X (Vpx) instead of Vpr (Accola et al., 1999). The pol ORF encodes the viral enzymes reverse transcriptase (RT), integrase (IN),

5 Introduction and protease (PR). Env encodes the viral envelope glycoprotein gp160, which is cleaved by a cellular protease into the subunits gp120 and gp41. Env forms trimers consisting of a cap made of three molecules of gp120 and a stem of three gp41 molecules.

Figure 1: Life cycle of HIV-1. Infection of a target cell starts with attachment and binding of the viral envelope glycoprotein (Env) to the CD4 receptor and one of the co-receptors CCR5 or CXCR4 (step 1). Binding to both receptors results in fusion of viral and cellular membranes (step 2) and release of the viral capsid into the cytoplasm. Within the cytoplasm, uncoating (step 3) of the viral capsid and reverse transcription of the genomic ssRNA into dsDNA (step 4) take place. The newly formed pre-integration complex (PIC) is actively transported into the nucleus through nuclear pores (step 5) where the viral cDNA is integrated into the host genome (step 6). Transcription of the provirus is mediated by the host RNA polymerase II (RNA pol II, step 7). Viral mRNAs are spliced and exported into the cytoplasm (step 8), where of new viral proteins occurs (step 9). Protein components are assembled to new viral particles and full-length RNA is incorporated into the particles (step 10). After budding and release of still immature particles (step 11 and 12), the viral protease is activated, resulting in mature, infectious viral particles which can infect new target cells (step 13). Several steps of the viral life cycle can be targeted by clinical inhibitors (white boxes) or cellular restriction factors (blue boxes). NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non- nucleoside reverse transcriptase inhibitor, NSTI, integrase strand transfer inhibitor; LTR, long terminal repeat. Adapted and modified from Engelman and Cherepanov, 2012.

To infect its target cells HIV enters the cell via interaction of Env with the CD4 receptor and one of the co-receptors CCR5 or CXCR4 (Fig. 1). Target cells include important cells of the host immune system like CD4+ T lymphocytes, macrophages (MACs), dendritic cells (DCs), monocytes, and microglia. After entry into the cell, reverse transcription of the genomic viral ssRNA into double-stranded (ds) complementary (c) DNA takes place. After nuclear import, the viral cDNA is integrated into the host genome. Transcription of the viral genome and export of newly synthesized mRNA into the cytoplasm is supported by the regulatory proteins Tat and Rev. Subsequently, translation and assembly of viral proteins into new viral particles

6 Introduction takes place. After budding, the viral protease is activated and cleaves the Gag and Gag-Pol polyproteins, resulting in mature, infective virions which can infect new target cells.

By now, no cure for HIV infection is available. However, according to the WHO, about 21 million infected individuals are currently receiving anti-retroviral therapy (ART) (http://www.who.int/hiv/en/). Due to this therapy, life expectancy of infected patients is increasing by supporting the immune system and preventing opportunistic infections. Anti- HIV drugs target different steps of the viral life cycle. Different classes include fusion inhibitors, co-receptor antagonists, nucleoside RT (NRTI) and non-nucleoside RT inhibitors (NNRTI), integrase inhibitors, and protease inhibitors. These are usually given as combination therapy to prevent the development of resistances. Until today, the design of an effective HIV-1 vaccine was not successful. Despite combination therapy, resistances to ART as well as the HIV reservoir in resting cells represent major problems. Furthermore, cells have evolved intrinsic mechanisms to protect themselves, including cellular restriction factors that are widely expressed.

2. Transposable elements

Approximately 45 % of the is derived from transposable elements (TEs) (Lander et al., 2001). TEs are classified into DNA transposons and retrotransposons. DNA transposons replicate by a ‘cut and paste’ mechanism with a DNA intermediate. Replication is mediated by the encoded enzyme transposase. DNA transposons comprise only about 3 % of the human genome (Lander et al., 2001). Retrotransposons replicate by a ‘copy and paste’ mechanism with an RNA intermediate that is reverse transcribed into cDNA and inserted into the genome at a new location. Retrotransposons are further divided into LTR and non-LTR retrotransposons. LTR retrotransposons are also known as ‘human endogenous retroviruses’ (HERVs) and resemble retroviruses in both their genome organization and mobility mechanism. They encode a minimum of two genes, gag and pol. Expression of both genes is sufficient for replication. Their coding region is flanked by two long-terminal repeats (LTRs), of which the 5’ LTR contains an internal promoter for the cellular RNA polymerase II. They comprise approximately 8 % of the human genome (Lander et al., 2001) and today are apparently immobile in humans. Non-LTR retrotransposons consist of two sub-types, long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs are autonomous elements that encode proteins to mediate their own mobility (reviewed in Beck et al., 2011). The most prominent representative is LINE-1 (L1), which comprises about 17 % of the human genome (Lander et al., 2001). In contrast, non-autonomous SINEs rely on proteins encoded by other elements to achieve replication within the host. The most frequent SINEs are Alu elements, which make up about 10 % of the human genome (Lander et al., 2001). Active Alu elements are approximately 280 bp in

7 Introduction length and end in an A-rich tail. The left monomer contains an internal RNA polymerase III promoter and is separated from the right monomer by an A-rich sequence (reviewed in Batzer and Deininger, 2002). In addition to Alu elements, up to 2700 copies of so called SINE/VNTR/Alu (SVA) elements exist within the human genome (Wang et al., 2005).

Retrotransposition-competent L1s are approximately 6 kb in length and contain a 5’ untranslated region (UTR), two ORFs in sense (ORF1 and 2), one ORF (ORF0) in antisense orientation, and a 3’ UTR followed by a poly(A) tail. Compared to retroviruses and LTR retrotransposons, which use an LTR promoter, the 5’ UTR of L1 harbors an internal RNA polymerase II promoter (Swergold, 1990). Moreover, the 5’ UTR also contains an antisense promoter that is responsible for transcription of adjacent cellular genes and the recently identified ORF0 (Denli et al., 2015; Speek, 2001). The primate-specific ORF0 is supposed to enhance L1 mobility. ORF1 encodes a 40 kDa protein (ORF1p) that is required for retrotransposition (Holmes et al., 1992). ORF1p contains nucleic acid chaperone activity, which is important for L1 integration into the genome (Martin and Bushman, 2001). The protein encoded by ORF2 (ORF2p) has a size of about 150 kDa and harbors endonuclease (EN) and reverse transcriptase (RT) activities that are critical for retrotransposition (Feng et al., 1996; Mathias et al., 1991; Moran et al., 1996). After transcription from the host genome and translation, ORF1p and ORF2p preferentially associate with their encoding RNA to form ribonucleoprotein (RNP) particles, a process that is known as cis preference (Kulpa and Moran, 2006). However, L1 encoded proteins can also act in trans to promote retrotransposition of truncated L1s, Alu elements, and other cellular mRNAs, thereby resulting in processed pseudogenes (Wei et al., 2001). After entry of the RNPs into the nucleus, retrotransposition occurs by a process called target-primed reverse transcription (TPRT) (Luan et al., 1993). The ORF2p-encoded EN induces a nick in the genomic target DNA, resulting in a free 3’ OH group that primes negative strand cDNA synthesis, whereas the retroelement RNA serves as template for the ORF2p-encoded RT. The other host DNA strand is cleaved by EN or host enzymes and serves as primer for second strand DNA synthesis. Either ORF2p-encoded RT or host enzymes synthesize the second strand and seal the gaps with the adjacent genomic DNA. During the process of TPRT, the L1 EN induces DNA double-strand breaks (DSBs) in the target DNA (Gasior et al., 2006) which can lead to activation of DNA damage responses and cell cycle arrest. Although initially most retrotransposition was thought to occur in the germline, somatic insertions of L1 were found in a variety of tumors and in the brain (reviewed in Beck et al., 2011; Hancks and Kazazian, 2016).

8 Introduction

Figure 2: LINE-1 retrotransposition cycle. Full-length LINE-1 (L1) (blue bar on the left ) is transcribed from the host genome and L1 mRNA is exported into the cytoplasm, where translation of ORF1p (green circle) and ORF2p (yellow circle) takes place. L1 proteins preferentially associate with their own mRNA to form ribonucleoprotein complexes (RNPs). L1 RNPs are transported into the nucleus where retrotransposition occurs by target-primed reverse transcription (TPRT), resulting in insertion of a new L1 copy at a new genomic location (dark red bar at the right chromosome). L1 replication can be blocked at various steps during the retrotransposition cycle by different cellular mechanisms (blue boxes) and host restriction factors (dark red boxes).

L1 is thought to be highly active during early embryogenesis, but epigenetic silencing by promoter methylation occurs early in development (Burden et al., 2005). In addition, L1 replication is regulated by RNA silencing (Yang and Kazazian, 2006), microRNAs like mir•128 (Hamdorf et al., 2015), post-translational modifications such as phosphorylation of ORF1p (Cook et al., 2015), and subcellular localization (Goodier et al., 2007) (Fig. 2). Up to 5 % of newborn children are estimated to have a new retrotransposition insertion (Cordaux et al., 2006) and 124 known human disease-causing insertions of L1, Alu, and SVA have been identified so far (reviewed in Hancks and Kazazian, 2016). Therefore, a better understanding of retrotransposition represents a crucial field of research to limit new disease-causing insertions. Within the last years, increasing numbers of host restriction factors have been identified that inhibit retrotransposition. In most cases, these host factors have been previously described to be active against exogenous retroviruses like HIV-1.

9 Introduction

3. Host restriction factors

During their replication cycle, retroviruses interact with both supportive and inhibitory host factors. As a first-line defense, host restriction factors represent inhibitory factors, which potently block HIV-1 replication by intervening with various replication steps. It has been suggested that restriction factors fulfill four hallmarks (Harris et al., 2012): (1) They have to directly and dominantly cause a significant decrease in viral infectivity, (2) they are counteracted by viral evasion mechanisms, (3) they show signs of rapid evolution, and (4) their expression can be strongly induced by (IFNs), thereby tightly linking their activity with the host’s immune system. However, some restriction factors are already constitutively expressed to high levels. Over the past years, several proteins with anti-HIV activity have been identified. Interestingly, many of them not only inhibit the replication of exogenous viruses like HIV-1, but were also found to restrict endogenous transposable elements. These factors include apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) family members, Tetherin, Mx2, SAMHD1, and others.

APOBEC3G (A3G) belongs to a cytidine deaminase family, which converts cytosine (C) to uracil (U). Editing during reverse transcription of the genomic HIV-1 RNA leads to G to A hypermutations in the newly synthesized minus-strand cDNA (Mangeat et al., 2003). The viral accessory protein Vif counteracts A3G and excludes it from virions by proteasomal degradation (Goila-Gaur and Strebel, 2008; Sheehy et al., 2002; Yu et al., 2003). Interestingly, all APOBEC3 family members also inhibit L1 retrotransposition to varying degrees, with APOBEC3A (A3A) and APOBEC3B (A3B) being most effective (Kinomoto et al., 2007; Koito and Ikeda, 2011). Intriguingly, the deaminase activity is not responsible for inhibition of retrotransposition (Horn et al., 2014; Kinomoto et al., 2007; Liang et al., 2016), indicating that APOBEC3 proteins inhibit HIV-1 and L1 replication through distinct mechanisms. Tetherin, also known as BST-2, is an IFN-inducible membrane protein that efficiently blocks release of virions by directly tethering them to the cell surface of the virus- producing cells. The antiretroviral activity of Tetherin is counteracted by the accessory protein Vpu of HIV-1 (Van Damme et al., 2008). Recently, Tetherin was found to reduce L1 retrotransposition in cell culture (Goodier et al., 2015). However, the mechanism of L1 restriction remains unknown. In addition to tethering viral particles to the plasma membrane, Tetherin was found to activate the NF-κB pathway (Tokarev et al., 2013). This signaling function of Tetherin might account for the inhibition of L1 by induction of pro-inflammatory cytokines. Mx2 is an IFN-induced GTPase that may target retroviral capsids or prevent the integration of proviral DNA into the host genome (Goujon et al., 2013; Kane et al., 2013). In addition to HIV-1, Mx2 has been shown to inhibit L1 retrotransposition (Goodier et al., 2015). For both the exact molecular mechanism of restriction is still unknown. Another important restriction factor for HIV-1 is the tripartite motif-containing protein 5α (TRIM5α). TRIM5α

10 Introduction recognizes incoming viral capsids and functions as a signal molecule that activates AP-1 and NF-κB signaling pathways (Pertel et al., 2011; Stremlau et al., 2004). TRIM5α proteins spontaneously assemble into hexagonal lattices on top of the viral capsid, thereby accelerating viral uncoating and blocking viral replication (Ganser-Pornillos et al., 2011). Interestingly, TRIM5α is not counteracted by any of the viral accessory proteins. Instead, viral escape occurs by adapting the amino acid composition of the capsid, thereby preventing binding of TRIM5α. Noteworthy, no restriction of transposable elements by TRIM5α has been reported so far. The SAM and HD domain-containing protein 1 (SAMHD1) has been identified as the major restriction factor for HIV-1 in non-dividing myeloid and resting CD4+ T cells, which is counteracted by the viral accessory protein Vpx of HIV-2 and some simian immunodeficiency viruses (SIVs) (Baldauf et al., 2012; Hrecka et al., 2011; Laguette et al., 2011). SAMHD1 is thought to inhibit reverse transcription of HIV-1 and other viruses by reducing the intracellular dNTP pool (Lahouassa et al., 2012). In addition to HIV-1, SAMHD1 was found to inhibit the replication of L1 and other retroelements (Hu et al., 2015; Zhao et al., 2013). Moreover, the zinc finger antiviral protein (ZAP), the ATP-dependent RNA helicase Moloney leukemia virus 10 (MOV10), the endoribonuclease RNaseL, and others were reported to have an impact on both HIV-1 and L1 replication. The growing list of cellular restriction factors that are able to protect the host from both exogenous and endogenous viral threats highlights their importance as part of the intrinsic immune response.

4. The restriction factor SAMHD1

SAMHD1 acts as a deoxynucleoside triphosphate triphosphohydrolase (dNTPase) that converts cellular dNTPs into the corresponding nucleosides (dNs) and inorganic triphosphate (Goldstone et al., 2011; Powell et al., 2011). Together with the de novo synthesis of dNTPs by the cellular enzyme ribonucleotide reductase, SAMHD1-mediated degradation of dNTPs contributes to the cell cycle-dependent regulation of intracellular dNTP levels (Franzolin et al., 2013). In non-cycling cells, SAMHD1 is thought to restrict retroviral infection by reducing the intracellular dNTP pool below the threshold that is necessary for efficient reverse transcription (Lahouassa et al., 2012). Human SAMHD1 comprises 626 amino acids and contains an N-terminal nuclear localization signal (NLS), a sterile alpha motif (SAM) and a histidine-aspartate (HD) domain, followed by a C-terminal Vpx-binding site (Fig. 3A).

11 Introduction

Figure 3: Structure of SAMHD1. (A) SAMHD1 consists of 626 amino acids and contains an N•terminal nuclear localization signal (NLS), a sterile alpha motif (SAM), and the enzymatic active site- containing histidine-aspartate (HD) domain. The C-terminal region of SAMHD1 harbors the important phosphorylation site at threonine 592 (T592), where SAMHD1 is phosphorylated by the cell cycle- dependent kinases (CDKs) 1 and 2 together with CyclinA2. Phosphorylation at T592 regulates its antiviral activity. Additionally, SAMHD1 contains a binding site for the accessory protein Vpx of HIV-2 and several SIVs, which induces proteasomal degradation of SAMHD1. (B) Enzymatically active SAMHD1 is thought to form homo-tetramers. Each monomer contains two allosteric and one catalytic substrate binding site within the HD domain. Binding of GTP/dGTP to allosteric site 1 and binding of any dNTP to allosteric site 2 induces tetramerization, followed by conformational changes in the catalytic site. Thereby, substrate dNTPs access the catalytic site, resulting in the cleavage of dNTPs into dNs and triphosphate. Occupation of both allosteric sites of each monomer is required for oligomerization and full enzymatic activity. Adapted from Herrmann et al., 2016.

The SAM domain is one of the most common protein-protein interaction domains. It is dispensable for retroviral restriction, but is required for maximal enzymatic activity (Beloglazova et al., 2013; White et al., 2013a). The HD domain of SAMHD1 mediates oligomerization and nucleic acid binding (Goldstone et al., 2011; Goncalves et al., 2012; Koharudin et al., 2014; Zhu et al., 2013). Additionally, the HD domain harbors the enzymatically active site of SAMHD1. Interestingly, the HD domain alone is sufficient to achieve potent HIV-1 restriction (White et al., 2013a). Nuclear localization of SAMHD1, which is mediated by the N-terminal NLS, is not required for its enzymatic or antiviral activity (Brandariz-Nunez et al., 2012; Hofmann et al., 2012).

The highly conserved amino acids histidine (H) and aspartate (D) at positions 206 and 207 within the enzymatically active site of the HD domain are critical for the dNTPase activity (Goldstone et al., 2011). In addition to its dNTPase activity, SAMHD1 has been shown to bind nucleic acids, displaying a preference for RNA over DNA (Goncalves et al., 2012; Tungler et al., 2013). According to Beloglazova and colleagues, SAMHD1 acts as a 3’-5’ exonuclease that exhibits a preference for nucleic acids with complex secondary structures such as viral ssRNA (Beloglazova et al., 2013). Later on, Ryoo and colleagues confirmed this

12 Introduction finding and reported that the RNase activity of SAMHD1 but not its dNTPase activity is essential for HIV-1 restriction and that SAMHD1 directly degrades genomic HIV-1 RNA (Ryoo et al., 2014). Controversially, the group of James Stivers confirmed that SAMHD1 indeed binds nucleic acids but does not contain any nuclease activity that is associated with the enzymatic active site (Seamon et al., 2015). Therefore, the exact mechanism of restriction and the enzymatic activities of SAMHD1 are still controversial. Noteworthy, SAMHD1 does not only restrict diverse retroviruses but also DNA viruses like type 1 (HSV-1), vaccinia virus (VACV), or hepatitis B virus (HBV) (Chen et al., 2014; Gramberg et al., 2013; Hollenbaugh et al., 2013; Kim et al., 2013; Sommer et al., 2016). Since DNA viruses also depend on intracellular dNTPs to replicate their genome inside the host cell, the dNTPase activity of SAMHD1 would explain this broad antiviral activity.

Enzymatically active SAMHD1 forms tetramers, most likely by tight association of two homodimers (Goldstone et al., 2011; Koharudin et al., 2014; Zhu et al., 2013) (Fig. 3B). In addition to dGTP, GTP acts as allosteric cofactor inducing the oligomerization (Amie et al., 2013; Ji et al., 2014; Zhu et al., 2015). The cofactor-induced tetramerization has been reported to be critical for the dNTPase activity and restriction of retroviral infection (Brandariz-Nunez et al., 2013; Hansen et al., 2014; Yan et al., 2013).

Although SAMHD1 is expressed in both, cycling and non-cycling cells, its antiviral activity is limited to non-cycling cells. SAMHD1 is phosphorylated at several residues, of which threonine at position 592 (T592) is important for the antiviral function (Cribier et al., 2013; Welbourn et al., 2013; White et al., 2013b). Phosphorylation of SAMHD1 at T592 is mediated by CDK1 and CDK2 together with CyclinA2 in a cell cycle-dependent manner (Cribier et al., 2013; St Gelais et al., 2014; White et al., 2013b). In dividing cells, SAMHD1 is phosphorylated and therefore unable to restrict HIV-1 infection. In non-dividing cells, this phosphorylation is lost whereby SAMHD1 becomes active against HIV-1. Therefore, T592- phosphorylation alters the ability of SAMHD1 to restrict viral infection. However, the consequences of this regulatory modification on the dNTPase activity are still controversially discussed.

In addition to exogenous viruses, SAMHD1 also inhibits replication of endogenous transposable elements like L1, Alu, and SVA (Hu et al., 2015; Zhao et al., 2013). Interestingly, this inhibition occurs in dividing cells where SAMHD1 was thought to be inactive. Zhao and colleagues reported that SAMHD1 inhibits L1 retrotransposition through inhibition of ORF2p-mediated RT activity by reducing ORF2p levels independently of its enzymatically active site (Zhao et al., 2013). Later, Hu and colleagues found that SAMHD1 indeed diminishes L1 protein level but identified the enzymatically active site to be important for restriction. Moreover, they characterized a second activity of SAMHD1 to be required for

13 Introduction restriction, which causes sequestration of L1 in large cytoplasmic foci co-localizing with cellular stress granules (Hu et al., 2015). Collectively, the exact molecular mechanism of SAMHD1-mediated inhibition of endogenous retroelements remains still unclear.

5. Involvement of SAMHD1 in autoimmunity and antiviral responses

Mutations within the samhd1 gene are associated with the rare autoimmune disease Aicardi- Goutières syndrome (AGS) (Rice et al., 2009). The disease was first defined in 1984 by Jean Aicardi and François Goutières (Aicardi and Goutieres, 1984) and represents a rare genetic autoimmune encephalopathy whose features phenocopy congenital viral infections. The disease is characterized by an early onset progressive encephalopathy concomitant with elevated type-I IFN (IFN-I) production. AGS patients develop signs of autoimmunity and exhibit constitutive upregulation of ISGs in the peripheral blood (Rice et al., 2013). Although it is not clear how the loss of functional SAMHD1 causes AGS, most AGS-related mutations lead to the loss of nuclear localization and involve conserved residues within the HD domain (Goncalves et al., 2012). In addition to SAMHD1, AGS is also caused by loss-of-function mutations in the nucleic acid-metabolizing genes three prime repair exonuclease 1 (Trex1), the three subunits of RNaseH2, and ADAR, as well as by gain-of-function mutations of the dsRNA sensor IFI-induced helicase C domain-containing protein 1 (IFIH1) (Crow et al., 2006a; Crow et al., 2006b; Rice et al., 2009; Rice et al., 2012). Since the recognition of nucleic acids by cellular receptors leads to the production of IFN-I, aberrant sensing of nucleic acids has been proposed as mechanism for the development of AGS and other autoimmune diseases. However, the nature of the endogenous nucleic acids that trigger the immune response as well as the innate sensor that is responsible or the immune induction are still uncertain. Currently, accumulation of retroelement-derived DNA as well as DNA replication or DNA damage-dependent by-products are discussed as trigger for aberrant autoimmune responses (Li et al., 2017; Stetson et al., 2008). The accumulation of self-DNA in the cytoplasm is normally avoided by coordinate action of cellular nucleases like Trex1 or ADAR. In case of Trex1, protein dysfunction, like it is the case in AGS, leads to the accumulation of endogenous nucleic acids, which can be sensed by innate immune receptors, resulting in the induction of an IFN-I response. In addition to AGS patients, SAMHD1 knockout (KO) mice display endogenously upregulated ISG levels compared to wild-type mice in the absence of viral infection (Behrendt et al., 2013; Rehwinkel et al., 2013). SAMHD1 KO mice are viable and fertile, representing an ideal model to study the role of nucleic acid sensing pathways in the development of autoimmune diseases.

While HIV-1 encodes Vpr, HIV-2 and many SIV strains encode the accessory protein Vpx instead of Vpr. Vpx directly binds to the C-terminus of SAMHD1 and induces its proteasomal degradation by linking it to an E3 ligase complex (Hrecka et al., 2011; Laguette et

14 Introduction al., 2011). The presence of Vpx enables infection of normally non-permissive, SAMHD1- expressing cells like resting CD4+ T cells or MACs with HIV-1 (Baldauf et al., 2012; Hrecka et al., 2011; Laguette et al., 2011). Since HIV-1 does not encode Vpx or another accessory protein with equal function, HIV-1 cannot circumvent SAMHD1-mediated restriction. HIV-1 is thought to avoid counteraction of SAMHD1, because lower viral replication in presence of SAMHD1 prevents the induction of a strong immune response through ineffective triggering of innate sensors (reviewed in Herrmann et al., 2016). However, the exact role of SAMHD1 in retroviral sensing is still not fully understood yet.

Recently, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as the cryptic sensor that activates the IFN-I pathway in response to HIV-1 infection (Gao et al., 2013; Sun et al., 2013). Interestingly, Gao and colleagues found cGAS- dependent sensing of HIV-1 in SAMHD1-expressing cells (Gao et al., 2013). However, other groups detected HIV-1-induced immune responses only in the absence of SAMHD1 (Lahaye et al., 2013; Manel et al., 2010; Sunseri et al., 2011). The viral capsid represents a key regulatory factor of innate sensing pathways through its interaction with host factors like Cyclophilin A (CypA) and the cleavage and polyadenylation specific factor CPSF6 in monocyte-derived macrophages (MDMs) (Lahaye et al., 2013; Rasaiyaah et al., 2013). On the one hand, CypA prevents HIV-1 sensing before nuclear entry even in the presence of SAMHD1. On the other hand, CPSF6 recruitment also impedes premature reverse transcription and recognition of viral cDNA. However, prevention of these specific interactions of HIV-1 with cellular co-factors (e.g. by virus mutation or depletion of host co- factor expression), HIV-1 cDNA triggers an immune response (Rasaiyaah et al., 2013). Moreover, Lahaye and colleagues reported that only mutated HIV-1 is capable of activating the innate immune system (Lahaye et al., 2013). In contrast, sensing in monocyte-derived dendritic cells (MDDCs) occurs exclusively in the absence of SAMHD1, with newly synthesized capsid proteins being the trigger for an antiviral immune response (Manel et al., 2010). Taken together, future studies are necessary to solve the questions (1) whether wild- type HIV-1 is sensed at all, (2) whether cGAS is the innate sensor responsible for HIV-1 sensing, and (3) whether the absence of SAMHD1 is required for this immune induction or not.

6. The cGAS/STING pathway of cytosolic DNA sensing

Innate immune responses constitute a first line of defense against invading pathogens. They are initiated through activation of so-called pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) (reviewed in Medzhitov, 2007). Nucleic acid sensing represents a very fundamental strategy employed by the innate immune system to detect the presence of pathogens. Several nucleic acid sensing

15 Introduction receptors exist in mammals that activate different signaling pathways, finally resulting in the expression of IFN-I and pro-inflammatory cytokines. Pro-inflammatory cytokines are important for the recruitment of specialized immune cells to the site of infection. Cell intrinsic IFN-I induction establishes an antiviral state by launching expression of ISGs including host restriction factors. There are two major non-redundant and complementary IFN-I-dependent nucleic acid recognition pathways present, toll-like receptors (TLRs) and cytoplasmic sensors (reviewed in Schlee and Hartmann, 2016). Noteworthy, TLRs are only expressed within endosomes of specialized immune cells like MACs, DCs, and B cells. Moreover, nucleic acid recognition does not involve direct viral infection of the recognizing cell, but requires uptake of nucleic acids from the outside, e.g. from cell debris of infected cells. In contrast to TLRs, cytosolic PRRs are ubiquitously expressed and contribute to the ability of virtually all cell types to respond to viral infection by IFN-I production. These sensors include RIG-I and MDA5, which recognize long dsRNA and short dsRNA with triphosphate or diphosphate at the 5’ end, as well as gamma--inducible protein 16 (IFI16) and cGAS, which both sense dsDNA.

The cyclic GMP-AMP synthase (cGAS) is a highly conserved sensor for cytosolic dsDNA, which exists in an auto-inhibited state in the absence of DNA (Li et al., 2013). It contains a nucleotidyl transferase and two major DNA-binding domains (Sun et al., 2013). After interaction with dsDNA, cGAS becomes active in a sequence-independent manner. In addition to dsDNA, ssDNA that forms internal duplex structures or Y-shaped ssDNA also activates cGAS (Herzner et al., 2015). DNA binding leads to ligand-induced dimerization, resulting in conformational changes, which enable catalytic activity (Civril et al., 2013; Li et al., 2013). Upon activation, cGAS produces the second messenger cyclic GMP-AMP (cGAMP), which comprises a unique 2’-5’ and a 3’-5’ phosphodiester linkage (Ablasser et al., 2013a) (Fig. 4). The resulting 2’3’-cGAMP molecule represents a novel class of second messengers, which can also be transferred from the producing to neighboring cells through gap junctions (Ablasser et al., 2013b) or is incorporated into newly formed viral particles (Bridgeman et al., 2015; Gentili et al., 2015). Both mechanisms enable rapid antiviral immunity in a transcription-independent, horizontal manner. Cyclic GAMP is a potent activator of the stimulator of IFN genes (STING), which is essential for triggering IFN-I and ISG expression (Ishikawa et al., 2009). Interestingly, STING has been reported to directly bind to dsDNA and ssDNA (Abe et al., 2013), leading to a cGAS-independent induction of IFN-I responses. Upon interaction with ligands, STING recruits and activates TANK-binding kinase 1 (TBK1) (Tanaka and Chen, 2012). TBK1 in turn phosphorylates STING, which becomes capable of interacting with the IFN regulatory factor 3 (IRF3), thereby enabling TBK1 to phosphorylate IRF3.

16 Introduction

Figure 4: The cGAS/STING pathway of cytosolic DNA sensing. Exogenous DNA from dead cells or invading pathogens as well as self DNA (DNA damage by-products, DNA from damaged mitochondria or accumulated cDNA of retroelements) binds to and activates cGAS. Upon activation, cGAS catalyzes the synthesis of the second messenger 2’3’-cGAMP from ATP and GTP, which is a potent activator of STING. Cyclic dinucleotides (CDNs) from bacteria can also directly activate STING. After activation, STING traffics from the ER to the ER-Golgi intermediate compartment (ERGIC) and the Golgi apparatus. STING then recruits and activates the kinases TBK1 and IKK. TBK1 phosphorylates STING, which then is able to recruit IRF3. IRF3 is activated by TBK1-mediated phosphorylation, dimerizes and enters the nucleus. Activation of IKK leads to phosphorylation and proteasomal degradation of the NF-κB inhibitor IκBα. NF-κB consisting of the two subunits p65 and p50 becomes active, enters the nucleus and functions together with IRF3 to turn on expression of type I IFNs, pro-inflammatory cytokines, and IFN-stimulated genes (ISGs). Adapted from Chen et al., 2016.

17 Introduction

Following activation, STING undergoes intracellular re-localization, trafficking from the endoplasmatic reticulum (ER) to the perinuclear compartments. Phosphorylated IRF3 dissociates from STING, followed by self-dimerization and translocation into the nucleus to regulate (Liu et al., 2015). STING also activates the IκB kinase (IKK), which phosphorylates IκB family of NF-κB inhibitors (Sharma et al., 2003). Phosphorylated IκB proteins are degraded, thereby releasing NF-κB, which enters the nucleus and induces pro- inflammatory cytokines. Since cGAS enables detection of a seemingly unlimited repertoire of pathogen-derived DNA as well as self-DNA, its activation has to be tightly regulated (reviewed in Chen et al., 2016). Expression of cGAS undergoes substantial induction after viral infection or stimulation with IFN-I, providing a positive feedback mechanism for its activation. Moreover, cGAS activity is regulated by post-translational modifications, such as phosphorylation, SUMOylation, and glutamylation (Cui et al., 2017; Seo et al., 2015; Xia et al., 2016). Activation of STING is also tightly regulated by post-translational modifications, like phosphorylation and ubiquitination, to prevent sustained innate immune activation (Konno et al., 2013; Liu et al., 2015; Qin et al., 2014; Zhang et al., 2012). Despite being regulated under normal conditions, aberrant activation of intracellular sensing pathways by endogenous DNA has been linked to the pathogenesis of autoimmune and inflammatory disorders (reviewed in Kretschmer and Lee-Kirsch, 2017; Qian et al., 2014; Smith and Jefferies, 2014).

18 Objectives

III. OBJECTIVES

1. The role of SAMHD1 in retroviral infection

Murine SAMHD1 has previously been shown to act as a HIV-1 restriction factor both in vitro and in vivo. The first aim of this study was the characterization of the endogenous as well as the HIV•1-induced immune responses in primary murine cells in the absence of SAMHD1. For this purpose, BMDCs isolated from wt, SAMHD1, IFNAR, and double KO mice were infected with an HIV-GFP reporter virus to unravel the full antiviral potency of SAMHD1 restriction in vitro. A transcriptome analysis of uninfected and infected cells was performed to answer the question whether HIV-1 infection induces a differential upregulation of ISGs compared to the autoimmune reaction in the absence of SAMHD1. Furthermore, this analysis was used to clarify whether the same or different genes are upregulated in wt compared to SAMHD1 KO cells upon HIV-1 infection. The cytoplasmic DNA sensor cGAS, which signals through the adaptor protein STING, might be a promising candidate for the induction of both IFN-I responses. Thus, the involvement of the cGAS/STING pathway in HIV-GFP infection was analyzed using BMDCs isolated from cGAS, STING, as well cGAS/SAMHD1 and STING/SAMHD1 double KO mice. Collectively, the results obtained from infection experiments and transcriptome analysis will provide further insights into the role of SAMHD1 in HIV-1 infection as well as its role in antiviral and autoimmune responses.

2. The role of SAMHD1 in the inhibition of endogenous retroelements

So far, the molecular trigger for the autoimmune reaction in AGS patients is not fully understood. Accumulation and aberrant sensing of nucleic acids derived from replicating endogenous retroelements in the absence of SAMHD1 is discussed as a potential source for the autoimmune response. SAMHD1 has recently been identified as a restriction factor for retroelements. However, the underlying mechanism is still ill-described and differs from what is known about SAMHD1-mediated HIV-1 restriction. Thus, the second aim of this thesis was the characterization of SAMHD1-mediated inhibition of endogenous retroelements, especially L1, in greater detail. For this purpose, different cell culture-based assays were used to determine the effect of SAMHD1 on retrotransposition. In addition, the impact of SAMHD1 on L1 expression and L1-encoded enzymatic functions was analyzed. The results obtained from these assays will help to better understand the role of endogenous retroelements as potential triggers for autoimmune reactions in humans. Moreover, these studies will help to gain further insights into the function and regulation of SAMHD1.

19

20 Material and Methods

IV. MATERIAL AND METHODS

1. Materials

1.1. Chemicals

Standard chemicals for laboratory use were purchased from Sigma Aldrich, Applichem, or Merck Chemicals GmbH.

1.2. Buffers, solutions, and media

1.2.1. Cell culture

Trypsin/EDTA: 0.25 % [w/v] trypsin, 140 mM NaCl, 5 mM KCl, 0.56 mM Na2HPO4, 5 mM glucose, 25 mM Tris-HCl, 0.01 % [w/v] EDTA, pH 7.0

Dulbecco’s Modified Eagle Medium (DMEM) and RPMI 1640 medium were purchased from Gibco (Life technologies) and supplemented with 10 % fetal calf serum (FCS, Sigma- Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM glutamine.

Erythrocytes lysis buffer: 1.5 M NH4Cl, 100 mM KHCO3, 1 mM EDTA, sterilized by filtration

1.2.2. Bacterial culture Luria Bertani (LB) medium: 10 g/l Bacto Tryptone, 5 g/l Bacto Yeast Extract, 8 g/l NaCl, 1 g/l glucose; pH 7.2; supplemented with 100 µg/ml ampicillin or kanamycin

Luria Bertani (LB) agar plates: 15 g/l Bacto Agar in LB medium; supplemented with 50 µg/ml ampicillin or 30 µg/ml kanamycin

SOC medium: 20 g/l Bacto Tryptone, 5 g/l Bacto Yeast Extract, 2.5 mM NaCl, 10 mM MgCl2,

10 mM MgSO4, 20 mM glucose in H2O; sterilized by filtration

1.2.3. Standard buffers and solutions

Phosphate-buffered saline (PBS): 138 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 1.5 mM

KH2PO4

HBS buffer (2x): 280 mM NaCl, 50 mM HEPES, 1.5 mM Na2HPO4; pH 7.07-7.15; sterilized by filtration

TAE buffer (1x): 40 mM Tris, 1 mM EDTA, 0.1 % [v/v] acetic acid

Genotyping lysis buffer: 0.1 M Tris-HCl, 0.2 M NaCl, 0.2 % [w/v] SDS, 5 mM EDTA, 0.1 mg/ml proteinase K, pH 8.5

21 Material and Methods

1.2.4. Protein methods

Western blot lysis buffer: 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.5 % [v/v] NP-40, 1x Halt Protease Inhibitor

Immunoprecipitation lysis buffer: 50 mM Tris-HCl (pH 7.5), 160 mM NaCl, 1 mM EDTA, 0.25 % [v/v] NP-40, 1x Halt Protease Inhibitor, 1 mM PMSF, RNaseOUT

SDS sample buffer (2x): 2 % [w/v] SDS, 50 mM EDTA, 10 mM Tris-HCl (pH 6.8), 5 % [v/v] β-mercaptoethanol, 2 % [w/v] bromphenol blue, 10 % [v/v] glycerol

SDS running buffer (1x): 25 mM Tris, 250 mM glycine, 0.1 % [w/v] SDS

Blotting buffer (1x): 25 mM Tris, 200 mM glycine

Washing buffer (PBS-T): 0.1 % [v/v] Tween-20 in PBSo.

Blocking buffer: 2.5 % [w/v] milk powder in PBS-T

ECL solution: 98.9 % [v/v] solution A, 1 % [v/v] solution B, 0.032 % [v/v] H2O2

ECL solution A: 0.1 M Tris (pH 8.6), 25 % [v/v] luminol

ECL solution B: 0.11 % [w/v] para-hydroxy coumarin acid in DMSO

1.2.5. Virus preparation TNE buffer: 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 100 mM NaCl

20 % sucrose solution: 20 % [w/v] sucrose diluted in TNE buffer, sterilized by filtration

1.2.6. LEAP assay

Lysis buffer: 10 mM Tris-HCl (pH 7.5), 0.5 % [w/v] CHAPS, 1 mM MgCl2, 1mM EGTA, 10 % [v/v] glycerol, 1 mM DTT, 1x Halt Protease Inhibitor

Sucrose stock solution (47 %): 80 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl (pH 7.5), 47 % [w/v] sucrose, sterilized by filtration

Sucrose working stock solution: 47 % stock solution, 1 mM DTT, 1x Halt Protease Inhibitor

Sucrose dilution buffer: 80 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 1x Halt Protease Inhibitor

17 % sucrose solution: 47 % working stock solution diluted with sucrose dilution buffer

8.5 % sucrose solution: 47 % working stock solution diluted with sucrose dilution buffer

22 Material and Methods

1.3. Vectors

1.3.1. Empty vectors pcDNA3.1 (+): Eucaryotic expression vector, multiple cloning site under control of the CMV promoter, neomycin and ampicillin resistance genes; purchased from Invitrogen. pcDNA6mycHis (A): Empty expression vector, multiple cloning site under control of a CMV promoter, C-terminal myc/His-tag, blasticidin and ampicillin resistance genes; purchased from Invitrogen.

1.3.2. SAMHD1 expression vectors

Human SAMHD1 variants used within this study were cloned into the pcDNA6mycHis (A) empty vector. pcDNA6mycHis_hucoSAMHD1: Expression vector for human codon-optimized wildtype SAMHD1, C-terminal myc/His-tag. pcDNA6mycHis_hucoSAMHD1_T592A: Expression vector for hucoSAMHD1 phosphorylation-defective mutant T592A, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_T592D: Expression vector for phosphomimetic mutant hucoSAMHD1 T592D, C-terminal myc/His-tag. pcDNA6mycHis_hucoSAMHD1_D207N: Expression vector for enzymatically inactive hucoSAMHD1 mutant D207N, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_D311N: Expression vector for enzymatically inactive hucoSAMHD1 mutant D311N, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_Q548A: Expression vector for RNase-defective hucoSAMHD1 mutant Q548A, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_D137A: Expression vector for cofactor-binding mutant D137A of hucoSAMHD1, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_L428S/Y432S: Expression vector for oligomerization- defective hucoSAMHD1 mutant L428S/Y432S, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_D207N/T592A: Expression vector for enzymatically inactive hucoSAMHD1 mutant D207N in the non-phosphorylated T592A background, C•terminal myc/his-tag

23 Material and Methods pcDNA6mycHis_hucoSAMHD1_D311N/T592A: Expression vector for enzymatically inactive hucoSAMHD1 mutant D311N in the non-phosphorylated T592A background, C•terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_Q548A/T592A: Expression vector for RNase-defective hucoSAMHD1 mutant Q548A in the non-phosphorylated T592A background, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_D137A/T592A: Expression vector for cofactor-binding mutant D137A of hucoSAMHD1 in the non-phosphorylated T592A background, C-terminal myc/his-tag. pcDNA6mycHis_hucoSAMHD1_L428S/Y432S/T592A: Expression vector for oligomerization-defective hucoSAMHD1 mutant L428S/Y432S in the non-phosphorylated T592A background, C-terminal myc/his-tag. pcDNA6mycHis_ΔNLS: Expression vector for cytoplasmic hucoSAMHD1, C-terminal myc/His-tag.

All murine SAMHD1 variants were cloned by Kathrin Friedrich, Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg. pcDNA6mycHis_mucoSAMHD1 Iso1: Expression vector for murine codon-optimized wildtype SAMHD1 isoform 1, C-terminal myc/his-tag. pcDNA6mycHis_mucoSAMHD1 Iso1 T603A: Expression vector for mucoSAMHD1 isoform 1 phosphorylation-defective mutant T603A, C-terminal myc/his-tag. pcDNA6mycHis_mucoSAMHD1 Iso1 T603D: Expression vector for mucoSAMHD1 isoform 1 phosphomimetic mutant T603D, C-terminal myc/his-tag. pcDNA6mycHis_mucoSAMHD1 Iso1 HD207/208AA: Expression vector for enzymatically inactive pcDNA6mycHis_mucoSAMHD1 isoform 1 mutant HD207/208AA, C-terminal myc/his-tag. pcDNA6mycHis_mucoSAMHD1 Iso1 D138A: Expression vector for cofactor-binding mutant D138A of mucoSAMHD1 isoform 1, C-terminal myc/his-tag. pcDNA6mycHis_mucoSAMHD1 Iso2: Expression vector for murine codon-optimized wildtype SAMHD1 isoform 2, C-terminal myc/his-tag. pcDNA6mycHis_mucoSAMHD1 Iso2 HD207/208AA: Expression vector for enzymatically inactive mucoSAMHD1 isoform 2 mutant HD207/208AA, C-terminal myc/his-tag.

24 Material and Methods pcDNA6mycHis_mucoSAMHD1 Iso2 D138A: Expression vector for cofactor-binding mutant D138A of mucoSAMHD1 isoform 2, C-terminal myc/his-tag.

1.3.3. Expression vectors for retroelements 99 PUR JM111 EGFP: Expression vector for retrotransposition-defective L1-RP, two missense mutations in ORF1p, EGFP reporter cassette, puromycin resistance gene (Ostertag et al., 2000); kind gift from John L. Goodier (John Hopkins University, Baltimore).

99 PUR RPS EGFP: Expression vector for retrotransposition-competent L1-RP, EGFP reporter cassette, puromycin resistance gene (Ostertag et al., 2000); kind gift from John L. Goodier (John Hopkins University, Baltimore). pAD2TE1: Expression vector for retrotransposition-competent L1-3.1, T7-tagged ORF1p, TAP-tagged ORF2p, neomycin reporter cassette (Doucet et al., 2010); kind gift from Nicolas Gilbert (Chinese Academy of Science, Beijing). pAD2TE1_ORF1-FLAG: Expression vector for retrotransposition-competent L1-3.1, FLAG- tagged ORF1p, TAP-tagged ORF2p, neomycin reporter cassette; cloned within this study. pcDNA3.1xLINE-1_ORF1-3’FLAG: Expression vector for C-terminally FLAG-tagged L1 ORF1p; cloned within this study. pAD500: Expression vector for TAP-tagged L1 ORF2p (Doucet et al., 2010); kind gift from Nicolas Gilbert (Chinese Academy of Science, Beijing). pAlutet: Expression vector for AluY retroelement, neomcycin reporter cassette (Dewannieux et al., 2003); kind gift from Nicolas Gilbert (Chinese Academy of Science, Beijing). pGL3-IAP92L23neoTNF: Expression vector for murine LTR retroelement IAP, neomycin reporter cassette (Dewannieux et al., 2004); kind gift from Sebastièn Nisole (Paris Descartes University). pCMVmus-6-DneoTNF: Expression vector for murine LTR retroelement MusD, neomycin reporter cassette (Esnault et al., 2005); kind gift from Sebastièn Nisole (Paris Descartes University).

L1 RP-luc: Expression of a luciferase reporter gene under control of the L1 RP promoter (Yu et al., 2001); kind gift from Gerald Schumann (Paul Ehrlich Institute, Langen).

1.3.4. Vectors for virus preparation pVSVG: Expression vector for the envelope protein of vesicular stomatitis virus; purchased from Addgene (#12259).

25 Material and Methods

NL4-3 E- CMV GFP: Expression vector for env-deficient HIV-1 based on proviral clone NL4- 3, GFP reporter gene under control of a CMV promoter instead of the viral nef gene (Zhang et al., 2008).

NL4-3 luc3E-: Expression vector for env-deficient HIV-1 based on proviral clone NL4-3, firefly luciferase reporter gene under control of a CMV promoter instead of the viral nef gene (Connor et al., 1995). pCMV deltaR8.9: Expression vector for HIV gag/pol, encodes Tat and Rev (Zufferey et al., 1997). pLKO.1-puro-shC: Lentiviral expression vector for scrambled shRNA, puromycin resistance gene; purchased from Addgene (#10870); kind gift from Henning Hofmann pLKO.1-puro-shSAMHD1: Lentiviral vector expressing a shRNA targeting SAMHD1; kind gift from Henning Hofmann (Robert Koch Institute, Berlin).

1.3.5. Others pcDNA4-huZAP (L)-HA: Expression vector for the long isoform of human ZAP, C-terminal HA-tag; purchased from Addgene (#45907) (Kerns et al., 2008). pmyc-MOV10: Expression vector for myc-tagged MOV10; purchased from Addgene (#10977) (Meister et al., 2005). pEGFP-N1: Expression vector for enhanced green fluorescence protein (EGFP) under control of a CMV promoter, neomycin and kanamycin resistance genes; purchased from Clonetech.

1.4. Oligonucleotides

All oligonucleotides were purchased from Biomers.net GmbH. All primer sequences are annotated in 5’ to 3’ orientation.

1.4.1. Cloning of SAMHD1 mutants 5‘ HindIII hucoSAMHD1: GGCCTCTAGACATTGGGTCGTCTTTGAACAGCTGCA

3‘ XbaI hucoSAMHD1nostop: GGCCAAGCTTCACCATGGACTCACTTTTGGGGTGTGGT GTC

5' overlap hucoSAMHD1 T592A: GCGATGTCATTGCCCCTCTGATCGCTCCACAGAAGAA AGAGTGG

3' overlap hucoSAMHD1 T592A: CCACTCTTTCTTCTGTGGAGCGATCAGAGGGGCAATG ACATCGC

26 Material and Methods

5' overlap hucoSAMHD1 T592D: GCGATGTCATTGCCCCTCTGATCGATCCACAGAAGAA AGAGTGG

3' overlap hucoSAMHD1 T592D: CCACTCTTTCTTCTGTGGATCGATCAGAGGGGCAATG ACATCGC

5‘ overlap hucoSAMHD1 D207N: GACTGTGCCACAACCTGGGCCATG

3‘ overlap hucoSAMHD1 D207N: CATGGCCCAGGTTGTGGCACAGTC

5‘ overlap hucoSAMHD1 D311N: GGGATCGACGTGAACAAATGGGA

3‘ overlap hucoSAMHD1 D311N: GTCCCATTTGTTCACGTCGATCCC

5‘ overlap hucoSAMHD1 Q548A: GAAATTCGCTGAAGCCCTGATCCG

3‘ overlap hucoSAMHD1 Q548A: CGGATCAGGGCTTCAGCGAATTTC

5’ overlap hucoSAMHD1 D137A: GGTCAGGATCATTGCTACACCACAGTTCC

3’ overlap hucoSAMHD1 D137A: GGAACTGTGGTGTAGCAATGATCCTGACC

5’ overlap hucoSAMHD1 L428S/Y432S: GACAACATTTTTTCCGAAATCCTGTCCAGTACT GACCCAAAG

3’ overlap hucoSAMHD1 L428S/Y432S: CTTTGGGTCAGTACTGGACAGGATTTCGGAAA AAATGTTGTC

5’ HindIII hucoSAMHD1 NLS mutant: GGCCAAGCTTCACCATG CAGAGAGCAGATAGCGAGCAGCCAAGT GCA GCA CCA GCC TGCGACGACAGCCC

1.4.2. Cloning of LINE-1 expression vectors L1ORF1-Not-for: CTACT GCGGCCGC AA CACC ATG GGG AAA AAA CAG AAC

L1ORF1-ApaI-FLAG-rev: CTACTGGGCCCAATTACTTATCGTCGTCATCCTTGTAGTCCATTTTGGCATGATTTTGCA GCGGCTGGTACCGGTTGTTCCTTTCC

5‘ Overlap L1•FLAG: GACTACAAGGATGACGACGATAAGTAAAGACCATCGAGACTAGG AAGAAACTGCATC

3‘ Overlap L1•FLAG: TTACTTATCGTCGTCATCCTTGTAGTCCATTTTGGCATGATTTTGC AGCGGCTGG

5‘ outer L1-FLAG: CCAGAATTTCATATCCAGCC

27 Material and Methods

3’ outer L1-FLAG: GTTACATATGTATACATGTGCCATC

1.4.3. LEAP assay LEAP (Ambion 3’ RACE): GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTT TVN

Linker (Ambion 3’RACE Outer): GCGAGCACAGAATTAATACGACT

L1 3’ end: GGGTTCGAAATCGATAAGCTTGGATCCAGAC

1.4.4. cDNA synthesis and qRT-PCR

Oligo dT: TTTTTTTTTTTTTTT endORF1_for: GAAGGAAGCGCTAAACATGG

T7-Tag_rev: CCCATTTGCTGTCCACCAG

Pydc4-for: CATTCCAGAACTTGCAGCTCGTG

Pydc4-rev: GTAAGTGGAGGAGGGCTGGATTC

Irg1-for: CGACCAGACTTCAGGCTCCCACC

Irg1-rev: GGTGGGTGGCAGGGTGCCATGTG

Ifi206-for: CTTACCTCCAGCCTGATGGAAGC

Ifi206-rev: CATGGTATCAGGTGATTCAGGG

Oas3-for: AGAACACTGGATAGATGTTAGCC

Oas3-rev: GGAGGGAGGAGTACACGTTGGG

ISG20-for: CCTGTATGACAAGTACATCCGAC

ISG20-rev: TGGCGTGGCCCTCACCATGTGC

Ifi44-for: GGCACATCTTAAAGGGCCACACTC

Ifi44-rev: CTGTCCTTCAGCAGTGGGTCATG

GAPDH-for: AAGGGGCGGAGATGATGAC

GAPDH-rev: GGTGCTGAGTATGTCGTGGAG

1.4.5. Genotyping SAMHD1-type-1: CAGTCCTGGTGCACACATAC

28 Material and Methods

SAMHD1-type-2: AAGACCTACAAAGAGGGCGG

SAMHD1-type-3: GGGTGTACAGAGGTTAGATGC

IFNAR1-DD-F1: AAGCCTCCCCGCAGTATTGATGAG

IFNAR1-DD-R1: TCTGCACAAACAATCACACCAGAC

IFNAR1-DD-F2: GTCTGTAATACGCATTTCTATCTC cGAS348: CCAAAGAAGCAGTCTAAGACTAGAGT cGAS349: TAGGTCGGCCAGGGTTCCATC cGAS350: GTATTATCAGCTACCAAGATGC

Sting Seq For: CTCCTTCCTGTACGATGAGAGAG

Sing Seq Rev: GCTCTGTATACTTACTGGCTGTCCG

1.5. Antibodies

1.5.1. Primary antibodies α-FLAG (M2): mouse monoclonal antibody directed against the FLAG tag epitope (DYKDDDDK) (Sigma-Aldrich). The antibody was diluted 1:1000 for western blot detection and 1:500 for immunofluorescence analysis.

α-DYKDDDDK (FLAG): rabbit polyclonal antibody directed against the FLAG tag epitope (DYKDDDDK) (). The antibody was diluted 1:1000 for western blot detection.

α-HA (16B12): mouse monoclonal antibody directed against the HA tag epitope (YPYDVPDYA) (Biolegends). The antibody was diluted 1:1000 for western blot detection.

α-myc (9B11): mouse monoclonal antibody directed against the myc tag epitope (EQKLISEEDL) (Cell Signaling). The antibody was diluted 1:1000 for western blot detection and 1:500 for immunofluorescence analysis.

α-myc (71D10): rabbit polyclonal antibody directed against the myc tag epitope (EQKLISEEDL) (Cell Signaling). The antibody was diluted 1:1000 for western blot detection.

α-T7: mouse monoclonal antibody directed against the T7 tag epitope (MASMTGGQQMG) (Novagen). The antibody was diluted 1:5000 for western blot detection.

α-T7: rabbit polyclonal antibody directed against the T7 tag epitope (MASMTGGQQMG) (abcam). The antibody was diluted 1:5000 for western blot detection.

29 Material and Methods

α-GAPDH: rabbit polyclonal antibody for detection of GAPDH (abcam). The antibody was diluted 1:500 for western blot detection.

α-Hsp90 α/β: mouse monoclonal antibody for detection of Hsp90 (Santa Cruz). The antibody was diluted 1:1000 for western blot detection.

α-G3BP1: rabbit polyclonal antibody for detection of G3BP1 (Proteintech). The antibody was diluted 1:1000 for immunofluorescence analysis.

α-SAMHD1 (3F5): mouse monoclonal antibody for detection of SAMHD1 (Novusbio). The antibody was diluted 1:500 for western blot detection.

α-SAMHD1 phosphoT592: rabbit polyclonal antibody for detection of phosphorylated SAMHD1 (pT592) (ProSci). The antibody was diluted 1:1000 for western blot detection.

1.5.2. Secondary antibodies α-rabbit-HRP and α-mouse-HRP: both HRP-coupled antibodies were purchased from Cell Signaling and diluted 1:2000 for western blot detection.

α-rabbit-HRP (conformation specific) (L27A9): the HRP-coupled antibody does not recognize denatured/reduced rabbit IgG or heavy chains. The antibody was purchased from Cell Signaling and diluted 1:2000 for western blot detection.

α-mouse-HRP (light chain specific) (D3V2A): the HRP-coupled antibody recognizes denatured/reduced mouse IgG light chains. The antibody was purchased from Cell Signaling and diluted 1:1000 for western blot detection.

α-mouse-Alexa488, α-mouse-Alexa555, and α-rabbit-Alexa647: fluorescent dye-coupled antibodies were purchased from Cell Signaling and diluted 1:1000 for immunofluorescence analysis.

1.5.3. Direct antibody α-myc-Alexa555 (9B11): mouse monoclonal antibody directed against the myc tag epitope (EQKLISEEDL) conjugated with the fluorescent dye Alexa555 (Cell Signaling). The antibody was diluted 1:250 for immunofluorescence analysis.

1.6. Enzymes

All restriction enzymes were purchased from New England Biolabs. T4 DNA ligase and Phusion DNA polymerase were purchased from Thermo Fisher. All enzymes were used according to the supplier’s manual with the respective buffers.

30 Material and Methods

1.7. Biological materials

1.7.1. Cell lines HEK 293T cells: human cell line derived from human embryonic kidney cells, which was transformed with sheared adenovirus 5 DNA and later with the large T antigen from SV40 (Shein and Enders, 1962). The cells grow adherent with an epithelial morphology and are cultivated in complete DMEM. HEK 293T cells expressing shRNA targeting SAMHD1 or control shRNA were generated by lentiviral transduction. Three days postinfection with shRNA-containing viral particles, cells were selected with 2.5 µg/ml puromycin. Efficient knockdown of SAMHD1 was confirmed by immunoblot. Stable shRNA expressing cells were cultivated in DMEM supplemented with 0.5 µg/ml puromycin.

HeLa cells: human cell line originally derived from cervical cancer cells (Scherer et al., 1953). The cells grow adherent and are cultivated in complete DMEM.

HeLa HA cells: a strain of HeLa cells that has been described previously to support retrotransposition of Alu retroelements (Hulme et al., 2007). The cells were a kind gift from John V. Moran (University of Michigan).

U2OS cells: human osteosarcoma epithelial cell line. The cells grow adherent and are cultivated in complete DMEM. Cells were purchased from DSMZ (ACC785).

THP-1 cells: human monocytic cell line derived from an acute monocytic leukemia patient (Tsuchiya et al., 1980). The cells grow in suspension in complete RPMI 1640 medium and can be differentiated by treatment with 50 ng/µl phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells. THP-1 cGAS KO and STING KO cells have been described previously (Mankan et al., 2014). In THP-1 shSAMHD1 cells, SAMHD1 expression is downregulated by shRNA to enable infection of differentiated cells. THP-1 wt, cGAS, and STING KO cells expressing shRNA targeting SAMHD1 or control shRNA were generated by lentiviral transduction. Three days postinfection, cells were selected with 2.5 µg/ml puromycin and efficient knockdown of SAMHD1 was confirmed by immunoblotting. Stable shRNA expressing cells were cultivated in RPMI 1640 medium supplemented with 0.5 µg/ml puromycin.

1.7.2. Bacterial strains

Chemical competent Escherichia coli DH10B are suitable for an efficient transformation of methylated DNA derived from eukaryotic sources or unmethylated DNA derived from PCR or cDNA. Genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 endA1 recA1 deoR Δ(ara,leu)7697 araD139 galU galK nupG rpsL λ- (Grant et al., 1990).

31 Material and Methods

1.7.3. Mouse strains

Homozygous mice from all strains were utilized between 8 and 12 weeks of age for bone marrow preparation. All strains were raised under specific pathogen-free conditions at the Franz Petzold Zentrum, Erlangen.

C57BL/6: wild-type mice were purchased from Charles River.

SAMHD1 KO: previously described in Behrendt, 2013

IFNAR KO: previously described in Kamphuis, 2006

IFNAR/SAMHD1 KO: previously described in Behrendt, 2013 cGAS KO: previously described in Schoggins, 2014 cGAS/SAMHD1: generated within this study by crossing SAMHD1 and cGAS KO mice.

STING KO: goldenticket allele, previously described in Sauer, 2011

STING/SAMHD1 KO: crossed within this study

2. Methods

2.1. Cell culture

In general, cells were cultivated at 37 °C, 5 % CO2, and 80 % humidity and passaged three times a week.

All cell lines were frequently tested for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Lonza) according to the supplier’s manual.

2.1.1. Bone marrow preparation and BMDC isolation

Femora and tibiae of one mouse were dissected, the flesh was removed, and the bones were stored in RPMI medium over night at 4 °C. The next day, the joints were removed, the bone marrow was flushed out with PBS + 0.5 % FCS using a syringe, and the cell suspension was homogenized and kept on ice. Cells were pelleted by centrifugation (1200 rpm, 5 min, 4 °C) and the supernatant was discarded. To minimize the number of erythrocytes, pellets were incubated in 1 ml erythrocytes lysis buffer for 1 min and the reaction was stopped by addition of 5 ml PBS + 0.5 % FCS. Cells were centrifuged (1200 rpm, 5 min, 4 °C), pellets were dissolved in 2 ml PBS + 0.5 % FCS and cell suspensions were passed through sterile cell strainers (Partec). After centrifugation (1200 rpm, 5 min, 4 °C) cells were re-suspended in cold RPMI medium and counted. To generate BMDCs, about 25-40 x 106 cells/T75 cell culture flask were cultivated in RPMI

32 Material and Methods supplemented with 10 ng/ml GM-CSF. Medium was exchanged after 3 days (supplemented with 10 ng/ml GM-CSF). Cells were differentiated for 8 days prior to infection.

2.2. Nucleic acid methods and transfections

2.2.1. Cloning and plasmid preparation

For overexpression of human and murine codon-optimized, 3’ mycHis-tagged SAMHD1 proteins, wildtype (wt) SAMHD1 or the indicated SAMHD1 mutants were cloned into the empty pcDNA6mycHis expression vector via the restriction sites HindIII and XbaI. Mutations were introduced by overlapping PCR mutagenesis. Primers used for generating SAMHD1 amplicons are listed in section 1.4. For expression of FLAG-tagged L1 ORF1, a 3’ FLAG-tag was attached to ORF1 from the L1-RP subfamily by PCR. The amplicon was ligated into the pcDNA3.1 (+) vector via ApaI and NotI. To generate the L1 reporter construct pAD2TE1_ORF1-FLAG, the T7 tag at the C-terminus of ORF1 in pAD2TE1 was replaced with a FLAG tag by overlapping PCR. The resulting amplicon was cloned into pAD2TE1 via AgeI and BstZ17I.

Plasmids were transformed into E. coli DH10B and plasmid DNA isolated through miniprep (GeneJET plasmid miniprep , Thermo Fisher). Positive clones were identified by test digestion. All constructs were validated by nucleotide sequencing (Macrogen) according to the manufacturer’s protocol.

Plasmid-transformed E. coli DH10B were cultured in 250 ml LB medium containing either 100 µg/ml ampicillin or 30 µg/ml kanamycin over night at 37 °C. To isolate plasmid DNA, the Pure Link™ HiPure Plasmid MaxiPrep Kit (Thermo Fisher) was used according to the supplier’s manual. For generation of cryo-stocks of the transformed bacteria, 500 µl bacteria suspension were mixed with an equal volume of 100 % [v/v] glycerol and stored at -80 °C.

2.2.2. RNA preparation

For total cellular RNA isolation, the NucleoSpin RNA® Kit (Macherey-Nagel) was used according to the supplier’s manual. Depending on the cell number, RNA was eluted in

30•50 µl nuclease-free H2O and concentration was determined using a NanoDrop1000 spectrometer (PeqLab) and stored at •20 °C.

2.2.3. cDNA synthesis and qRT-PCR

Reverse transcription of isolated cellular RNA was performed with an oligodT primer (500 µg/ml) using SuperScript® II Reverse Transcriptase (Thermo Fisher) according to the manufacturer’s protocol. Quantitative PCR was performed with 40 ng (mouse ISGs) or 100 ng cDNA (L1) in a total volume of 11.5 µl per reaction. 12.5 µl 2 x SYBR Green Mix (Thermo Fisher) together with 0.5 µl of 30 mM 5’ and 3’ primer and 1 µl ROX per 500 µl were

33 Material and Methods added to each well and analyzed in an Applied Biosystems 7500 Real Time PCR system. To analyze ISG expression levels, the ΔΔCt (Relative Quantification) method with GAPDH as endogenous control was used. Finally, transcript levels were evaluated using the corresponding software SDS (sequence detection system; version 1.9). Values obtained for uninfected samples were set to 1, values of the corresponding infected samples were normalized on the uninfected control.

8 1 To determine L1 cDNA copies, the standard method with log10 serial dilutions (10 - 10 molecules) of the L1 expression plasmid pAD2TE1 were used. Finally, L1 copies were evaluated by the corresponding software SDS (sequence detection system; version 1.9) using the internal standard values.

Table 1: Standard qRT-PCR program.

Stage Time Temperature Repeats 1 2 min 50°C 1 2 10 min 95°C 1 0.15 min 95°C 3 40 1 min 50°C 0.15 min 95°C 1 min 60°C 4 1 0.15 min 95°C 0.15 min 60°C

2.2.4. Transfections Calcium phosphate transfection: HEK 293T cells were seeded in DMEM. The next day, the medium was removed and replaced by transfection medium (DMEM without penicillin/streptomycin and FCS). DNA was diluted in H2O, 2.5 M CaCl2 was added and mixed. Afterwards, cold 2x HBS buffer (pH 7.05 – 7.15) was added drop-wise and the transfection mix was incubated for 5 min at RT. The transfection mix was drop-wise added to the cells, which were then incubated at 37°C. Five to six hours posttransfection, the medium was replaced by complete DMEM. The amounts of the different reagents used for transfection are listed in Table 2.

Table 2: Amounts cells, DNA, CaCl2, and 2x HBS puffer for Ca2PO4 transfection. T175 flask 10 cm dish 6-well 48-well Cells 8 x 106 in 30 ml 2 x 106 in 10 ml 2 x 105 in 2 ml 3 x 104 in 0.25 ml DNA 90 µg 30 µg 4 µg 0.4 µg

adH2O 1830 µl 610 µl 70 µl 8.75 µl

CaCl2 (2.5 M) 270 µl 90 µl 10 µl 1.25 µl 2x HBS 2100 µl 700 µl 80 µl 10 µl

34 Material and Methods

Lipofectamine2000 transfection: HeLa cells were seeded in DMEM and transfected the following day using Lipofectamine® 2000 transfection reagent (Thermo Fisher). Per 6-well, 3 µg DNA and 6 µl Lipofectamine® 2000 were diluted in 125 µl transfection medium, mixed and incubated for 5 min at RT. Afterwards, 250 µl transfection mix were added to the cells.

FuGENE HD transfection: HeLa HA and U2OS cells were seeded in DMEM and transfected the following day using FuGENE® HD transfection reagent (Promega). Per 6-well, 4 µg DNA were diluted in transfection medium, 12 µl FuGENE were added and the samples were incubated for 15 min at RT. Afterwards, the transfection mix was added to the cells.

2.3. Virus preparations

2.3.1. HIV-GFP

For HIV-GFP reporter virus production, 8 x 106 HEK 293T cells were seeded in T175 cell culture flasks and cotransfected with 72 µg of the env-deficient reporter virus plasmid pNL4•3-E-CMV-GFP that encodes CMV-driven GFP gene instead of the nef gene and 18 µg of an expression plasmid for the glycoprotein of the vesicular stomatitis virus (pVSV-G) using calcium phosphate. Cell culture supernatant was harvested two days posttransfection, passed through 0.4 µm pore size filters, purified through a 20 % sucrose cushion, and stored at -80 °C. Three days postinfection, the virus was titrated on HEK 293T cells to determine infectivity by flow cytometry.

2.3.2. HIV-Luc

For HIV-Luc reporter virus production, 2 x 106 HEK 293T cells were seeded in 10 cm dishes and cotransfected with 24 µg of an env-deficient reporter virus plasmid (NL4-3 luc3E-) that encodes a CMV-driven luciferase reporter gene instead of the viral nef gene and 6 µg of the pVSV-G expression plasmid using the calcium phosphate method. Cell culture supernatant was harvested two days posttransfection, passed through 0.4 µm pore size filters and stored at -80 °C. Three days postinfection, the virus was titrated on HEK 293T cells to quantify luciferase activity in cps (counts per second).

2.3.3. shRNA-encoding lentiviral particles:

To obtain shRNA-encoding viral particles, 2 x 106 HEK 293T cells were seeded in 10 cm dishes and cotransfected with 6 µg of the pVSV-G expression plasmid, 12 µg of the HIV packaging plasmid pCMV deltaR9.8, and 12 µg of the lentiviral vector pLKO.1-puro encoding shRNA targeting SAMHD1 (shSAMHD1) or scrambled control shRNA (shCtrl). Cell culture supernatant was harvested two days posttransfection, passed through 0.4 µm pore size filters and stored at -80 °C. Viral particles were quantified by p24 ELISA.

35 Material and Methods

2.4. Infection assays

2.4.1. Infection of THP-1 cells with HIV-GFP

For infection of PMA-differentiated THP-1 cells, 6 x 105 cells per 6-well were seeded in 2 ml cell culture medium and differentiated with 50 ng/µl PMA for 24 h. For infection of undifferentiated THP-1 cells, 4 x 105 cells were seeded per 6-well and infected immediately. The cells were spin-inoculated with a VSV-G-pseudotyped HIV-GFP (MOI = 1) for 2 h at 1500 rpm at RT. Subsequently, the cells were incubated for 4 h at 37 °C before 500 µl medium were removed and 2 ml fresh cell culture medium were added. The cells were harvested in 1 ml PBS three days postinfection with a cell scraper in case of PMA- differentiated THP1 cells or only washed in PBS in case of undifferentiated cells. The cell suspension was transferred into a 5 ml FACS tube, washed with PBS, and re-suspended in 2 % paraformaldehyde (PFA) in PBS. GFP-positive cells were determined by flow cytometry.

2.4.2. Infection of THP-1 cells with HIV-Luc

To infect different THP-1 cell lines with HIV-Luc, 5 x 104 cells were seeded in 96-wells in 100 µl cell culture medium supplemented with 50 ng/µl PMA. The next day, the cells were infected with 1 x 105 cps of a VSV-G-pseudotyped HIV-Luc reporter virus by spin-inoculation for 2 h at 1500 rpm. Afterwards, the cells were incubated for 4 h at 37 °C, before 50 µl new cell culture medium were added per well. After three days, the cell culture medium was discarded, and cells were lysed in 100 µl lysis buffer (cell culture lysis 5x reagent, Promega; diluted in H2O) for 5 min at RT. Luciferase expression of 50 µl whole cell lysate was determined with 50 µl luciferase substrate solution per well (luciferase assay system, Promega).

2.4.3. Infection of mBMDCs with HIV-GFP

After eight days of differentiation, 1.5 x 106 mBMDCs were spin-inoculated with an HIV-GFP reporter virus (MOI = 1) in triplicates for 2 h at 1200 rpm. Afterwards, cells were incubated for 4 h at 37 °C, before 500 µl fresh cell culture medium were added per well. Cells were harvested for flow cytometry two days postinfection, washed in PBS, and fixed in 250 µl 2 % PFA in PBS.

2.4.4. Infection of mBMDCs for RNA isolation

For RNA isolation, 3 x 106 mBMDCs were seeded in 6-well plates and spin-inoculated with an HIV-GFP reporter virus at a MOI of 1.5 for 2 h at 1200 rpm. Cells were harvested for RNA isolation 16 hours postinfection and washed twice in PBS. Total cellular RNA was isolated using the NucleoSpin RNA® Kit (Macherey-Nagel) according to the supplier’s manual. RNA isolated from uninfected cells served as control for following experiments.

36 Material and Methods

2.5. Transcriptome analysis of murine BMDCs

For transcriptome analysis, BMDCs of three wt, SAMHD1, IFNAR, and IFNAR/SAMHD1 double KO mice isolated. After eight days of stimulation, 1.5 x 106 cells were spin-inoculated with HIV-GFP in 6-well plates at a MOI of 1.5 for 2 h at 1200 rpm. Uninfected cells were used as control. Total RNA of uninfected and infected mBMDCs was isolated and purified 16 hpi using the NucleoSpin RNA® Kit (Macherey-Nagel) according to the supplier’s manual. RNA was eluted in 40 µl nuclease-free H2O. The concentration was determined using a NanoDrop1000 spectrometer (PeqLab) and stored at -20 °C. IlluminaRNA sequencing was performed by Dr. Rayk Behrendt (TU Dresden). Bioinformatic analysis was carried out by Dr. Andreas Dahl (NGS facility CRTD, Dresden). Reads were aligned and mapped to mouse reference transcriptome (mm9) using pBWA and BEDtools software. Normalization was performed on a sample to sample basis by applying Pearson’s correlation coefficient using a DESeqR software package (v.1.6.1.). Differential expression was calculated with DESeq (negative binominal distribution) and p values were calculated using a false discovery rate of 10 %.

2.6. Retrotransposition assays

2.6.1. GFP-based retrotransposition assay

The L1-GFP reporter plasmids 99 PUR L1RP EGFP and 99 PUR JM111 EGFP (negative control; retrotransposition-defective due to two missense mutations in ORF1p) and the corresponding retrotransposition assay have been described previously (Ostertag et al., 2000). Both plasmids contain a CMV-GFP reporter cassette interrupted by a γ-globin in sense orientation in the opposite transcriptional orientation within the 3’ UTR of L1. Therefore, GFP is expressed only after splicing of full length L1 mRNA, reverse transcription, and insertion into the host genome. The L1 reporter plasmid and the indicated SAMHD1 expression plasmids or empty vector were transfected into HEK 293T cells (3 x 105 cells/6•well) at a ratio of 3:1 µg using calcium phosphate. Cells were selected by addition of 2.5 µg/ml puromycin two days posttransfection. After three days of selection, cells were harvested, and GFP-positive cells were quantified by flow cytometry.

2.6.2. Neomycin-based retrotransposition assays

For neomycin (neo)-based retrotransposition assays, reporter plasmids for L1 (pAD2TE1), IAP (pGL3-IAP92L23neoTNF) or MusD (pCMVmus-6-DneoTNF) were transfected into HeLa cells (4 x 105 cells/6-well) together with the indicated SAMHD1 expression plasmids or empty vector at a mass ratio of 2:1 µg using Lipofectamine2000 (Thermo Fisher). For the Alu retrotransposition assay, HeLa HA cells (4.5 x 105 cells/6-well) were transfected with 1.5 µg of the AluY reporter plasmid (pAlutet), 1.5 µg of an ORF2p expression plasmid (pAD500), together with 1 µg of the different SAMHD1 expression plasmids or empty vector using

37 Material and Methods

FuGENE HD transfection reagent (Promega). Similar to the L1-GFP reporter construct, the neomycin gene is interrupted by an intron in the opposite transcriptional orientation and is expressed only after successful retrotransposition. One day posttransfection, cells were detached using trypsin and transferred into 10 cm dishes. One or two days later, cells were selected for successful retrotransposition events by addition of 500 µg/ml G418. After complete selection (about 10 days), cells were washed three times with PBS, fixed with 1 % PFA in PBS, and G418-resistant foci were probed with 0.1 % crystal violet in 10 % ethanol. Foci were quantified using ImageJ software (NIH).

2.7. LINE-1 promoter assay

To determine the effect of SAMHD1 on L1 promoter activity, a L1 promoter construct driving luciferase reporter gene expression was used (Yu et al., 2001). One day prior to transfection, 3 x 104 HEK 293T cells were seeded in 48 wells. Cells were transfected with 0.3 µg of the L1 promoter reporter plasmid (L1RP-luc) together with 0.1 µg of empty vector (pcDNA6mycHis) or SAMHD1 expression plasmids using calcium phosphate. Two days posttransfection, cells were lysed in 250 µl 1x Cell Culture Lysis reagent (Promega). 50 µl lysate were transferred into a 96-well plate and luciferase activity was quantified using commercially available components (Promega).

2.8. LEAP assay

To determine L1 ORF2p-encoded reverse transcriptase (RT) activity in vitro, the L1 amplification protocol (LEAP), which has been described previously (Kulpa and Moran, 2006; Viollet et al., 2016), was used. This assay mimics the initial steps of target-primed reverse transcription of L1 RNA. For this purpose, 1 x 107 HEK 293T cells were seeded into T175 cell culture flasks. The next day, cells were cotransfected with 25 µg of the full-length L1 expression plasmid pAD2TE1 and 25 µg of expression plasmids for SAMHD1, ZAP (L) or empty vector (pcDNA6mycHis) using calcium phosphate. Two days posttransfection, cells were harvested using trypsin, washed twice with cold PBS, transferred into a 1.5 ml reaction tube and lysed in 1 ml cold LEAP assay lysis buffer by for 15 min at 4 °C. Lysates were centrifuged for 15 min at 20000 x g. Supernatant was transferred into a new tube and 10 µl were taken off as input control for western blot analysis. Lysates were mixed with 4 ml of the 8.5 % sucrose solution and layered over 6 ml of the 17 % sucrose solution. Centrifuge tubes were balanced with 8.5 % sucrose solution in pre-chilled SW41 Ti buckets. L1 ribonucleoprotein complexes (RNPs) were isolated by ultracentrifugation at 168 000 x g and 4 °C for at least 2 h. Depending on the pellet size, precipitates were re-suspended in 50-80 µl nuclease-free water supplemented with Halt Protease Inhibitor over night at 4 °C. Protein concentrations were determined with Bradford reagent (Sigma-Aldrich) and adjusted to 1.5 mg/ml. To control for successful precipitation of L1 RNPs, 30 µg of the RNP samples

38 Material and Methods were analyzed by western blot. RNA was isolated from 50 µg of the RNP samples using the NucleoSpin RNA® Kit (Macherey-Nagel) according to the supplier’s manual. 1 µg of the extracted RNA was treated with DNaseI using the DNA-free™ Kit (Thermo Fisher) according to the supplier’s manual in a 10 µl reaction. RNA was reverse transcribed using a 3’ RACE primer and M-MLV RT (Promega) for 1 h at 42 °C. To analyze ORF2p-mediated reverse transcription, 5 µg of RNP samples were incubated with 50 mM Tris-HCl (pH 7.5), 50 mM

KCl, 5 mM MgCl2, 10 mM DTT, 0.4 µM 3’ RACE primer, 20 U RNaseOUT, 0.2 mM dNTPs, and 0.05 % [v/v] Tween-20 for 1h at 37 °C. After reverse transcription, MLV RT and LEAP products were amplified by PCR using Phusion DNA polymerase (Thermo Fisher) together with the 5’ L1 primer (binding to the 3’ end of L1) and the 3’ RACE outer primer (binding to the linker region of the 3’ RACE primer used for reverse transcription).

Table 3: PCR program used to amplify cDNA of MLV-RT and LEAP reactions. Temperature [°C] Time Cycles 94 3 min 94 30 sec 56 30 sec 35 x 72 30 sec 72 7 min 12 hold

Amplified PCR products were separated on 2 % agarose gels and visualized with the QUANTUM ST5 imaging system (Peqlab).

2.9. Protein methods 2.9.1. Preparation of cell lysates In general, cells were harvested by centrifugation (3500 rpm, 5 min), washed with PBS and lysed in western blot lysis buffer for at least 30 min at 4 °C. Protein concentration was determined based on the Bradford assay (Sigma-Aldrich). Photometric readout was performed in a bio photometer (Eppendorf) at 560 nm. Protein samples were stored at •20 °C.

2.9.2. Co-immunoprecipitation

To analyze interaction of SAMHD1 and L1 encoded proteins, HEK 293T cells were lysed 48 h posttransfection in 300 µl immunoprecipitation lysis buffer for 30 min at 4 °C. RNase inhibitors were omitted from samples treated with 15 µg/ml RNaseA (Life technologies). Per sample, 50 µl magnetic Dynabeads (Thermo Fisher) were coupled with 2 µg of monoclonal anti-T7 or anti-FLAG antibodies in PBS-T for 20 min at RT. Afterwards, Dynabeads were incubated with cell lysates for 20 min at RT and washed three times with PBS-T. Protein

39 Material and Methods complexes were eluted 25 µl 50 mM glycine (pH 2.6) for 10 min at RT, supplemented with SDS sample buffer and analyzed by SDS-PAGE and western blot.

For immunoprecipitation of SAMHD1 with subsequent LEAP reaction (LEAP-IP), HEK 293T cells were lysed two days posttransfection and myc-tagged SAMHD1 was precipitated with 2 µg of a monoclonal anti-myc antibody coupled to 50 µl magnetic Dynabeads per reaction. Immunoprecipitation was performed as described above. Protein complexes were eluted in 50 µl 1x MLV-RT buffer (Promega). For the LEAP reaction, 5 µl of the eluate were directly subjected to the reaction as described in section 2.8. Samples for MLV-RT reactions were incubated for 10 min at 95 °C to denature ORF2p-encoded reverse transcriptase activity and 5 µl were used for MLV-RT-mediated cDNA synthesis as described in section 2.8.

2.9.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blot

To determine the expression of the proteins of interest, cell lysates were separated by SDS- PAGE and detected by immunoblot analysis. Depending on the protein of interest, 20-30 µg protein lysates were denatured in SDS sample buffer for 5 min at 95 °C. Samples were separated by SDS-PAGE in 10 % polyacrylamide gels. Separated proteins were transferred onto Immobilon PVDF membranes (Merck). Membranes were incubated in blocking buffer to avoid unspecific antibody binding and probed with different primary and the corresponding secondary antibodies.

2.10. Indirect immunofluorescence staining

For immunofluorescence analysis, HEK 293T, HeLa HA, or U2OS cells were grown on coverslips in 24-well dishes. In case of HEK 293T cells, coverslips were pre-coated with 50 µg/ml poly-D-lysine (Millipore). HEK 293T cells were transfected with Lipofectamine2000, HeLa HA and U2OS cells were transfected using FuGENE HD. In general, cells were transfected with a total amount of 0.4 µg DNA. Cells were transfected with pAD2TE1_ORF1- FLAG together with expression vectors for SAMHD1 or empty vector (pcDNA6mycHis) at a ratio of 3:1 µg. Two days posttransfection, cells were fixed with 4 % PFA in PBS at 37 °C for 15 min and permeabilized using 0.4 % saponin in PBS for 20 min at 4 °C. Antibodies were diluted in PBS containing 0.4 % saponin and 1 % FCS. Antibodies used for intracellular staining of SAMHD1-myc, L1 ORF1p-FLAG, and G3BP1 and the corresponding secondary antibodies are listed in section 1.5 of the used materials. Coverslips were mounted in Vectashield mounting medium containing DAPI (Vector laboratories) and analyzed by confocal microscopy using a Leica TCS SP5 confocal laser scanning microscope equipped with a 63x1.4 HCX PL APO CS oil immersion objective lens (Leica Microsytems). Images were analyzed using LAS AF software (Leica Microsystems) and processed with Adobe® Photoshop CS5.

40 Material and Methods

2.11. Intracellular dNTP quantifications

Intracellular dNTP levels were quantified by two different approaches, liquid chromatography- tandem analysis and dNTP incorporation assay. In both cases, HEK 293T shSAMHD1 cells were transfected with the different SAMHD1 mutants using the calcium phosphate method. Quantification of dNTPs by liquid chromatography-tandem mass spectrometry was performed by Dominique Thomas and Nerea Ferreirós (Goethe University Frankfurt) has been described previously (Thomas et al., 2015; Wittmann et al., 2015). Here, 1 x 106 cells were pelleted, lysed and the analytes were extracted by protein precipitation. Samples were chromatographically separated using an anion exchange HPLC column and analyzed in a 500 QTrap mass spectrometer. The calibration ranges in the injected solution were 4-1000 ng/ml for dTTP and dCTP, 2-500 ng/ml for dATP, and 4 - 500 ng/ml for dGTP. Quantification by the dNTP incorporation assay was performed by Caitlin Shepard and Baek Kim (Emory University Atlanta) as described previously (Diamond et al., 2004; Gramberg et al., 2013). Briefly, the lysates of 2 x 106 transfected cells were incubated with 5’-32P-labeled 23-mer oligonucleotides annealed to one of four distinct 24-mer templates with a single nucleotide overhang (A, C, G, or T). The template/primer was incubated with extracted cellular dNTPs and purified HIV-1 RT. Reactions were resolved by 20 % Urea-PAGE and single nucleotide incorporation was quantified by analyzing 32P-containing oligomers.

2.12. Genotyping

Mouse tail biopsies were lysed in 400 µl lysis buffer supplemented with 0.1 mg/ml proteinase K per tail and incubated for at least 4 h at 55 °C. Residual hair and bone fragments were pelleted by centrifugation (5 min, 14000 rpm) and supernatants were transferred into new tubes. Lysates were mixed with 500 µl isopropanol, incubated for 3 min at RT and DNA was precipitated by centrifugation (5 min, 14000 rpm). Supernatants were removed, precipitated DNA was washed with 1 ml 70 % ethanol per tube, and again centrifuged for 5 min at 14000 rpm. Supernatants were removed, and DNA pellets were dried for 10 min at RT. Afterwards, 50-100 µl pre-warmed TE buffer were added and DNA was resolved by shaking at 55 °C for at least 15 min. DNA concentrations were determined and 200 ng per sample were used for genotyping by PCR with the Phusion DNA polymerase (Thermo Fisher) according to the supplier’s manual and the respective primers (table 2). PCR products were separated on a 2 % agarose gel and visualized with the QUANTUM ST5 imaging system (Peqlab). STING mice were genotyped by single nucleotide sequencing (Macrogen) according to the manufacturer’s protocol.

41 Material and Methods

Table 4: PCR program used for genotyping of genomic mouse DNA. Temperature [°C] Time Cycles 95 4 min 95 30 sec 64 30 sec 35 x 72 45 sec 72 10 min 12 hold

Table 5: Size of DNA fragments obtained from genotyping PCR.

Gene wt del SAMHD1 245 bp 385 bp IFNAR 143 bp 376 bp cGAS 390 bp 312 bp STING 406 bp 406 bp

42 Results

V. RESULTS

1. The role of SAMHD1 in retroviral restriction and sensing

1.1. SAMHD1 and the cGAS/STING pathway restrict HIV-GFP infection in non-cycling human THP-1 cells

The antiviral activity of SAMHD1 was found to be regulated by cell cycle-dependent phosphorylation, enabling SAMHD1 to restrict HIV-1 infection only in non-dividing cells (reviewed in Herrmann et al., 2016). To confirm the cell cycle-dependent inhibition of HIV-1, the monocytic human cell line THP-1 was used. THP-1 cells can be differentiated with phorbol 12-myristate 13-acetate (PMA) to resting macrophage-like cells. Undifferentiated (•PMA) and PMA-differentiated (+PMA) THP-1 cells expressing a SAMHD1-specific (shSAMHD1) or control shRNA (shCtrl) were infected with a single-round infection HIV-1 pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G), which allows infection of a large variety of cell types. Additionally, the virus encodes GFP instead of the viral nef gene (HIV-GFP) to monitor viral infection. In •PMA THP-1 cells no difference in HIV-GFP infection between shSAMHD1 and shCtrl cells was found (Fig. 5A). This is likely due to the inactivation of endogenous SAMHD1 as a result of CDK-mediated phosphorylation at T592 in cycling cells (Cribier et al., 2013; Welbourn et al., 2013; White et al., 2013). In +PMA cells, the absence of SAMHD1 resulted in a 20-fold higher infection compared to SAMHD1-expressing shCtrl cells (Fig. 5A). Notably, the infection rate of differentiated THP-1 cells was generally lower than that of undifferentiated cells, most likely due to the induced cell cycle arrest. In summary, these data confirm that SAMHD1 restricts HIV-1 infection only in differentiated THP-1 cells.

The cytosolic DNA sensor cGAS was found to be involved in the sensing of HIV-1 cDNA in the cytoplasm of infected THP-1 cells (Gao et al., 2013). Upon binding of dsDNA, cGAS produces the second messenger cGAMP, which binds to and activates the downstream adaptor protein STING. STING in turn recruits and activates TBK1, which leads to phosphorylation of the transcription factor IRF3. In addition, STING induces the IκB kinase, resulting in the activation of NF-κB. Thus, IFN-I and pro-inflammatory cytokines are induced upon sensing of cytoplasmic DNA. However, the role of SAMHD1 in cGAS-mediated sensing of HIV-1 cDNA is still unclear. While Gao and colleagues showed that HIV-1 infection is recognized by cGAS in SAMHD1-containing cells, Lahaye and colleagues reported sensing of HIV-1 only in the absence of SAMHD1 (Gao et al., 2013; Lahaye et al., 2013).

43 Results

A B wt cGAS KO STING KO 80 shCtrl shSAMHD1

60 cells [%] cells

+ 40 SAMHD1 20

HIV-GFP Hsp90 0

mock -PMA +PMA HIV-GFP C D

60 21005 shCtrl shCtrl shSAMHD1 shSAMHD1

40 11005

cells [%] cells +

20 51004 HIV-GFP

0 0 Luciferase activity [RLU/s] Luciferaseactivity wt wt

cGAS KO STING KO cGAS KO STING KO HIV-GFP HIV-Luc

Figure 5. SAMHD1, cGAS, and STING impair HIV-GFP infection in differentiated THP-1 cells. (A) Undifferentiated (-PMA) and PMA-differentiated (+PMA) THP-1 shCtrl and shSAMHD1 cells were infected with VSV-G-pseudotyped HIV-GFP reporter virus at a MOI of 1. HIV-GFP positive cells were quantified by flow cytometry three days postinfection and are plotted as average of triplicate infections. Error bars represent the standard deviation. (B) SAMHD1 expression in wild-type (wt), cGAS KO, and STING KO THP-1 cells expressing SAMHD1-specific shRNA (shSAMHD1) or control shRNA (shCtrl) was analyzed by western blot analysis using an anti-SAMHD1 antibody. Hsp90 was stained as loading control. (C) PMA-differentiated THP-1 wild-type (wt), cGAS KO, or STING KO cells expressing either a shRNA targeting SAMHD1 (shSAMHD1) or a control shRNA (shCtrl) were infected with a VSV-G- pseudotyped HIV-GFP reporter virus at a MOI of 1. Three days postinfection, GFP-positive cells were quantified by flow cytometry. The percentage of HIV-GFP positive cells is shown as average of triplicate infections. Error bars indicate the standard deviation. One out of three independent experiments is shown. (D) PMA-differentiated THP-1 wt, cGAS KO, or STING KO cells stably expressing shRNA targeting SAMHD1 (shSAMHD1) or control shRNA (shCtrl) were infected with 1 x 105 counts per second (cps) of a VSV-G-pseudotyped HIV-Luc reporter virus. Infectivity was determined three days posttransfection by luciferase assay and is depicted in relative light units per second (RLU/s) as average of quadruplicate infections. Error bars represent the standard deviation. One out of two independent experiments is shown.

To analyze the role of cGAS and STING in HIV-1 infection, THP-1 cGAS and STING knockout (KO) cells were used. THP-1 cells are competent for sensing cytoplasmic DNA and have been previously used to study cGAS/STING-mediated detection of HIV-1 infection (Gao et al., 2013; Lahaye et al., 2013). Both cell lines were generated during a study of Mankan and colleagues using the CRISPR/Cas9 method and have been described previously (Mankan et al., 2014). To elucidate the role of SAMHD1 in the cGAS/STING pathway,

44 Results

SAMHD1 was depleted using a SAMHD1-specific shRNA (shSAMHD1). A vector encoding a scrambled shRNA (shCtrl) was used as control. The knockdown of SAMHD1 in the different cell lines was confirmed by western blot analysis (Fig. 5B). Twenty-four hours prior to infection, cells were differentiated with PMA and infected with either a VSV-G-pseudotyped HIV-GFP (Fig. 5C) or HIV-Luc reporter virus (Fig. 5D). Infected cells were quantified by flow cytometry or a luciferase-based reporter assay three days postinfection. Infection with both viruses yielded very similar results. As shown before (Fig. 5A), the infection rate of wt THP-1 cells depleted of SAMHD1 (wt shSAMHD1) was approximately 8-times higher compared to the corresponding control cells (shCtrl) (Fig. 5C and 5D). Knockout of cGAS resulted in up to 7.5-fold more infected cells compared to the wt control (Fig. 5C), indicating that cGAS impedes HIV-1 infection of SAMHD1-competent cells. Moreover, in the absence of SAMHD1, the infection of cGAS KO cells was further enhanced compared to cells lacking only SAMHD1 or cGAS (Fig. 5C and 5D). Infection of cGAS KO shSAMHD1 cells was almost 10•fold higher compared to cGAS single KO cells and up to 5-fold enhanced compared to wt shSAMHD1 cells. These findings demonstrate that SAMHD1 impedes a productive infection of cGAS-deficient THP-1 cells. In SAMHD1-competent but STING-deficient cells, the frequency of infected cells was approximately 3-fold higher compared to wt cells (Fig. 5C and 5D). In cells lacking both SAMHD1 and STING, more infected cells were found after infection with HIV-Luc (Fig. 5D), but not upon infection with HIV-GFP compared to wt shSAMHD1 cells (Fig. 5C). However, compared to STING single KO cells, the knockdown of SAMHD1 resulted in almost 6-times more infected cells (Fig. 5D). Notably, overall infection of cells lacking both STING and SAMHD1 was lower compared to cells deficient for cGAS and SAMHD1. In summary, a higher infection rate of differentiated THP-1 cGAS and STING KO compared to wt cells was found, which was further enhanced in the absence of SAMHD1. These findings indicate that the restrictive activity of SAMHD1 covers sensing of HIV-1 infection through the cGAS/STING pathway in monocytic human cells.

1.2. SAMHD1, signaling through the IFN-I receptor, and the cGAS/STING pathway impair HIV-GFP infection in primary murine BMDCs

To validate that SAMHD1 covers sensing of HIV-1 infection by the cGAS/STING pathway in an ex vivo model, primary cells isolated from different knockout mouse strains were used. In contrast to permanent cell lines, these primary cells represent a heterogenic mixture of different cell types, which secrete diverse cytokines and growth factors. These signal molecules can influence the sensing of HIV-1 infection and the establishment of an antiviral state.

45 Results

A B 60 10 n = 4 n = 5 8 40

6

cells [%] cells [%] cells

+ + 4 20

2

HIV-GFP HIV-GFP 0 0

wt wt

IFNAR KO STING KO SAMHD1 KO SAMHD1 KO

IFNAR/SAMHD1 KO STING/SAMHD1 KO C D 40 20 n = 3 n = 4

30 15

cells [%] cells [%] cells + 20 + 10

10 5

HIV-GFP HIV-GFP 0 0

wt wt

cGAS KO cGAS KO SAMHD1 KO SAMHD1 KO

cGAS/SAMHD1 KO cGAS/SAMHD1 KO Figure 6. The increased HIV-GFP infectivity in SAMHD1 KO BMDCs is further enhanced in the absence of IFNAR, or cGAS and STING. Infection of bone marrow-derived dendritic cells (BMDCs) of wild-type (wt), SAMHD1 knockout (KO), IFNAR KO, SAMHD1/IFNAR double KO (A), STING KO, STING/SAMHD1 double KO (B), cGAS KO, and cGAS/SAMHD1 double KO mice (C and D). Eight days poststimulation, cells were infected with a HIV-GFP reporter virus at a MOI of 1. After three days, the fraction of GFP-positive cells was determined by flow cytometry. The average of HIV-GFP-positive cells of triplicate infections is depicted. Horizontal lines represent the mean of GFP-positive cells of BMDCs from one genotype. Each symbol is representative for the mean of triplicate infections of BMDCs obtained from one mouse.

Murine SAMHD1 has previously been shown to act as an HIV-1 restriction factor both in vitro and in vivo (Behrendt et al., 2013; Rehwinkel et al., 2013; Wittmann et al., 2015). To elucidate the role of SAMHD1 on the IFN-I receptor (IFNAR)-mediated positive feedback loop during HIV•1 infection, bone marrow-derived dendritic cells (BMDCs) were isolated from wt, SAMHD1, IFNAR, and IFNAR/SAMHD1 double knockout (KO) mice. Subsequently, the cells were infected with VSV-G-pseudotyped HIV-GFP and two days postinfection, GFP- positive cells were quantified by flow cytometry. Quantification of GFP-positive cells isolated from IFNAR KO mice revealed that the cells were about 10-times more susceptible to HIV-

46 Results

GFP infection than wt cells (Fig. 6A). In addition, a nearly 28-fold enhancement of HIV-GFP infectivity in SAMHD1 KO BMDCs compared to wt cells was found, thereby confirming that murine SAMHD1 acts as a restriction factor for HIV-1 (Behrendt et al., 2013; Rehwinkel et al., 2013; Wittmann et al., 2015). Intriguingly, a nearly 70-fold higher infection rate was observed for IFNAR/SAMHD1 double KO compared to wt cells (Fig. 6A), demonstrating that the presence of IFNAR-mediated signaling further increases SAMHD1-dependent HIV-1 restriction in primary murine BMDCs. To address the role of the cGAS/STING pathway ex vivo, cGAS and STING KO mice were crossed with SAMHD1-deficient mice. BMDCs of cGAS and STING as well as the respective SAMHD1 double KO mice were isolated and infected with HIV-GFP reporter virus. Quantification of HIV-GFP-positive cells two days postinfection revealed no difference between wt and STING KO cells (Fig. 6B). Again, an almost 6-fold enhanced infection rate of SAMHD1 KO compared to wt cells was observed. Infection of BMDCs obtained from STING/SAMHD1 double KO mice lead to an approximately 12-fold enhanced infectivity compared to wt cells. However, the percentage of HIV-GFP-positive cells was decreased in STING/SAMHD1 double KO compared to SAMHD1 KO cells. Moreover, the absence of cGAS alone had none or only a slight effect on HIV-GFP infection (Fig. 6C, 6D). Interestingly, a greatly enhanced infection of cells lacking both SAMHD1 and cGAS compared to cGAS single KO cells was detected (Fig. 6D). These results suggest that the absence of essential modulators of the cGAS/STING pathway in presence of SAMHD1 is not sufficient to achieve an enhanced HIV-1 infection in vitro. Since only cells isolated from cGAS/SAMHD1 double KO mice displayed an enhanced infection compared to SAMHD1 KO BMDCs, SAMHD1 covers cGAS-dependent sensing of HIV-1 infection. Conclusively, these in vitro experiments demonstrate that the increased HIV-1 infectivity in SAMHD1 KO BMDCs is further enhanced in the absence of the IFN-I receptor, cGAS, or STING. Thus, these data highlight the role of SAMHD1 in covering HIV-1-induced cGAS/STING-dependent immune responses.

1.3. HIV-GFP infection induces the expression of different genes in wt and SAMHD1 KO BMDCs.

Loss of SAMHD1 in mice has been shown to result in a spontaneous upregulation of interferon-stimulated genes (ISGs) in the absence of viral infection, resembling the autoimmune reaction that is found in AGS patients (Behrendt et al., 2013). Therefore, SAMHD1 KO mice represent an ideal system to study the basic principles that lead to this IFN-I signature. So far, it is still unclear how the spontaneous immune response in the absence of SAMHD1 is triggered. Furthermore, it still remains elusive whether the spontaneous and the HIV-1-induced immune responses in SAMHD1-deficient cells are detected through the same receptor and if similar or different genes are regulated. For this

47 Results purpose, the transcriptome of uninfected and HIV-GFP infected BMDCs from three wt, SAMHD1, IFNAR, and IFNAR/SAMHD1 double KO cells was analyzed by Illumina-based RNA sequencing 16 h postinfection. Resulting hits were aligned and mapped to a mouse reference transcriptome (mm9). Normalization was performed on a sample to sample basis by applying the Pearson’s correlation coefficient.

To answer the question which genes are differentially regulated in wt and SAMHD1 KO BMDCs upon infection, transcriptomes of uninfected and HIV-GFP-infected cells of both genotypes were examined. An up- or downregulation of log2-fold change of 1.5, corresponding to an absolute change of 2.8-fold, was used as a commonly accepted threshold. Analysis of significantly upregulated genes revealed that 250 transcripts were enhanced in wt cells (Table S1), whereas only 162 elevated genes were found for SAMHD1 KO cells (Fig. 7A, Table S2). Additionally, slightly more genes were downregulated in wt than in SAMHD1 KO BMDCs after infection (Fig. 7A). To determine the number of ISGs that were upregulated or downregulated in BMDCs obtained from wt and SAMHD1 KO mice after infection, the interferome.org online database was used. This database contains IFN-I, IFN- II, and IFN-III-induced ISGs, which are manually curated from publicly available microarray data that were significantly up- or downregulated upon IFN treatment relative to control samples. A default limit of 2-fold change in the expression upon IFN-treatment was used as search criterion. Moreover, filters for in vitro data and the species Mus musculus were applied. For wt BMDCs, 150 of the 250 (60 %) upregulated genes were found to be ISGs (Fig. 7A). In BMDCs of SAMHD1 KO mice, 114 out of 162 (70 %) elevated transcripts were identified as ISGs (Fig. 7A). Analysis of genes with decreased expression revealed 183 for wt (Table S3) and 135 transcripts for SAMHD1 KO cells (Table S4). However, no differences in the percentage of ISGs between wt and SAMHD1 KO cells were found. For both genotypes, the percentage of ISGs identified from downregulated transcripts was about 70 % (Fig. 7A). Although the expression of fewer transcripts is regulated in response to HIV-1 infection in SAMHD1-deficient cells, this infection induces a slightly higher percentage of ISGs.

48 Results

A 300 total genes 200 ISGs

100

0

-100 Number of up-/downregulated of Number genes upon HIV-GFP infection genesuponHIV-GFP -200 wt SAMHD1 KO wt SAMHD1 KO

B wt +HIV SAMHD1 KO +HIV Upregulation

Downregulation

Figure 7. Differential regulation of genes upon HIV-GFP infection in wt and SAMHD1 KO BMDCs. Bone marrow-derived dendritic cells (BMDCs) of three wild-type (wt) and SAMHD1 KO mice were infected with VSV-G-pseudotyped HIV-GFP reporter virus at a MOI of 1.5. Total cellular RNA was harvested 16 hpi and analyzed by transcriptome sequencing. RNA of uninfected cells served as control. (A) Upregulation (log2 > 1.5-fold; p-value < 0.05) or downregulation (log2 < -1.5-fold; p < 0.05) of transcripts upon infection was compared to uninfected control cells. ISGs were determined using the interferome.org database. (B) Genes that were up- or downregulated upon infection (+HIV) were analyzed for clustering of biological terms using the online DAVID Functional Annotation Tool (https://david.ncifcrf.gov/home.jsp; category GOTERM_BP_DIRECT).

49 Results

To analyze differences in gene clustering between HIV-1-infected wt and SAMHD1 KO compared to uninfected control cells, significantly up- or downregulated genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool. This online database provides a comprehensive gene ontology system in which genes are classified into a set of predefined clusters depending on their functional characteristics. For this purpose, gene ontology covers three different domains: (1) cellular compartments, (2) molecular functions, and (3) biological processes. To analyze differences in gene clustering of HIV-GFP-infected wt and SAMHD1 KO cells, the search was limited to biological processes. Using the DAVID online platform, 221 out of 250 genes for wt cells and 149 out of 162 genes for SAMHD1 KO cells were found within the database. Comparison of both genotypes revealed that more hits related to innate immune, inflammatory response, and defense response to viruses were found for SAMHD1 KO compared to wt cells (Fig. 7B). Analysis of downregulated genes resulted in 171 out of 183 for wt and 128 out of 135 DAVID IDs for SAMHD1 KO BMDCs. Comparison of both cell types displayed only minor differences in the clustering of downregulated transcripts (Fig. 7B). Taken together, infection of BMDCs isolated from SAMHD1 KO mice results in the upregulation of more genes that are associated with antiviral immune responses and inflammation compared to infected wt cells. This finding leads to the hypothesis that SAMHD1 impedes the induction of an anti-HIV-1 response in vitro.

1.4. Spontaneously elevated transcripts are further upregulated upon HIV-GFP infection in the absence of SAMHD1

To answer the question whether the same or different genes are upregulated during the spontaneous and the HIV-GFP-induced immune responses in the absence of SAMHD1, global gene expression profiles of uninfected wt and SAMHD1 KO BMDCs were compared to the transcriptome of HIV-GFP-infected cells. In the absence of viral infection, 166 genes were at least 2.8-fold upregulated in SAMHD1 KO cells compared to wt cells (Table S5). With the help of the database interferome.org, 45 of the 166 upregulated genes were identified as ISGs, which resembles the spontaneous IFN-I signature that is endogenously induced in the absence of SAMHD1. Interestingly, 107 of the originally identified 166 genes were further upregulated in SAMHD1-deficient cells upon HIV-GFP infection (Fig. 8).

50 Results

40000

30000 wt 20000 wt +HIV SAMHD1 15000 SAMHD1 +HIV

10000

5000

number of readsnumber of 1500

1000

500

clu irg1 il1b six1 ppbp vcan cxcl1 ccl17 h2-m2apol7c slc2a6dusp5 ifi27l2ah2-eb2 eps8l2 adam23 gm8221 mab21l3 zmynd15 Figure 8. HIV-GFP infection results in the additional upregulation of spontaneously elevated transcripts in SAMHD1 KO BMDCs. Bone marrow-derived dendritic cells (BMDCs) of three wild-type (wt) and SAMHD1 KO mice were infected with a VSV-G-pseudotyped HIV-GFP reporter virus at a MOI of 1.5. RNA was harvested 16 hpi and analyzed by transcriptome sequencing. RNA of uninfected cells served as control. The number of reads per mRNA transcript of uninfected and infected cells (+HIV) is plotted for the top 20 genes that showed the highest upregulation in SAMHD1 KO BMDCs upon infection.

The most prominent upregulation in SAMHD1 KO cells was observed for irg1 (immune responsive gene 1) and ppbp (pro-platelet basic protein), which transcript levels were almost 40-fold (irg1) and 8-fold (ppbp) enhanced upon HIV-GFP infection, respectively (Fig. 8). Irg1 acts as an enzyme that produces itaconic acid, which exhibits antimicrobial effects (Michelucci et al., 2013). Ppbp, which is also known as CXCL7, belongs to the C-X-C motif ligand family and represents a potent chemoattractant and activator of neutrophils (Di Stefano et al., 2009).

Collectively, this finding demonstrates an additional, virus-induced upregulation of spontaneously elevated transcripts in the absence of SAMHD1. Since 65 % of spontaneously upregulated genes were further enhanced upon HIV-1 infection in SAMHD1 KO BMDCs, indicating that most of the genes play a role in both, the autoimmune reaction as well as the antiviral response in the absence of SAMHD1.

1.5. Analysis of highly upregulated genes in SAMHD1 KO BMDCs upon HIV-GFP infection To address the question, which genes were highly upregulated in SAMHD1 KO BMDCs upon HIV-1 infection, the transcriptome of infected SAMHD1 KO cells was compared to that of infected wt cells. The comparison revealed 37 genes that are at least 2.8-fold upregulated in the absence of SAMHD1 (Fig. 6A). These genes most likely result from more HIV-RT

51 Results products which are sensed in the absence of SAMHD1. The most prominent difference was obtained for ppbp, which transcript levels were more than 8-fold higher in BMDCs from SAMHD1 KO mice, followed by oasl1 (oligoadenylate synthase-like 1) and dnah2 (dynein axonemal heavy chain 2), which were more than 4-fold upregulated (Fig. 9A and B). Oasl1 belongs to the Oas family of oligoadenylate synthases but has no enzymatic activity. Nevertheless, it has a potent antiviral activity due to its coactivating role in the RIG-I pathway of RNA sensing (Zhu et al., 2014).

A wt +HIV SAMHD1 KO +HIV ppbp 1614,2 13048,9 oasl1 928 3730,8 dnah2 345 1386,4 ly6a 260,8 891,5 h2-eb2 92,4 614,9 egr1 232,3 612,6 ccl17 76 437 nxpe4 94,9 414,6 apol7c 68,6 414,4 mgl2 82 392 mylk 98,9 356,8 lad1 49,2 296,8 asgr2 12,8 193,3 apol10b 42,4 149,7 stxbp6 24,6 110,6

low high transcript levels

52 Results

B ppbp oasl1 dnah2 cxcl10 10.0 5.0 5.0 3.0

8.0 4.0 4.0 2.0 6.0 3.0 3.0

4.0 2.0 2.0 1.0 2.0 1.0 1.0

0.0 0.0 0.0 0.0

ifit1bl1 pydc4 ms4a4c ifit1 3.0 3.0 3.0 2.5

2.0 GFP GFP infection

- 2.0 2.0 2.0

1.5 HIV 1.0 1.0 1.0 1.0

0.5 upon upon

wt 0.0 0.0 0.0 0.0

ifi206 ifi44 isg15 oas3 2.5 2.0 2.0 2.0

2.0 1.5 1.5 1.5

compared to to compared 1.5 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5

0.0 0.0 0.0 0.0 upregulation upregulation

isg20 irg1 fold 1.5 1.5 ppbp 10 wt 1.0 1.0 8 SAMHD1 KO IFNAR KO 0.5 60.5 IFNAR/SAMHD1 KO

4 0.0 0.0 2

Figure 9. Infection with HIV-GFPupregulation+HIV results in an upregulation of ISGs in SAMHD1 KO compared to wt BMDCs. Bone marrow-derived0 dendritic cells (BMDCs) of three wild-type (wt), SAMHD1, IFNAR, and IFNAR/SAMHD1 double knockout (KO) mice were infected with a VSV-G-pseudotyped HIV-GFP reporter virus at a MOI of 1.5. RNA was harvested 16 hpi and analyzed by transcriptome sequencing. RNA of uninfected cells served as control. (A) Comparison of SAMHD1 KO with wt cells revealed 37 genes that were strongly enhanced in SAMHD1 KO compared to wt cells. The number of reads per mRNA transcript for the top 15 genes that showed higher induction in SAMHD1 KO over wt cells upon infection (+HIV) is depicted. The colored scale indicates low (blue) or high (red) numbers of mRNA reads. (B) Obtained hits were compared to candidate genes already published within the literature. The fold upregulation relative to the wt upon HIV-GFP infection is shown and depicted as bar graphs.

To validate the results of the RNA sequencing assay, obtained hits were compared to candidate genes that were already reported within the literature (Fig. 9B). In agreement with previous results, these genes were upregulated in SAMHD1 KO compared to wt cells upon HIV-GFP infection (Johnson et al., 2018; Maelfait et al., 2016). Notably, except dnah2 (dynein axonemal heavy chain 2), which was also elevated over wt level in the absence of the IFN-I receptor, all selected genes were downregulated in BMDCs obtained from mice

53 Results lacking IFNAR (Fig. 9B). This indicates that dnah2 is directly induced upon infection, whereas the other genes require the IFN-I feedback loop for higher expression. Taken together, SAMHD1 covers an IFN-I-dependent transcriptional upregulation of ISGs upon HIV-1 infection in primary murine BMDCs.

1.6. SAMHD1- and IFNAR-dependent gene regulation in HIV-GFP infected BMDCs

HIV-GFP infection of BMDCs isolated from SAMHD1 and IFNAR/SAMHD1 double KO mice revealed a nearly 3-fold enhancement of HIV-GFP infectivity in double KO compared to SAMHD1 single KO cells (Fig. 6A). To identify genes that are causing the inhibitory effect, significant transcript upregulation of at least 2.8-fold in SAMHD1 KO versus IFNAR/SAMHD1 double KO cells was analyzed. Comparison of RNA level in both cell types revealed an enhanced expression of 92 genes in SAMHD1 KO cells (Table S6, Fig. 10A). The most prominent upregulation was found for oas1g (oligoadenylate synthase 1g), which transcript level was almost 2000-fold enhanced in SAMHD1 KO cells. Oas1 catalyzes the synthesis of the second messenger 2’-5’ oligoadenylate, which activates RNaseL and thus leads to a degradation of viral . Moreover, the expression of Ifi44 (interferon-induced protein 44), a protein which suppresses the HIV-1 LTR promoter (Power et al., 2015), was nearly 750•fold enhanced (Fig. 10A). To obtain candidate genes that are highly upregulated only in the absence of SAMHD1, enhanced genes were compared to transcripts that were upregulated upon HIV-GFP infection in wt compared to IFNAR KO cells (Fig. 10B). Most of the genes that were only enhanced in SAMHD1 compared to double KO cells showed a decreased expression in SAMHD1-competent cells (wt vs. IFNAR KO). This finding was most obvious for ifi44, the CD20 homolog ms4a4c (membrane-spanning 4 domains subfamily A member C), and the very large interferon-inducible GTPase 1 (vlig1). However, five of the identified genes are also upregulated in cells expressing SAMHD1, namely oas1a, rtp4 (receptor-transporting protein 4), ifi206 (interferon-inducible gene 206), ifit3b (interferon-induced protein with tetratricopoeptide repeats 3b), and pydc4 (pyrin domain-containing 4) (Fig 10B). Interestingly, Ifi206 represents the murine ortholog to the human DNA-sensor IFN-γ-inducible protein 16 (Ifi16), which is also responsible for the sensing of HIV-1 infection.

54 Results

A IFNAR/SAMHD1 KO SAMHD1 KO B SAMHD1 KO vs. upregulation wt vs. IFNAR KO +HIV +HIV IFNAR/SAMHD1 KO oas1g 0 509,7 1978,2 oas1g 1978,2 354,6 ifi44 0 210,8 744,4 ifi44 744,4 76,1 oas1a 6,3 1518,3 243,9 oas1a 243,9 568,1 rtp4 4,7 1266,4 242,2 rtp4 242,2 699,4 ifit1bl1 0,4 118,6 232,3 ifit1bl1 232,3 67,6 ifi206 1,5 359,7 187,4 ifi206 187,4 249,0 ms4a4c 1,2 199,9 152,2 ms4a4c 152,2 23,9 ifit3b 1,2 212,7 139,1 ifit3b 139,1 229,1 oasl2 44,6 5484,5 119,4 oasl2 119,4 95,0 ifit2 59,8 4874 79,9 ifit2 79,9 76,6 vlig1 1,2 102 79,9 vlig1 79,9 11,6 ifi27l2a 5,8 582,3 75,6 ifi27l2a 75,6 41,1 oas3 67,5 5042,1 70,0 oas3 70,0 37,0 pydc4 2,3 239,5 69,1 pydc4 69,1 304,4 cmpk2 29,3 928,2 29,9 cmpk2 29,9 25,1 irgb10 11,6 370,9 27,9 irgb10 27,9 18,9 irf7 157,1 4184,6 27,3 irf7 27,3 24,1 usp18 49,6 1510,3 24,6 usp18 24,6 14,2 zbp1 47,8 1219,8 21,3 zbp1 21,3 10,0 oasl1 187,5 3730,8 17,4 oasl1 17,4 12,6 mnda 45,2 644,9 16,0 mnda 16,0 8,9

ifi205 15,3 244 15,6 ifi205 15,6 14,1 GFP infection infection GFP

xaf1 61,5 1074,9 15,3 - xaf1 15,3 10,7 slfn5 586,1 7656,4 14,4 slfn5 14,4 10,3 slfn8 147,9 2108,9 13,0 slfn8 13,0 7,9 rsad2 561,1 6765,5 12,9 rsad2 12,9 19,4 slfn1 47,6 769,3 12,9 slfn1 12,9 6,0 mx2 20,4 316,5 12,4 mx2 12,4 7,0 ly6a 60,6 891,5 11,8 ly6a 11,8 4,2 ifi47 116,2 1262,6 10,6 ifi47 10,6 6,7 cxcl10 48,4 552,8 10,6 cxcl10 10,6 5,9 ifi203 121 1254,6 9,6 ifi203 9,6 7,1 ifit1 278,6 2778,8 9,5 ifit1 9,5 19,7 phf11d 28 258,7 9,3 HIV upon upregulation phf11d 9,3 4,5 h2-t24 196,3 1695,7 8,2 h2-t24 8,2 6,9 ifitm3 301,5 3219,7 8,1 ifitm3 8,1 4,9 phf11a 15,6 147,5 7,9 phf11a 7,9 9,4 ddx60 221 1843,7 7,4 ddx60 7,4 4,7 ifi27 201,4 1580,2 7,3 ifi27 7,3 6,4 isg15 265,1 1884 7,2 isg15 7,2 6,8 oas1b 37,3 259,2 6,4 oas1b 6,4 7,0 phf11b 72 538,2 6,4 phf11b 6,4 3,1 mgl2 55 392 6,1 mgl2 6,1 3,3 ppbp 1846 13048,9 5,9 ppbp 5,9 1,7 ddx58 566,9 3351,3 5,8 ddx58 5,8 4,4 trim30d 345,4 2215,7 5,7 trim30d 5,7 4,3 siglec1 151,6 963,3 5,7 siglec1 5,7 2,1 gbp7 80,7 407,6 5,3 gbp7 5,3 7,5 pyhin1 309 1827,8 5,1 pyhin1 5,1 4,0 isg20 37,6 198,4 5,1 isg20 5,1 4,1

slightly upregulated highly upregulated

Figure 10. SAMHD1- and IFNAR-dependent transcript upregulation upon HIV-GFP infection in murine BMDCs. Bone marrow-derived dendritic cells (BMDCs) of three wild-type (wt), SAMHD1 KO, IFNAR KO, and IFNAR/SAMHD1 double knockout (KO) mice were infected with a VSV-G- pseudotyped HIV-GFP reporter virus at a MOI of 1.5. RNA was harvested 16 hpi and analyzed by transcriptome sequencing. RNA isolated from uninfected cells served as control. Genes that were at least 2.8-fold upregulated in SAMHD1 KO cells over double KO cells upon infection were analyzed (p < 0.05), resulting in a number of 98 genes. (A) The reads per gene obtained for double KO and SAMHD1 KO cells after infection as well as the resulting upregulation are depicted. (B) Genes that were enhanced in SAMHD1 KO over IFNAR/SAMHD1 KO BMDCs were compared to their upregulation in wt over IFNAR KO cells. The fold upregulation upon infection is depicted. The 50 most highly upregulated genes out of 98 are shown. The colored scale indicates slightly upregulated (white to light red) or highly upregulated (dark red) genes.

55 Results

Taken together, these 92 transcripts that were upregulated in SAMHD1 over IFNAR/SAMHD1 double KO cells represent candidate genes which might be responsible for the enhanced infection of IFNAR/SAMHD1 double KO compared to SAMHD1 KO BMDCs.

1.7. Analysis of genes which are directly induced upon HIV-GFP infection in murine BMDCs To answer the question which genes are directly upregulated in BMDCs upon HIV-1 infection, transcripts that were significantly enhanced more than 2.8-fold in infected compared to uninfected IFNAR/SAMHD1 double KO cells were examined. This analysis revealed 92 genes (Table S7). To identify SAMHD1-dependent genes, the upregulation of these candidate genes was compared to the transcriptome of IFNAR KO BMDCs (Fig. 11). Most of the genes that were enhanced in double KO cells were also highly upregulated in IFNAR KO cells upon infection. Interestingly, this comparison also identified genes that were upregulated in double KO compared to IFNAR KO cells, for example ifit1 (interferon-induced protein with tetratricopeptide repeats 1), oasl1, and ddit4 (DNA damage-inducible transcript 4). The greatest difference was observed for ifit1, which was over 20-fold enhanced in double KO cells and has been reported to inhibit HIV production (Nasr et al., 2017). Collectively, over 90 genes were identified to be directly upregulated upon HIV-GFP infection. Analysis using the interferome.org online database revealed that 54 of these genes are ISGs. This finding indicates that HIV-1 infection itself results in the expression of antiviral genes that do not require the positive IFNAR-dependent feedback loop for their induction. These genes include several members of the C-X-C motif chemokine ligand family, including CXCL1 and CXCL3, as well as other chemokines, for example IL-1β and TNF. Upregulation of these signaling molecules could provide an early defense mechanism against HIV-1 infection.

56 Results

IFNAR/SAMHD1 KO IFNAR KO tmem236 62,2 64,0 gramd1c 48,5 53,1 irg1 36,0 41,9 ifit1 28,4 6,1 karma 21,1 20,4 ccdc141 17,9 21,4 susd2 16,1 18,9 gm19325 13,0 15,0 cxcl3 12,6 18,8 cxcl2 11,4 9,4 oasl1 10,6 2,8 cxcl5 10,2 11,0 cxcl1 10,0 8,5 ptgs2 9,6 13,2 ednrb 9,2 12,7 ddit4l 8,7 4,3

serpinc1 8,5 14,5 GFP infection infection GFP

- cp 8,3 9,1 tnf 8,2 6,1 HIV slpi 7,7 8,3 fam181b 7,3 14,4

fam71f2 6,9 19,8 upon upon tlcd2 6,5 12,0 gm15832 6,5 11,7 fam212b 6,4 2,7 gm12689 6,3 8,2 clec4e 6,0 6,1 slc6a9 5,4 7,1 cdo1 5,3 9,8

old upregulation upregulation old rsad2 5,2 2,7 f isg15 5,2 3,5 gtse1 5,0 3,1 rp24-492o4.8 4,9 5,9 h2-m2 4,7 5,2 sarg 4,6 6,4 il20rb 4,4 5,0 pcx 4,4 5,5 psrc1 4,4 3,1 sod2 4,3 4,7 tnfrsf10b 4,3 2,3 lgr4 4,2 5,8 il1b 4,1 3,6 six4 4,1 12,5 mss51 4,0 9,1 fosl1 4,0 3,9 hmcn2 3,9 3,6 orm1 3,8 4,5 saa3 3,8 5,0 slc19a2 3,8 2,9 treml4 3,8 4,5

slightly highly upregulated upregulated

Figure 11. Influence of SAMHD1 on directly upregulated genes upon HIV-GFP infection in the absence of the IFN-I receptor. Bone marrow-derived dendritic cells (BMDCs) of three wild-type (wt), SAMHD1, IFNAR, and IFNAR/SAMHD1 double knockout (KO) mice were infected with a VSV-G- pseudotyped HIV-GFP reporter virus at a MOI of 1.5. RNA was harvested 16 hpi and analyzed by transcriptome sequencing. RNA isolated from uninfected cells served as control. Genes that were upregulated in IFNAR/SAMHD1 KO BMDCs upon HIV-GFP infection (log2 > 1.5-fold; p < 0.05) were compared to the genes of IFNAR KO cells. The 50 most highly upregulated genes out of 92 transcripts are shown. The fold upregulation upon infection compared to uninfected control cells is depicted. Transcripts that are differentially regulated in both genotypes are highlighted with bold frames. The colored scale indicates slightly upregulated (white to light red) or highly upregulated (dark red) genes.

57 Results

1.8. Validation of transcriptome analysis results by quantitative RT-PCR In addition to transcriptome sequencing, upregulation of selected genes was confirmed by quantitative RT-PCR experiments in uninfected and HIV-GFP-infected BMDCs isolated from wt, SAMHD1, IFNAR, and IFNAR/SAMHD1 double KO mice. More transcripts for pydc4, irg1, and ifi206 were detected in SAMHD1 KO compared to wt cells 16 h postinfection (Fig. 11A). A B 120 100 pydc4 80 irg1 60 ifi206 oas3 isg20 50 40 ifi44 40 pydc4

30 20

20 fold upregulation +HIV upregulation fold

10

0 upregulation +HIV fold 0

wt wt

IFNAR KO IFNAR KO SAMHD1 KO SAMHD1 KO

IFNAR/SAMHD1 KO IFNAR/SAMHD1 KO Figure 11. Validation of the RNA sequencing results on the differential regulation of selected ISGs by quantitative PCR. (A) Bone marrow-derived dendritic cells (BMDCs) of three wild-type (wt), SAMHD1, IFNAR, and IFNAR/SAMHD1 double knockout (KO) mice were infected with a VSV-G- pseudotyped HIV-GFP reporter virus at a MOI of 1.5. After 16 h, total RNA of infected and uninfected cells was isolated. Quantitative RT-PCR was performed with 40 ng cDNA input per reaction using the ΔΔCt method with gapdh as endogenous control. Upregulation of transcripts for pydc4, irg1, ifi206, oas3, and isg20 of infected compared to uninfected cells is depicted as average of three mice derived each from triplicate reactions. Error bars represent the standard error of the mean. The qRT-PCR analysis was performed by Franziska Kühner, Institute of Clinical and Molecular Virology, Friedrich- Alexander University Erlangen-Nürnberg. (B) BMDCs of three wt, SAMHD1 KO, IFNAR KO, and double-KO mice were infected with HIV-GFP at a MOI of 0.5. RNA of infected and uninfected cells was isolated 24 hpi. Quantitative RT-PCR was performed using the ΔΔCt method with gapdh as endogenous control. The upregulation of transcripts for ifi44 and pydc4 in infected compared to uninfected cells is depicted. The experiment was performed by Sabine Wittmann, Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg.

Notably, this upregulation was lost in the absence of functional IFN-I signaling, thereby confirming the results obtained from the transcriptome analysis (Fig. 8). However, irg1 represented one exception, since transcript levels for this gene were also enhanced in IFNAR and double KO cells, indicating that this gene is directly upregulated upon infection. Indeed, the transcriptome analysis identified irg1 to be highly upregulated in all cell types (Fig. 10B). Moreover, no difference in the transcript levels of oas3 and isg20 between wt and SAMHD1 KO cells was detected (Fig. 11A), thereby confirming the results obtained from the RNA sequencing experiment, in which only a slight increase in transcripts for both genes was

58 Results observed (Fig. 8). Analysis of ifi44 and pydc4 transcripts 24 h postinfection revealed that both genes are upregulated in SAMHD1 KO compared to wt cells upon HIV-GFP infection (Fig. 11B). Surprisingly, pydc4 was also found to be upregulated in IFNAR/SAMHD1 double KO cells to a level comparable to that of wt cells, which contradicts the results obtained from the qRT-PCR experiment performed 16 h postinfection (Fig. 11A and B). This finding suggests that differences in the regulation of ISG transcripts might exist which depend on the time point after infection.

In summary, this transcriptome analysis revealed candidate genes that are induced upon HIV-GFP infection in a SAMHD1- and IFNAR-dependent manner in primary murine BMDCs. These primary hits include genes that have been previously described within the literature in context of SAMHD1 or HIV-1 infection, thus validating the experimental approach. Moreover, new target genes were identified, thereby providing further insights into the HIV-1-induced innate immune response in the absence of the restrictive activity of SAMHD1.

2. The role of SAMHD1 in the inhibition of endogenous retroelements

2.1. Inhibition of LINE-1, but not HIV-1, is regulated by phosphorylation of SAMHD1 at T592 in cycling cells

Accumulation of endogenous retroelements has been discussed as a potential trigger for autoimmune reactions such as AGS by aberrant activation of cytosolic nucleic acid sensors. Recently, SAMHD1 has been shown to inhibit LINE-1 (L1) retrotransposition in cell culture (Hu et al., 2015; Zhao et al., 2013). Therefore, the inhibitory effect of SAMHD1 on L1 retrotransposition and its role in maintaining genome integrity to prevent accumulation of endogenous retroelements was analyzed. To identify the underlying mechanism of L1 restriction, a well-established L1 retrotransposition assay was used (Ostertag et al., 2000). The retrotransposition-competent L1-GFP reporter plasmid (99 PUR RPS EGFP) contains a GFP reporter cassette under the control of a CMV promoter inserted into the L1 3’ untranslated region (UTR) in antisense orientation. The GFP gene is interrupted by a γ•globin intron in sense orientation. Therefore, GFP is expressed only after splicing of full•length L1 mRNA and successful retrotransposition (Fig. 13A). As a negative control, a retrotransposition-defective mutant, which contains two miss-sense mutations within ORF1 (99 PUR JM111 EGFP, JM111) was used. Both plasmids encode a puromycin resistance gene as selection marker. The L1 reporter plasmids were transfected into HEK 293T cells and two days posttransfection, successfully transfected cells were selected by addition of puromycin to the cell culture medium. After 2-3 days of selection, successful retrotransposition events were quantified by flow cytometry gating for GFP-positive cells (Fig. 13B). To analyze the effect of SAMHD1 on L1 retrotransposition, L1-GFP reporter plasmids together with control vector (pcDNA) or increasing amounts of a SAMHD1

59 Results expression plasmid were transfected into HEK 293T cells. Analysis of retrotransposition events by flow cytometry revealed no GFP-positive cells for the defective JM111 control. Transfection of L1-GFP together with the empty control vector (pcDNA) resulted in approximately 7 % GFP-positive cells, showing that successful retrotransposition is a rather infrequent event. Coexpression of increasing amounts of SAMHD1 resulted in a dose- dependent inhibition of L1 retrotransposition up to 50 % at maximum (Fig. 13C). Since transfection of more than 1 µg of the SAMHD1 expression plasmid did not further decrease the number of L1-GFP-positive cells, a ratio of 3:1 µg L1 to SAMHD1 expression plasmid was used for the following experiments.

L1 Promoter A SD SA B

Plasmid DNA 5‘ UTR ORF1 ORF2 3‘ UTR GFP Intron E Transfection ofHEK 293T cells

Transcription Promoter CMV Splicing L1 Promoter 2 d

mRNA 5‘ UTR ORF1 ORF2 3‘ UTR EGFP Puromycinselection

DMEM 2,5 µg/ml puro CMV Promoter CMV

TPRT L1 Promoter 2-3 d

Genomic DNA 5‘ UTR ORF1 ORF2 3‘ UTR EGFP FACS analysis of

GFP+ cells CMV Promoter CMV

C GFP+ cells 8

6

cells [%] cells 4 +

2 L1-GFP 0

1 µg 2 µg JM111 pcDNA 0.5 µg 1.5 µg SAMHD1

LINE-1

Figure 13. Human SAMHD1 inhibits LINE-1 retrotransposition in a dose-dependent manner. (A) Illustration of the LINE-1 (L1) retrotransposition assay. (B) Schematic depiction of the L1-GFP reporter construct. L1 consists of a 5‘- and 3‘-UTR and two open reading frames (ORFs). An EGFP reporter cassette under the control of a CMV promoter is inserted into the 3‘-UTR in antisense orientation. The EGFP gene is interrupted by a sense orientation γ-globin intron (Intron). Splice donor (SD) and splice acceptor (SA) sites are indicated. EGFP is only expressed from its own CMV promotor after successful splicing, reverse transcription and integration into chromosomal DNA. TPRT: target- primed reverse transcription. (C) HEK 293T cells were transfected with a retrotransposition-competent or a retrotransposition-defective (JM111) L1-GFP reporter plasmid together with empty vector (pcDNA) or increasing amounts of SAMHD1. GFP-positive cells were quantified by flow cytometry five days posttransfection. The percentage of L1-GFP positive cells is shown as average of triplicate transfections. Error bars represent the standard deviation.

60 Results

SAMHD1 is phosphorylated at threonine 592 (T592) by the cyclin-dependent kinases (CDKs) 1 and 2 together with Cyclin A2 (Cribier et al., 2013; St Gelais et al., 2014; White et al., 2013b), which regulates its antiviral activity. Moreover, the dNTPase activity associated with the HD domain of SAMHD1 is critical for HIV-1 restriction (Goldstone et al., 2011; Lahouassa et al., 2012; Powell et al., 2011). The previous study from Zhao and colleagues did not show a difference in L1 restriction between the non-phosphorylated SAMHD1 mutant T592A and the phosphomimetic mutant T592D. Furthermore, the enzymatically inactive SAMHD1 mutant tested in their studies (D311A) still inhibited retrotransposition (Zhao et al., 2013).

A 10 B

8

6 cells [%] cells

+ pSAMHD1-T592 4 SAMHD1 (myc)

2 L1-GFP Hsp90 0

wt

JM111 pcDNA T592A T592D D207N LINE-1 C D 5

4

3

cells [%] cells + 2 SAMHD1 1 L1-GFP GAPDH 0

wt wt

pcDNA T592AD207N T592AD207N FLAG-wt w/o tag myc tag

LINE-1

Figure 14. Restriction of LINE-1 retrotransposition is regulated by phosphorylation at T592 and requires enzymatically active SAMHD1. (A) HEK 293T cells were cotransfected with a retrotransposition-competent or a retrotransposition-defective (JM111) LINE-1 (L1)-GFP reporter plasmid and either empty vector (pcDNA), SAMHD1 wt, the non-phosphorylated (T592A), the phosphomimetic (T592D), or the enzymatically inactive mutant (D207N). GFP-positive cells were analyzed by flow cytometry five days posttransfection. The percentage of L1-GFP positive cells is depicted as average of triplicate transfections. Error bars represent the standard deviation. (B) HEK 293T cells were transfected with empty vector (pcDNA) or expression plasmids for the indicated SAMHD1 mutants. Lysates were analyzed two days post transfection by immunoblotting using myc- and T592 phosphorylation-specific antibodies. Hsp90 was probed as loading control. One out of three independent experiments is shown. (C) HEK 293T cells were transfected with the L1-GFP expression plasmid together with empty vector (pcDNA), SAMHD1 wt, T592A, or D207N either with a C-terminal myc tag or without any tag sequence, or an N-terminally FLAG-tagged SAMHD1 wt (FLAG-wt). Five days posttransfection, GFP-positive cells were quantified by flow cytometry. The percentage of L1-

61 Results

GFP positive cells is shown as average of triplicate transfections with error bars indicating the standard deviation. (D) HEK 293T cells were transfected with the indicated SAMHD1 variants. Cell lysates were analyzed for SAMHD1 expression two days posttransfection by immunoblotting using an anti-SAMHD1 antibody. GAPDH was stained as loading control. One representative result out of three independent experiments is shown.

Since these findings stand in contrast to what is known for the SAMHD1-mediated restriction of HIV-1, the role of T592-phosphorylation and the enzymatically active site of SAMHD1 in L1 inhibition were examined. Therefore, HEK 293T cells were cotransfected with L1-GFP or the JM111 negative control together with either empty vector (pcDNA), SAMHD1 wt, the non- phosphorylated T592A, the phosphomimetic T592D, or the enzymatically inactive D207N mutant. The expression of the different SAMHD1 constructs compared to wt as well as their phosphorylation status at T592 was analyzed by western blotting. Analysis of L1-GFP- positive cells revealed a 20 % reduction of L1 retrotransposition in the presence of SAMHD1 wt (Fig. 14A). However, cotransfection of the non-phosphorylated T592A mutant almost completely abolished retrotransposition, reducing successful retrotransposition events to more than 90 %. In contrast, the phosphomimetic T592D mutant as well as the enzymatically inactive D207N mutant were not active against L1 (Fig. 14A). Analysis of the expression levels revealed that both phosphorylation mutants as well as the enzymatically inactive mutant are expressed to similar levels compared to wt SAMHD1 (Fig. 14B). Investigation of the phosphorylation status at T592 confirmed that only SAMHD1 wt and D207N are phosphorylated at this residue, whereas the phosphomimetic T to D exchange was not recognized by the antibody. Taken together, these results show that the inhibition of L1 retrotransposition is regulated by phosphorylation of SAMHD1 at T592. Wt SAMHD1 is only slightly active against L1 due to its phosphorylation at T592 in cycling HEK 293T cells. In addition, mutation of the enzymatically active site (D207N) prevented the anti-L1-activity, demonstrating that an enzymatic activity is required for L1 restriction.

To exclude potential effects of the C-terminal myc tag, SAMHD1 wt, T592A, and D207N were expressed as tagged and untagged versions. Flow cytometry analysis revealed that the SAMHD1 constructs without a tag sequence were generally more active against L1 than C- terminally myc-tagged SAMHD1 variants, displaying a 2-fold higher anti-L1 activity (Fig. 14C). Untagged SAMHD1 wt reduced retrotransposition to 70 %, whereas SAMHD1- myc only inhibited L1 replication to about 30 %. In addition, untagged SAMHD1 D207N retained half of its anti-L1 activity, whereas D207N-myc completely lost its ability to inhibit L1. However, analysis of the non-phosphorylated T592A mutant revealed that the restriction of L1 by untagged SAMHD1 is also regulated by phosphorylation at T592. Untagged SAMHD1 T592A further decreased retrotransposition to the remaining 30 % compared to the wt (Fig. 14C). Thus, the anti-L1 activity of untagged SAMHD1 is still regulated by phosphorylation and requires enzymatically active SAMHD1. Western blot analysis of both

62 Results myc-tagged and untagged SAMHD1 proteins indicated that the enhanced restrictive activity cannot be explained by higher protein expression of the untagged SAMHD1 variants (Fig. 14D). In addition, none of the tested constructs was as active against L1 as SAMHD1- FLAG, which was far more active than C-terminally myc-tagged SAMHD1 (Fig. 14C). Interestingly, FLAG-SAMHD1 inhibited retrotransposition more effectively than untagged SAMHD1. Since the amino acid sequences of all SAMHD1 wt proteins used within this assay were identical, these results indicate that tag sequences at both termini of SAMHD1 either positively or negatively influence its anti-L1 activity.

To further address the role of the T592 phosphorylation, the L1-GFP retrotransposition assay was performed in the presence of CDK inhibitors to prevent CDK-mediated phosphorylation of SAMHD1, or the DNA polymerase inhibitor aphidicolin to induce cell cycle arrest. To inhibit cellular CDKs, two different inhibitors were used. Olomoucine II is an ATP-competitive purine derivate which generally inhibits all CDKs and induces cell cycle arrest at G to M transition. In addition, a CDK2-specific inhibitor was used. Our group previously showed that treatment of cells with either inhibitor leads to dephosphorylation of SAMHD1 (Wittmann et al., 2015). Aphidicolin is a tetracyclic diterpene antibiotic isolated from Cephalosporum aphidicola. It specifically and reversibly inhibits DNA polymerase α and δ in eukaryotic cells and thereby induces cell cycle arrest at the early S phase. To determine the effect of CDK inhibitors on L1 replication, HEK 293T cells were transfected with the L1-GFP reporter plasmid and either empty vector (pcDNA) or an expression plasmid for SAMHD1. GFP positive cells were determined by flow cytometry four days posttransfection. Coexpression of SAMHD1 only slightly further reduced the number of L1-GFP-positive cells. In case of Olomoucine, SAMHD1 expression reduced the number of L1-GFP positive cells to 6 % compared to the empty vector control. Incubation with the specific CDK2 inhibitor after SAMHD1 overexpression further decreased the efficacy of retrotransposition by about 15 % compared to the respective pcDNA control (Fig. 15A). Taken together, no enhanced anti-L1 activity of wild-type SAMHD1 could be detected after treatment with CDK inhibitors.

63 Results

A B 10 8

8 6 pcDNA

6 cells [%] cells cells [%] cells SAMHD1

+ 4 + 4

2

2

L1-GFP L1-GFP 0 0 0 1 2 3 Aphidicolin [µg/ml] DMSO

Olomoucine LINE-1 CDK2 inhibitor LINE-1

Figure 15. Inhibition of cellular CDKs or aphidicolin-induced cell cycle arrest prevents LINE-1 retrotransposition. HEK 293T cells were transfected with the LINE-1 (L1)-GFP reporter plasmid together with either empty vector (pcDNA) or SAMHD1 wt. GFP-positive cells were analyzed by flow cytometry four days posttransfection. The percentage of L1-GFP positive cells is depicted as average of triplicate transfections. Error bars represent the standard deviation. (A) Six hours posttransfection, cells were treated with 3 µM Olomoucine II or 3 µM of a CDK2-specific inhibitor for four days. DMSO was used as solvent control. (B) Two days posttransfection, cells were treated with 1, 2 or 3 µg/ml Aphidicolin for two days. Untreated cells served as control.

Treatment of HEK 293T cells with aphidicolin for two days almost completely abolished L1 retrotransposition, most likely due to the induced cell cycle arrest (Fig. 15B). Therefore, no further restriction of L1 in presence of SAMHD1 could be detected. Collectively, both methods were not suitable to analyze the effect of SAMHD1 dephosphorylation during L1 retrotransposition. Incubation of HEK 293T cells with both, CDK inhibitors and aphidicolin, reduced the efficacy of retrotransposition already in the absence of SAMHD1. For this reason, no enhancing effects of the treatments on the anti-L1 activity of wild-type SAMHD1 could be detected.

Phosphorylation of SAMHD1 at T592 is thought to inactivate its anti-HIV-1 activity in cycling cells (Fig. 14B). In resting cells, however, phosphorylation at T592 is lost due to lack of CDK activity or activation of a phosphatase, thus enabling SAMHD1-mediated restriction of HIV-1 (Cribier et al., 2013; Welbourn et al., 2013; White et al., 2013b).

64 Results

80 pcDNA wt 60 T592A

D207N cells [%] cells

+ 40

20 HIV-GFP 0 0.1 0.3 0.7 MOI

Figure 16. SAMHD1 T592A does not restrict HIV-1 in cycling cells. HEK 293T cells were transfected with empty vector (pcDNA), wt SAMHD1, the non-phosphorylated (T592A), or the enzymatically inactive mutant (D207N). Two days posttransfection, cells were infected with a VSV-G- pseudotyped HIV-GFP reporter virus at the indicated MOIs. GFP-positive cells were determined three days postinfection by flow cytometry. The average of HIV-GFP positive cells of triplicate infections is depicted. Error bars indicate the standard deviation. One out of three independent experiments is shown.

To investigate whether overexpressed non-phosphorylated SAMHD1 T592A is able to restrict HIV-1 in cycling cells, HEK 293T cells were transfected with an empty vector (pcDNA), or plasmids encoding SAMHD1 wt, T592A, or D207N. Two days posttransfection, cells were infected with increasing amounts of VSV-G-pseudotyped HIV-GFP reporter virus ranging from 0.1 to 0.7 multiplicities of infection (MOI) (Fig. 16). HIV-GFP-positive cells were quantified three days postinfection by flow cytometry. No restriction of HIV-GFP could be detected in infected cells expressing wt SAMHD1 compared to the empty vector control at any MOI tested (Fig. 16). Furthermore, overexpression of the enzymatically inactive D207N mutant did not reduce the number of HIV-GFP-infected cells. Surprisingly, even overexpression of the non-phosphorylated T592A mutant had any effect on HIV-GFP infection, neither at low nor at high MOIs. Although restriction of both, L1 and HIV-1, is regulated by phosphorylation at T592 and requires enzymatically active SAMHD1, SAMHD1 T592A restricts only L1, but not HIV-1, in cycling HEK 293T cells. Therefore, SAMHD1 most likely controls both threats by a similar yet distinct mechanism.

65 Results

A B 3 shCtrl shSAMHD1

2

cells [%] cells +

pSAMHD1-T592 1

SAMHD1 L1-GFP Hsp90 0

wt

pcDNA D207N

LINE-1

Figure 17. Endogenous SAMHD1 does not inhibit LINE-1 retrotransposition. (A) The shRNA- mediated knockdown of endogenous SAMHD1 as well as its phosphorylation status was analyzed by immunoblot using a SAMHD1- and a T592 phosphorylation-specific antibody. Hsp90 was stained as loading control. One out of three independent experiments is shown. (B) HEK 293T shCtrl or shSAMHD1 cells were cotransfected with a LINE-1 (L1)-GFP reporter plasmid and either empty vector (pcDNA), SAMHD1 wt, or the enzymatically inactive D207N mutant. Five days posttransfection, GFP- positive cells were determined by flow cytometry. The percentage of L1-GFP positive cells is depicted as average of triplicate transfections. Error bars represent the standard deviation.

To determine the role of endogenous SAMHD1 in L1 retrotransposition, SAMHD1 knockdown cells were generated by transduction of HEK 293T cells with lentiviral vectors encoding either a shRNA targeting SAMHD1 (shSAMHD1) or a control shRNA (shCtrl). Efficient knockdown of endogenous SAMHD1 was confirmed by western blot analysis (Fig. 17A). The L1 retrotransposition assay was performed in shCtrl and shSAMHD1 cells transfected with the L1-GFP reporter plasmid and empty vector (pcDNA), SAMHD1 wt, or the inactive mutant D207N. Quantification of L1-GFP positive cells revealed no increase of retrotransposition in shSAMHD1 compared to shCtrl cells (Fig. 17B), indicating that endogenous SAMHD1 is not active against L1. Since SAMHD1 is phosphorylated at T592 in cycling HEK 293T cells (Fig. 17A), this finding provides an explanation for the lack of an anti- L1 activity of endogenous SAMHD1.

2.2. SAMHD1 inhibits various transposable elements in cycling cells

To exclude the possibility of cell type-specific effects in HEK 293T cells, additional L1 retrotransposition assays in HeLa cells were performed. Therefore, a L1 reporter plasmid, in which the EGFP reporter gene is replaced by a neomycin (neo) resistance gene interrupted by an intron (L1-neo), was used (Fig. 18A). L1-neo was cotransfected with empty vector (pcDNA), SAMHD1 wt, the highly active T592A, or the enzymatically inactive D207N mutant into HeLa cells. Two days posttransfection, cells were incubated in geneticin (G418)- containing medium to select for successful retrotransposition events. Retrotransposition- positive cells expand during selection and form G418-resistant foci, which can be stained with crystal violet and quantified by automated image analyses. Similar to the L1-GFP assay,

66 Results wt SAMHD1 and the inactive mutant D207N did not inhibit L1-neo. In contrast, expression of the unphosphorylated mutant T592A reduced the number of G418-resistant foci up to 90 % (Fig. 18B). These results show that the anti-L1 activity of SAMHD1 in HeLa cells is also regulated by phosphorylation at T592 and depends on a functional enzymatically active site. To answer the question whether SAMHD1 inhibits non-autonomous non-LTR retroelements as well, a retrotransposition assay with an Alu element was performed. Alu elements are short interspersed nuclear elements (SINEs) that depend on the enzymatic functions encoded by L1 ORF2p for successful retrotransposition (Fig. 18A). To investigate the role of SAMHD1 during Alu replication, an Alu reporter plasmid of the Y-subfamily (AluY) was used, which accounts for the vast majority of disease-associated insertions in humans (Carroll et al., 2001). The AluY-neo reporter construct was transfected into HeLa HA cells together with an expression plasmid for L1 ORF2p and plasmids encoding SAMHD1 wt, non- phosphorylated T592A, inactive D207N, or an empty vector (pcDNA). HeLa HA cells represent a distinct sub-cell line of HeLa cells, which supports retrotransposition of Alu elements (Hulme et al., 2007). Interestingly, SAMHD1 wt was more active against AluY-neo compared to L1-neo. In case of AluY, wt SAMHD1 reduced the number of G418-resistant foci by approximately 40 %, while it had no effect on L1-neo (Fig. 18B, C, and F). Furthermore, overexpression of the non-phosphorylated T592A mutant completely abolished AluY replication, whereas SAMHD1 D207N was not able to block retrotransposition (Fig. 18C). Together, the results show that SAMHD1-mediated restriction of AluY is regulated by phosphorylation at T592 and requires an enzymatically active HD domain. Since SAMHD1 inhibits AluY elements, which rely on L1 ORF2p for their replication, SAMHD1-mediated restriction most likely occurs on the level of ORF2p or an ORF2p- mediated enzymatic activity.

To analyze whether the SAMHD1-mediated inhibition of L1 and AluY is ORF2p-specific or if SAMHD1 can block other transposable elements as well, the inhibition of two murine LTR- containing retrotransposons, intracisternal A particle (IAP) and MusD, was analyzed (Fig. 18A). Reporter plasmids for IAP-neo or MusD-neo were cotransfected into HeLa cells together with either empty vector (pcDNA) or expression plasmids for SAMHD1 wt, T592A, or D207N.

67 Results

A Promoter SD SA

LINE-1 5‘ UTR ORF1 ORF2 3‘ UTR O Intron NE Promoter

Promoter SD SA NE O O NE

Alu A B Left An Right monomer Intron

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T592A D207N pcDNA6

D207N

Figure 18. SAMHD1 T592A inhibits LINE-1, AluY, IAP, and MusD replication in HeLa cells. (A) Schematic depiction of the LINE-1 (L1), Alu, IAP, and MusD-neomycin (neo) reporter constructs. All reporter elements encode a neo retrotransposition indicator cassette interrupted by an intron in reverse transcriptional orientation. Splice donor (SD) and splice acceptor (SA) sites are indicated.

68 Results

Resistance gene is expressed only after successful retrotransposition. UTR: untranslated region; ORF: open reading frame; A/B: A- and B-box Pol III promoter; LTR: long terminal repeat; gag: group-specific antigen; pro: protease; pol: polymerase. (B) HeLa cells were transfected with a L1-neo reporter plasmid together with either an empty vector (pcDNA), SAMHD1 wt, the highly active, non- phosphorylated T592A mutant, or the enzymatically inactive D207N mutant. (C) HeLa HA cells were cotransfected with an AluY-neo reporter plasmid, an L1 ORF2p expression plasmid, and either empty vector (pcDNA), or the indicated SAMHD1 variants. (D) HeLa cells were transfected with an IAP-neo reporter plasmid together with an empty vector (pcDNA) or indicated SAMHD1 expression plasmids. (E) HeLa cells were transfected with a MusD-neo reporter plasmid and either empty vector (pcDNA) or the indicated SAMHD1 variants. (F) To exclude cytotoxic effects of long-termed SAMHD1 expression, HeLa cells were cotransfected with pcDNA3.1, which encodes a neomycin resistance gene and an empty vector (pcDNA), or expression plasmids for different SAMHD1 mutants. In general, cells were cultured in geneticin (G418)-containing medium for 10 days. After complete selection, cells were fixed and stained with crystal violet. Stained foci were counted using ImageJ software. The number of G418-resistant foci is shown as average of triplicate transfections with error bars representing the standard deviation. (G) Representative crystal violet-stained G418-resistant foci of transfected cells for all reporter plasmids. One of three independent experiments is depicted.

Analysis of G418-resistant foci showed that SAMHD1 wt is slightly active against IAP, reducing the number of G418-resistant foci by about 30 %, but not against MusD (Fig. 18D, E, and F). However, expression of SAMHD1 T592A potently inhibited both LTR retrotransposons, whereas the enzymatically active site mutant D207N lost its restrictive activity (Fig. 18D and E). Cytotoxic effects of SAMHD1 were excluded by cotransfection of a plasmid encoding a neo resistance gene (pcDNA3.1) and SAMHD1 wt, T592A, D207N or the empty vector pcDNA6, which does not encode a neo resistance gene. Quantification of G418-resistant foci revealed that overexpression of the highly active T592A mutant slightly decreased the number of G418-resistant foci (Fig. 18F). However, this very weak reduction of G418-resistant colonies does not explain the strong inhibitory effect of SAMHD1 T592A on all transposable elements tested. Taken together, these results show that, in addition to the non-LTR retroelements L1 and Alu, SAMHD1 T592A efficiently blocks the replication of the murine LTR retrotransposons IAP and MusD. Since SAMHD1 D207N is not active against all transposable elements tested, these results indicate that the enzymatic activity of SAMHD1 is required for a broadly acting restriction of a variety of different transposable elements.

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2.3. Restriction of LINE-1 depends on an enzymatically active SAMHD1 and an intact allosteric binding sites

To identify the underlying mechanism of L1 restriction, a variety of SAMHD1 mutants that have been described previously in context of HIV-1 inhibition were tested for their anti-L1 activity.

A B HEK 293T HEK 293T shSAMHD1 15 2.5

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L428S/Y432S L428S/Y432S LINE-1 LINE-1 C

pSAMHD1-T592

SAMHD1 (myc)

Hsp90

Figure 19. Mutational analysis of human SAMHD1. HEK 293T (A) or HEK 293T shSAMHD1 cells (B) were transfected with a LINE-1 (L1)-GFP reporter plasmid together with either empty vector (pcDNA), SAMHD1 wt, the non-phosphorylated T592A mutant, the phosphomimetic T592D mutant, two enzymatically inactive mutants (D207N and D311N), the RNase mutant (Q548A), the allosteric site mutant (D137A), or the oligomerization mutant (L428S/Y432S). GFP positive cells were determined by flow cytometry five days posttransfection. The percentage of L1-GFP positive cells is shown as average of triplicate transfections. Error bars indicate the standard deviation. (C) SAMHD1 protein expression and its phosphorylation status were analyzed two days posttransfection by immunoblot using T592 phosphorylation-specific and myc-specific antibodies. Endogenous Hsp90 was stained as loading control. One out of three independent experiments is depicted.

As shown before, expression of wt SAMHD1 only slightly inhibited L1 retrotransposition to approximately 30 % (Fig. 19A). The non-phosphorylated T592A mutant almost completely abolished retrotransposition, whereas the phosphomimetic variant T592D was not able to restrict L1. Both enzymatically inactive SAMHD1 mutants (D207N, D311N) lost the ability to inhibit L1. In addition to its dNTPase activity, SAMHD1 has been postulated to inhibit HIV-1

70 Results infection by directly degrading HIV-1 RNA (Ryoo et al., 2014). Mutation of glutamine at position 548 to alanine (Q548A) was shown to inactivate the postulated RNase activity (Ryoo et al., 2014). Inducing this mutation in SAMHD1 completely abolished its anti-L1 activity (Fig. 19A), indicating that the RNase activity of SAMHD1 is important for L1 restriction. Co- factor induced tetramerization of SAMHD1 has been shown to be critical for dNTPase activity and restriction of retroviral infection (Brandariz-Nunez et al., 2013; Yan et al., 2013). Interfering with the allosteric dGTP/GTP-binding site (D137A), which prevents co-factor induced tetramerization, or mutating the oligomerization interface between the monomers (L428S/Y432S) impaired the anti-L1 activity of SAMHD1. To exclude possible side effects of endogenous SAMHD1 or oligomerization of the transfected mutants with the endogenously expressed SAMHD1, the assay was also conducted in HEK 293T shSAMHD1 cells (Fig. 19B). Analysis of L1 retrotransposition revealed very similar results in the absence of endogenous SAMHD1. Only SAMHD1 T592A potently inhibited L1. All other features of SAMHD1 tested in this assay seem to be important for inhibition of retrotransposition. Mutations of these features abrogate the ability of SAMHD1 to inhibit L1 replication, including the RNase activity and the oligomerization of SAMHD1. Interestingly, phosphorylation of SAMHD1 at T592 was diminished in SAMHD1 D137A and L428S/Y432S mutants, as indicated by immunoblot analysis using a phosphorylation-specific antibody (Fig. 19C). This indicates that tetramerization of SAMHD1 is required for efficient phosphorylation by CDK1/2 and Cyclin A2 and restriction of L1.

Except the unphosphorylated mutant T592A, all other SAMHD1 mutants did not show great differences compared to the wt protein regarding their anti-L1 activity. To further examine differences between the mutants, all SAMHD1 mutations were combined with the activating T592A amino acid substitution (Fig. 20A). In the T592A-background, both enzymatically inactive mutants (D207N/T592A and D311N/T592A) were not able to block L1 replication. This demonstrates that the highly active T592A mutant relies on the enzymatic active site of SAMHD1 to keep L1 in check. In agreement with the results for the single mutants, the allosteric site mutant in the T592A background (D137A/T592A) did not restrict retrotransposition. Surprisingly, the RNase (Q548A/T592A) as well as the oligomerization mutant (L428S/Y432S/T592A) were still able to inhibit L1 when combined with the activating T592A mutation, reducing the number of GFP-positive cells up to 70 % (Q548A/T592A) and almost 90 % (L428S/Y432S/T592A), respectively (Fig. 20A).

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A HEK 293T B HEK 293T shSAMHD1 6 3

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pcDNA T592AD207ND311NQ548AD137A pcDNA T592AD207ND311NQ548AD137A

L428S/Y432S L428S/Y432S T592A T592A

LINE-1 LINE-1 C T592A

SAMHD1 (myc)

Hsp90

Figure 20. Human SAMHD1 mutants in a constitutively active T592A background differentially restrict LINE-1 replication. HEK 293T (A) or HEK 293T shSAMHD1 cells (B) were transfected with a LINE-1 (L1)-GFP reporter plasmid together with either empty vector (pcDNA), SAMHD1 wt, the non- phosphorylated T592A mutant, the enzymatically inactive D207N/T592A and D311N/T592A mutants, the RNase mutant Q548A/T592A, the allosteric site mutant D137A/T592A, or the oligomerization mutant L428S/Y432S/T592A. GFP positive cells were determined by flow cytometry five days posttransfection. The percentage of L1-GFP positive cells is shown as average of triplicate transfections. Error bars indicate the standard deviation. (C) SAMHD1 protein expression was analyzed two days posttransfection by immunoblot using an anti-myc antibody. Endogenous Hsp90 was stained as loading control. One out of three independent experiments is depicted.

To exclude possible side effects of endogenous SAMHD1, these mutants were also tested for their anti-L1 activity in HEK 293T shSAMHD1 cells (Fig. 20B). Quantification of L1-GFP- positive cells in this cell line revealed no difference compared to the results obtained in wild- type cells, excluding an impact of endogenous SAMHD1 on L1 retrotransposition. Additionally, the expression level of the different mutants was analyzed (Fig. 20C). Immunoblot analysis revealed that all mutants are expressed equally to the wild type, except SAMHD1 D137A/T592A. Taken together, the results in the T592A-background demonstrate that the potential RNase activity and the oligomerization of SAMHD1 are dispensable for L1 restriction, whereas the allosteric cofactor binding-site is important for L1 inhibition.

72 Results

2.4. Murine SAMHD1 potently restricts retrotransposition of human LINE-1

In addition to human SAMHD1, the anti-L1 activity of murine SAMHD1 was analyzed. Due to alternative splicing, murine SAMHD1 exists in two isoforms, which differ in their C-terminal domain. Isoform 2 (Iso2) is seven amino acids shorter than isoform 1 (Iso1) and lacks the regulatory phosphorylation site at T603, which is equivalent to T592 in human SAMHD1 (Wittmann et al., 2015).

A 8

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pcDNA T603AT603DHD/AAD138A HD/AAD138A Iso1 Iso2

LINE-1 B 15

human murine

10 cells [%] cells + pSAMHD1-T592 5

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wt Iso1 Iso2 pcDNA T592A human murine LINE-1

Figure 21. Murine SAMHD1 potently inhibits LINE-1 retrotransposition. (A) HEK 293T cells were cotransfected with a L1-GFP reporter plasmid and either empty vector (pcDNA), isoform 1 (Iso1) or isoform 2 (Iso2) of murine SAMHD1, the non-phosphorylated T603A or the phosphomimetic T603D mutant of Iso1, the enzymatically inactive HD/AA mutants, or the allosteric site mutants (D138A). (B) HEK 293T cells were cotransfected with a L1-GFP reporter plasmid and the empty vector (pcDNA), human SAMHD1 or the non-phosphorylated T592A mutant, and murine SAMHD1 Iso1 or Iso2. GFP- positive cells were determined by flow cytometry five days posttransfection. The percentage of L1- GFP positive cells is shown as average of triplicate transfections. Error bars indicate the standard deviation. Two days posttransfection, SAMHD1 expression was analyzed by immunoblot using T592 phosphorylation- or myc-specific antibodies. Endogenous Hsp90 was stained as loading control. One out of three independent experiments is depicted.

73 Results

To examine the anti-L1 activity of murine SAMHD1, HEK 293T cells were transfected with the L1-GFP reporter plasmid together with an empty vector (pcDNA), or both isoforms of murine SAMHD1. Beyond the phosphorylation mutants T603A and T603D of Iso1, enzymatically inactive mutants (HD/AA), or allosteric binding site mutants (D138A) were analyzed. The phosphorylation status of Iso1 at T603 was investigated by western blot analysis using the human T592 phosphorylation-specific antibody. Detection of L1-GFP positive cells revealed that both murine isoforms efficiently blocked retrotransposition by more than 90 % (Fig. 21A). In agreement with the findings obtained for human SAMHD1, the non-phosphorylated T603A mutant of Iso1 was more active against L1 than wt, whereas the phosphomimetic mutant T603D did not restrict L1-GFP (Fig. 21A). However, the T603D mutant maintained a residual anti-L1 activity, which still reduced the number of L1-GFP positive cells by approximately 50 %. This finding indicates that the anti-L1 activity of murine SAMHD1 is not as tightly regulated as the inhibition mediated by human SAMHD1. Similar to what was found for human SAMHD1, mutations of the enzymatically active site (HD/AA) or the allosteric cofactor-binding site (D138A) interfered with the anti-L1 activity of both isoforms (Fig. 21A). However, the D138A mutant of Iso2 remained some of its inhibitory activity, restricting L1 retrotransposition up to 60 %. Interestingly, mutation of the allosteric site of Iso1 completely abolishes phosphorylation at T603 (Fig. 21A), indicating that cofactor- induced tetramerization of murine SAMHD1 is required for its phosphorylation at T603. Taken together, restriction of L1 replication by murine SAMHD1 is also regulated by phosphorylation and relies on the enzymatically active site as well as the allosteric cofactor- binding sites.

To directly compare the anti-L1 activity of human and murine SAMHD1, the L1-GFP reporter plasmid was cotransfected with human SAMHD1 wt or the non-phosphorylated T592A mutant, or both isoforms of murine SAMHD1 in HEK 293T cells. In parallel, the phosphorylation status as well as the expression level of the constructs was analyzed by immunoblotting. Quantification of L1-positive cells showed that non-phosphorylated Iso2 had a stronger effect on L1 retrotransposition than phosphorylated Iso1 (Fig. 21B). Interestingly, the phosphorylated Iso1 was far more active against L1 than the phosphorylated human wt, which again reduced the efficacy of retrotransposition to only 20 %. Moreover, non- phosphorylated Iso2 restricted L1 as efficient as the human T592A mutant (Fig. 21B). In summary, murine SAMHD1 controls retrotransposition more efficiently than human SAMHD1 despite being phosphorylated at T603 as shown for Iso1.

74 Results

2.5. Nuclear localization of SAMHD1 is not required for LINE-1 inhibition

Since nuclear localization of SAMHD1 is not required for HIV-1 restriction (Brandariz-Nunez et al., 2012), the impact of the subcellular localization of SAMHD1 on L1 restriction was examined. For this purpose, an NLS-mutant of SAMHD1 (SAMHD1 ΔNLS), in which the NLS-sequence KRPR within the C-terminus was mutated to AAPA, was generated (Hofmann et al., 2012). Cytoplasmic localization of the ΔNLS mutant was confirmed by immunofluorescence staining of transfected HeLa cells compared to wt SAMHD1 or T592A, which are located within the nucleus (Fig. 21A).

A DAPI SAMHD1 Merge B 6

wt

4

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wt NLS  pcDNA T592A LINE-1 ΔNLS

C

Figure 22. Nuclear localization of SAMHD1 is not required for LINE-1 restriction. (A) HeLa cells were transfected with the indicated SAMHD1 expression vectors. Two days posttranfection, cells were fixed, permeabilized, and SAMHD1 (red) was stained with an anti-myc and a corresponding Alexa555 antibody. Nuclear DNA was stained with DAPI (blue). Subcellular localization of SAMHD1 was analyzed by confocal microscopy. (B) HEK 293T cells were cotransfected with a LINE-1 (L1)-GFP reporter plasmid and either empty vector (pcDNA), SAMHD1 wt, the constitutively active, non- phosphorylated T592A mutant, or an NLS mutant (ΔNLS). GFP-positive cells were determined by flow cytometry five days posttransfection. The percentage of L1-GFP positive cells as average of triplicate transfections is shown with error bars indicating the standard deviation. (C) HEK 293T cells were transfected with an empty vector (pcDNA) or expression vectors for SAMHD1 wt or the NLS mutant (ΔNLS). Two days posttransfection, SAMHD1 expression and its phosphorylation status were analyzed by immunoblotting using myc- and T592 phosphorylation-specific antibodies.

75 Results

To analyze the anti-L1 activity of this ΔNLS mutant, HEK 293T cells were transfected with the L1-GFP reporter plasmid and empty vector (pcDNA), SAMHD1 wt, the highly active T592A, or the cytoplasmic ΔNLS mutant. Analysis of GFP-positive cells revealed that expression of the ΔNLS mutant potently restricts L1 retrotransposition, almost as efficient as the non-phosphorylated T592A mutant, with only approximately 10 % L1 retrotransposition remaining (Fig. 22B). Since CDK1/2 and Cyclin A2 are predominantly located within the nucleus, the phosphorylation status of the ΔNLS mutant was examined by western blot analysis. Interestingly, the SAMHD1 ΔNLS mutant was phosphorylated at T592 similar to the wt (Fig. 22C). Taken together, cytoplasmic SAMHD1 is far more active against L1 than nuclear SAMHD1, despite being phosphorylated at T592.

2.6. The dNTPase activity of SAMHD1 is not sufficient for LINE-1 restriction

The dNTPase activity of SAMHD1 is thought to be the main mechanism for controlling HIV-1 infection (Goldstone et al., 2011; Lahouassa et al., 2012; Powell et al., 2011). Since the mutational analysis of SAMHD1 in the L1 retrotransposition assay revealed that an enzymatic activity of SAMHD1 is required for restriction, it was conceivable to test the ability of the different mutants to degrade cellular dNTPs. HEK 293T shSAMHD1 cells were transfected with either empty vector (pcDNA), SAMHD1 wt, or the different SAMHD1 mutants. Empty vector transfected HEK shCtrl cells served as control to determine the dNTP level in the presence of endogenous SAMHD1. Intracellular dNTP levels were quantified either by HPLC (Fig. 23A) or by a single nucleotide incorporation assay (Fig. 23B) two days posttransfection. Both assays yielded very similar results. The intracellular dNTP level of control vector (pcDNA) transfected shSAMHD1 cells were about 30-40 % higher than those of shCtrl cells, demonstrating that endogenous SAMHD1 of cycling HEK 293T cells acts as a dNTP hydrolase. All mutants except the enzymatically inactive D207N/T592A mutant efficiently depleted the intracellular dATP and dGTP level. In cells expressing SAMHD1 D207N/T592A the level of dATP and dGTP were almost as high as the ones obtained from empty vector-transfected cells. In case of dCTP and dTTP, the results were less clear. Quantification of dATP, dGTP, and dTTP revealed no difference between the presence of wt SAMHD1, the non-phosphorylated T592A, or the phosphomimetic T592D mutant. Only in case of dCTP, the T592D mutant showed an impaired hydrolase activity. These results indicate that the phosphorylation of SAMHD1 at T592 regulates its anti-L1, but not its dNTPase activity in cycling HEK 293T cells. However, the lack of dNTP degradation of the enzymatically inactive D207N/T592A mutant correlates with its failure to restrict L1. Together, these findings suggest that the dNTPase activity of SAMHD1 is necessary but not sufficient for efficient inhibition of L1. Moreover, the ΔNLS mutant was found to degrade cellular dNTPs as efficient as the wt, indicating that the subcellular localization of SAMHD1 is not important for its ability to degrade dNTPs.

76 Results

A B HPLC Single nucleotide incorporation assay 15 10

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77 Results

C 2.5 pcDNA 2.0 wt T592A

1.5

cells [%] cells + 1.0

0.5 L1-GFP 0.0 0 1 1.5 2 2.5 nucleosides [mM] LINE-1

Figure 23. The dNTPase activity of SAMHD1 is necessary, but not sufficient for LINE-1 restriction. (A and B) HEK 293T shCtrl and shSAMHD1 cells were transfected with empty vector (pcDNA) or expression plasmids for SAMHD1 wt, the non-phosphorylated T592A, the phosphomimetic T592D, the enzymatically inactive D207N/T592A, the RNase mutant Q548A/T592A, the allosteric site mutant D137A/T592A, the oligomerization mutant L428S/Y432S/T592A, or the ΔNLS mutant of SAMHD1. Cells were lysed two days posttransfection and intracellular dNTP levels were determined by HPLC analysis (A) or a single dNTP incorporation assay (B). The average of triplicate measurements for dATP, dGTP, dCTP, and dTTP in µM is shown. Error bars indicate the standard deviation. (C) HEK 293T cells were transfected with a LINE-1 (L1)-GFP reporter plasmid and either empty vector (pcDNA), SAMHD1 wt, or the constitutively active T592A mutant. Cell culture medium was supplemented with nucleosides at the indicated concentrations during puromycin selection. GFP- positive cells were analysed four days posttransfection by flow cytometry. The percentage of L1-GFP positive cells is depicted as average of duplicate transfections.

To directly examine the effect of the dNTPase activity on L1 replication, a retrotransposition assay with increasing amounts of exogenously added nucleosides (dNs) was performed, which are intracellularly phosphorylated to nucleoside triphosphates (dNTPs). In case of HIV–1 infection, addition of dNs has been shown to counteract the inhibitory effect of SAMHD1 (Baldauf et al., 2012; Lahouassa et al., 2012). HEK 293T cells were transfected with the L1-GFP reporter plasmid and either empty vector (pcDNA), SAMHD1 wt, or the non- phosphorylated, highly active T592A mutant. Increasing amounts of dNs were added to the cell culture medium two days posttransfection for two days. L1-GFP positive cells were determined four days posttransfection by flow cytometry. Notably, addition of more than 1.5 mM dNs resulted in a reduction of retrotransposition efficacy to about 50 %, most likely due to cytotoxic effects of ammonium hydroxide, the solvent of deoxyguanosine (Fig. 23C). Interestingly, the difference in L1 inhibition between the slightly active SAMHD1 wt and the highly active T592A mutant became less prominent with increasing amounts of nucleosides. These results indicate that the dNTPase activity of SAMHD1 might be involved in L1 restriction. However, due to the reduced retrotransposition efficacy, these findings are inconclusive.

78 Results

2.7. SAMHD1 does not affect LINE-1 expression

Next, it had to be clarified at which step of the L1 life cycle the SAMHD1-mediated restriction occurs. Therefore, the influence of SAMHD1 on L1 promoter activity, L1 transcription, and L1 protein expression was examined.

A B

6 ORF1p (T7) 10 8 1.510 T592A SAMHD1 (myc) D207N ZAP (HA) 6 ZAP (L) 10 1.010 6 Hsp90

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E F LINE-1 LINE-1 T592A D207N T592A D207N SAMHD1 (myc) SAMHD1 (myc)

ORF1p (T7) ORF2p (FLAG)

Hsp90 Hsp90

Figure 24. SAMHD1 T592A does not affect LINE-1 expression. (A) HEK 293T cells were cotransfected with a LINE-1 (L1) promoter construct driving luciferase reporter gene expression (L1 promoter-Luc) and either empty vector (pcDNA) or the indicated SAMHD1 expression plasmids. Cells were lysed two days posttransfection and the luciferase activity (relative light units, RLU) was determined in quadruplicates. The average RLU/s of three independent transfections is depicted. Error bars indicate the standard deviation. (B) HEK 293T cells were cotransfected with a full-length L1 expression plasmid (pAD2TE1) and plasmids for constitutively active (T592A), enzymatically inactive SAMHD1 (D207N), or the long isoform of the zinc finger antiviral protein (ZAP (L)). Total cellular mRNA was isolated at the indicated time points posttransfection and analyzed by qRT-PCR with primers targeting the T7 tag sequence fused to ORF1. A serial dilution of pAD2TE1 served as standard curve. The average number of L1 copies per 100 ng RNA input from triplicate reaction is shown. Error bars indicate the standard deviation. Two days posttransfection, protein expression was analyzed by immunoblot using T7- (ORF1p), myc- (SAMHD1), and HA-specific antibodies (ZAP). (C and D) HEK 293T cells were transfected with the L1 reporter plasmid pAD2TE1 encoding T7- tagged ORF1p or an ORF2p-3xFLAG expression plasmid together with plasmids encoding GFP, or the human SAMHD1 proteins wt SAMHD1, T592A, T592D, or D207N. Two days posttransfection, immunoblot analysis was performed to analyze SAMHD1-myc, ORF1p-T7, and ORF2p-FLAG

79 Results expression levels. (E and F). HEK 293T cells were cotransfected with the full-length L1 reporter plasmid pAD2TE1 (ORF1p-T7) or an ORF2-3xFLAG expression plasmid together with increasing amounts of SAMHD1 T592A or D207N. A GFP expression plasmid was cotransfected to ensure a constant amount of transfected DNA. Cells were lysed two days posttransfection and immunoblot analysis was performed to determine SAMHD1-myc, ORF1p-T7 (C), or ORF2p-FLAG (D) expression levels. Endogenous Hsp90 was used as loading control. One of at least three independent experiments is shown.

To analyze the effect of SAMHD1 on L1 promoter activity, HEK 293T cells were cotransfected with a L1 promoter construct together with empty vector (pcDNA), SAMHD1 wt, T592A, or D207N mutant. The promoter construct encodes the L1 promoter followed by a luciferase reporter gene. Two days posttransfection, the luciferase activity in whole cell lysates was determined as marker for L1 promoter activity. No difference in the luciferase activity between SAMHD1 wt, T592A, or D207N compared to the empty vector control was detected (Fig. 24A). This result demonstrates that SAMHD1 does not restrict L1 retrotransposition by interfering with its promoter activity. Previously, SAMHD1 has been shown to bind and degrade HIV-1 RNA (Beloglazova et al., 2013; Ryoo et al., 2014). Thus, the influence of SAMHD1 on L1 mRNA level was analyzed. HEK 293T cells were transfected with an L1 expression plasmid that encodes T7-tagged ORF1p together with the highly active SAMHD1 T592A or the inactive D207N mutant. Cotransfection of the long isoform of the zinc finger antiviral protein (ZAP (L)), which has been reported to degrade L1 RNA (Goodier et al., 2015; Moldovan and Moran, 2015), served as positive control. Total RNA was isolated at 12, 24, 36, and 48 hours posttransfection. Additionally, expression levels of L1 ORF1p, SAMHD1, and ZAP were analyzed by immunoblot analysis 48 h posttransfection (Fig. 24B, inset). Quantitative RT-PCR was performed with primers targeting the T7 tag fused to ORF1p to exclude detection of endogenously expressed L1 RNA or cDNA. Quantification of L1 mRNA copies revealed that overexpression of ZAP (L) reduced the RNA level over time between 50 and 70 % compared to pcDNA transfected samples (Fig. 24B). In contrast, expression of the highly active SAMHD1 T592A or the inactive D207N mutant had no effect on L1 mRNA level (Fig. 24B). This finding strongly suggests that SAMHD1 does not restrict L1 by degrading its RNA. Within the next step, the protein levels of L1 ORF1p and ORF2p in the presence of different SAMHD1 mutants were analyzed by western blot analysis. HEK 293T cells were transfected with either a full-length L1 expression vector encoding T7-tagged ORF1p (Fig. 24C) or an expression plasmid for FLAG-tagged ORF2p (Fig. 24D) together with expression plasmids for wt SAMHD1, T592A, T592D, or D207N. No differences in L1 protein expression in presence of the different SAMHD1 mutants was detected. In contrast, higher protein levels for both, ORF1p and ORF2p were found upon expression of SAMHD1. To rule out the possibility that minor effects were missed and to exclude saturation, the experiment was repeated with increasing amounts of SAMHD1 T592A and D207N. Analysis of L1 protein expression two days posttransfection showed that neither ORF1p (Fig. 24E) nor ORF2p (Fig. 24F) levels were decreased in the presence of

80 Results

SAMHD1 T592A compared to control transfection or coexpression of inactive SAMHD1 D207N. Thus, SAMHD1 does not degrade L1 proteins or induces their degradation.

2.8. SAMHD1 does not impair LINE-1 reverse transcription in vitro

Zhao et al. postulated that SAMHD1 prevents ORF2p-mediated reverse transcription of L1 RNA (Zhao et al., 2013). To analyze ORF2-encoded RT activity in presence of SAMHD1, the well-established L1 element amplification protocol (LEAP), which mimics the initial stages of L1 target-primed reverse transcription (TPRT), was used (Kulpa and Moran, 2006). HEK 293T cells were cotransfected with a L1 reporter plasmid encoding T7-tagged ORF1p and empty vector (pcDNA), T592A, D207N, or ZAP (L), which degrades L1 RNA and thus prevents ORF2p-mediated reverse transcription (Goodier et al., 2015; Moldovan and Moran, 2015).

A

RNP LEAP primer

ORF1 ORF1 ORF2 5‘ Poly(A)-3‘ + 3‘-Poly(T) 5‘ ORF1

RT

cDNA 5‘ ORF2 Poly(T)

5‘ Poly(A)-3‘

PCR L1 3‘ end primer Linker to LEAP primer

3‘ 5‘ cDNA

B LINE-1 C LINE-1

ORF1p (T7)

SAMHD1 (myc) MLV-RT

Input ZAP (HA) Hsp90 LEAP RNPs ORF1p (T7)

Figure 25. SAMHD1 T592A does not impair ORF2p-mediated reverse transcription in vitro. (A) LINE-1 (L1) RNPs were isolated from whole cell lysates of transfected HEK 293T cells by ultracentrifugation through a sucrose cushion. L1 mRNA from isolated RNPs was reverse transcribed using a LEAP primer containing an oligo(dT) and a linker sequence. Reverse transcription occurred by either ORF2p (LEAP reaction) or exogenously added MLV-RT. Resulting cDNA was amplified by PCR using primers binding to the linker sequence of the LEAP primer and the 3’ end of L1 cDNA. (B) HEK 293T cells were cotransfected with a full-length L1 expression plasmid (pAD2TE1) and either empty vector (pcDNA) or expression plasmids coding for constitutively active SAMHD1 (T592A), enzymatically inactive SAMHD1 (D207N), or the long isoform of the zinc finger antiviral protein

81 Results

(ZAP (L)). RNPs were isolated two days posttransfection. Protein expression in whole cell lysates (input) was confirmed by immunoblot detecting ORF1p-T7, SAMHD1-myc, or ZAP (L)-HA. Endogenous Hsp90 served as loading control. The amount of ORF1p in isolated RNP samples after ultracentrifugation was analyzed by immunoblot with a T7-specific antibody. (C) cDNA synthesized by either exogenously added MLV-RT or ORF2p-encoded L1 RT was analyzed by PCR using primers binding to the linker sequence of the LEAP primer and the 3’ end of L1. Amplification products were visualized on a 2 % agarose gel.

Two days posttransfection, L1 ribonucleoprotein complexes (RNPs) were isolated by ultracentrifugation through a sucrose cushion from whole cell lysates (Fig. 25A). L1 RNA was isolated from purified RNPs and reverse transcription was performed using a LEAP primer containing an oligo(dT) followed by a linker sequence with exogenously added MLV- RT. Complementary DNA (cDNA) synthesis mediated by the endogenous ORF2-encoded RT was performed with isolated RNPs in presence of the LEAP primer (Fig. 25A). Amplification of both, MLV-RT and ORF2p-RT synthesized cDNA was conducted by standard PCR using primers that specifically bind to the 3’ end of L1 and the linker sequence of the LEAP primer. Additionally, cell lysates were controlled for efficient protein expression and purified RNP samples were analyzed for their ORF1p content by immunoblotting (Fig. 25B). ORF1p expression could be detected in RNP samples of control- and SAMHD1- containing samples, thereby confirming efficient precipitation of L1 RNPs. Since ZAP degrades L1 RNA (Goodier et al., 2015; Moldovan and Moran, 2015), only a weak ORF1p signal was detected after coexpression of ZAP (L). Amplification of MLV-RT generated cDNA resulted in a constant L1 RNA signal from control and SAMHD1-containing samples (Fig. 25C, upper panel). However, a weaker MLV-RT signal was observed for the ZAP (L)- containing sample due to L1 RNA degradation. These results demonstrate that SAMHD1 does not affect the L1 RNA content in RNPs. Furthermore, no differences in amount of the LEAP products between SAMHD1 T592A and D207N compared to the empty vector control (pcDNA) could be detected (Fig. 25C, lower panel). On the contrary, expression of the control protein ZAP (L) resulted in a reduced LEAP signal. In summary, SAMHD1 does not affect ORF2p-mediated L1 RT activity in vitro.

2.9. SAMHD1 directly interacts with ORF2p in functional LINE-1 RNPs

To analyze a possible interaction of SAMHD1 with L1-encoded proteins, co- immunoprecipitation experiments were performed. For this purpose, myc-tagged SAMHD1 wt, T592A or T592D were coexpressed together with L1 expressing T7-tagged ORF1p in HEK 293T cells. Two days posttransfection, ORF1p-T7 was precipitated from whole cell lysates with a T7 monoclonal antibody (mAb) coupled to magnetic beads.

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A LINE-1 + - + + + 150 SAMHD1 - T592A T592A wt T592D SAMHD1 (myc) 100 IP α-T7 ORF1p (T7) 50 SAMHD1 (myc)

WCL intensity normalized 0 wt T592A T592D ORF1p (T7)

B pcDNA T592A

SAMHD1 (myc) 150

IP α-FLAG ORF2p (FLAG) 100 ORF1p (FLAG)

50 SAMHD1 (myc)

WCL ORF2p (FLAG) intensity normalized 0 ORF1p ORF2p ORF1p (FLAG)

C D T592A pcDNA T592A MOV10 LINE-1 - + + + + - + + LINE-1 RNase A - - + - + - - + SAMHD1 (myc)

IP α-T7 MOV10 (myc) MLV-RT

ORF1p (T7) IP α-myc LEAP SAMHD1 (myc)

WCL MOV10 (myc) SAMHD1 (myc) WCL ORF1p (T7) ORF1p (T7) Figure 26. SAMHD1 directly interacts with ORF2p in LINE-1 RNPs. (A) HEK 293T cells were cotransfected with plasmids encoding full-length LINE-1 (L1), SAMHD1 wt, constitutively active SAMHD1 (T592A), or the phosphomimetic mutant (T592D). Cells were lysed two days posttransfection and ORF1p was precipitated using anti-T7 antibody coupled to magnetic beads. The resulting precipitates were analyzed by immunoblot detecting SAMHD1 mutants with an anti-myc antibody. SAMHD1 wt, T592A, and T592D signals were quantified using the AIDA Image Analyzer Software. Signals were normalized on the ORF1p signal in the IP (bait) and the SAMHD1 signal in the WCL (input). The experiment was performed by Sabine Wittmann, Institute for Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg. (B) HEK 293T cells were cotransfected with expression vectors coding for ORF1p-FLAG or ORF2p-3xFLAG and SAMHD1 T592A or empty vector (pcDNA). Cells were lysed two days posttransfection and both L1 proteins were precipitated using an anti-FLAG antibody coupled to magnetic beads. Coprecipitated SAMHD1 T592A protein was detected using an anti-myc antibody. The T592A signal of one representative co-immunoprecipitation was analyzed using the AIDA Image Analyzer Software. T592A signals were normalized on FLAG

83 Results signals (IP) and WCL (input). (C) HEK 293T cells were transfected with a full-length L1 expression plasmid and either empty vector (pcDNA), SAMHD1 T592A or MOV10. Two days posttransfection, cells were lysed and whole cell lysates were treated with RNase inhibitor or 15 µg/ml RNaseA to degrade L1 RNA prior to ORF1p precipitation using magnetic beads coated with an anti-T7 antibody. Co-precipitated SAMHD1 T592A and MOV10 were detected by immunoblot using an anti-myc antibody. (D) HEK 293T cells were transfected with empty vector (pcDNA), an expression vector for full-length L1 and plasmids encoding wt SAMHD1 or the phosphorylation mutants T592A or T592D. Two days posttransfection, cells were lysed and SAMHD1 was precipitated using magnetic beads coated with an anti-myc antibody. After precipitation, LEAP- and MLV-RT reactions were performed and amplified PCR products were visualized on a 2 % agarose gel (inverted picture). Protein expression was controlled by immunoblot. One out of three independent experiments is depicted. WCL: whole cell lysate. IP: immunoprecipitation.

After pulldown of ORF1p, a stronger signal for SAMHD1 T592A was detected compared to wt or T592D. (Fig. 26A). Densitometric analysis using the AIDA Image Analyzer Software confirmed this observation, showing that phosphorylation at T592 reduces the coprecipitation of SAMHD1 with ORF1p by approximately 75 %. These findings indicate that phosphorylation of SAMHD1 at T592 weakens the interaction with L1 RNPs. To determine whether SAMHD1 directly interacts with ORF1p or with ORF2p, HEK 293T cells were transfected with either an empty vector (pcDNA) or SAMHD1 T592A together with expression plasmids for ORF1p-FLAG or ORF2p-3xFLAG. Immunoprecipitation of L1 proteins with an anti-FLAG mAb coupled to magnetic beads was performed two days posttransfection. Interestingly, SAMHD1 T592A precipitated with both L1 proteins (Fig. 26B). However, coprecipitation of T592A with ORF2p resulted in a 6-fold stronger precipitation signal although it is expressed to a lower level than ORF1p (densitometric analysis using AIDA Image Analyzer). This finding shows that SAMHD1 most likely directly interacts with ORF2p. Since an interaction of SAMHD1 with ssRNA has been reported within the literature (Goncalves et al., 2012), the interaction with L1 RNA was analyzed. For this purpose, HEK 293T cells were transfected with a full-length L1 expression plasmid encoding T7-tagged ORF1p together with either an empty vector (pcDNA) or myc-tagged SAMHD1 T592A. Since MOV10 directly interacts with L1 RNA (Goodier et al., 2012), an expression plasmid for myc- tagged MOV10 was cotransfected together with L1 as a positive control. Whole cell lysates were treated with either RNaseA to degrade L1 RNA or the RNase inhibitor RNaseOUT prior to immunoprecipitation. Although MOV10 decreased ORF1 protein levels, it was efficiently precipitated together with ORF1p in the presence of the RNase inhibitor. However, treatment of lysates with RNaseA prevented MOV10 precipitation, thereby confirming that MOV10 directly interacts with L1 RNA. In contrast, SAMHD1 T592A was efficiently precipitated under both conditions, demonstrating that SAMHD1 indeed directly interacts with L1 proteins and that the presence of L1 RNA is not required for this interaction.

To answer the question whether SAMHD1 interacts with ORF2p as part of functional L1 RNPs, the immunoprecipitation protocol was combined with the LEAP assay protocol described above (Fig. 25A). HEK 293T cells were cotransfected with expression plasmids for

84 Results myc-tagged SAMHD1 wt, T592A, or T592D with a full-length L1 vector coding for T7-tagged ORF1p. Efficient protein expression was confirmed by immunoblotting of whole cell lysates two days posttransfection (Fig. 26D, lower panel). The different SAMHD1 mutants were precipitated with an anti-myc mAb coupled to magnetic beads. Afterwards, MLV-RT reaction was performed with heat-inactivated protein-containing beads to inactivate the ORF2p- encoded L1 RT and the LEAP primer. LEAP reactions were directly performed with the L1 ORF2p-RT, which was supposed to be coprecipitated with SAMHD1, and the LEAP primer. Amplification of both resulting cDNA products by PCR was performed with primers binding to the 3’ end of L1 and the linker sequence of the LEAP primer used for reverse transcription (Fig. 26D, upper panel). MLV-RT reactions revealed that a similar amount of L1 RNA was precipitated together with all SAMHD1 proteins, thereby confirming that SAMHD1 does not degrade L1 RNA. Moreover, LEAP products were detected in all samples in which SAMHD1 was pulled down, indicating that SAMHD1 indeed interacts with functional L1 RNPs. This finding confirms the results obtained from the previously performed LEAP assay (Fig. 25C) and demonstrates that SAMHD1 interacts with functional L1 RNPs. Interestingly, slightly more LEAP PCR products were obtained for the non-phosphorylated T592A mutant compared to the phosphorylated SAMHD1 wt. This finding further supports the hypothesis that phosphorylation at T592 regulates the interaction with L1 RNPs. In summary, SAMHD1 directly interacts with functional L1 RNPs in an RNA-independent but phosphorylation- dependent manner.

2.10. SAMHD1 does not promote the accumulation of LINE-1 RNPs in cytoplasmic stress granules

Recently, Hu and colleagues reported that SAMHD1 prevents successful retrotransposition by promoting the assembly and accumulation of L1 RNPs in cellular stress granules (SGs) (Hu et al., 2015). SGs are dense aggregations consisting of proteins and RNAs in the cytosol. They are thought to protect cellular RNAs under harmful conditions. After disappearance of stress conditions, these RNAs might be either degraded or a re-initiation of translation takes place.

85 Results

A DAPI SAMHD1 G3BP1 Overlay B DAPI SAMHD1 G3BP1 Overlay

pcDNA pcDNA

pcDNA pcDNA

pcDNA + As2O3 pcDNA + As2O3

pcDNA + T592A pcDNA + T592A

pcDNA + T592A pcDNA + T592A

pcDNA + D207N

C DAPI SAMHD1 G3BP1 Overlay

pcDNA

pcDNA + As2O3

pcDNA + T592A

pcDNA + T592A

Figure 27. SAMHD1 T582A does not promote stress granule formation. HEK 293T (A), HeLa HA (B), or U2OS cells (C) were transfected with an empty vector (pcDNA), the non-phosphorylated SAMHD1 T592A-myc, or the enzymatically inactive D207N-myc mutant. As a positive control for stress granule (SG) induction, pcDNA-transfected cells were treated with 0.5 mM As2O3 for 1 h at 37 °C prior to fixation. Cells were stained with antibodies targeting the myc tag (red) or endogenous G3BP1 (cyan) as a SG marker two days posttransfection. Cellular DNA was stained with DAPI (blue). Samples were analyzed by confocal microscopy.

86 Results

A DAPI ORF1 SAMHD1 G3BP1 Overlay

L1 ORF1-FLAG + pcDNA

L1 ORF1-FLAG + pcDNA

L1 ORF1-FLAG + T592A

L1 ORF1-FLAG + T592A

L1 ORF1-FLAG + D207N

B DAPI ORF1 SAMHD1 G3BP1 Overlay

ORF1-FLAG + pcDNA

ORF1-FLAG + pcDNA

ORF1-FLAG + T592A

ORF1-FLAG + T592A

ORF1-FLAG + D207N

87 Results

C D DAPI ORF1 SAMHD1 G3BP1 Overlay

L1 ORF1-FLAG + 120 SG-negative SG-positive pcDNA

100 L1 ORF1-FLAG + 80 pcDNA

60 L1 ORF1-FLAG + 40 T592A

Number of cells of Number 20

0 L1 ORF1-FLAG + T592A

pcDNAAs2O3T592AD207NpcDNAT592AD207NpcDNAT592AD207N L1 ORF1-FLAG + pcDNA L1 ORF1-FLAG ORF1-FLAG D207N

Figure 28. SAMHD1 does not promote the sequestration of LINE-1 in cytoplasmic stress granules. HEK 293T cells were transfected with either a full-length LINE-1 (L1) expression vector enconding FLAG-tagged ORF1 (A) or an expression vector for ORF1-FLAG (B) together with empty vector (pcDNA), highly active SAMHD1 T592A-myc or enzymatically inactive D207N-myc. Two days posttransfection, cells were stained with antibodies targeting ORF1-FLAG (green), SAMHD1-myc (red) or endogenous G3BP1 (cyan) as a stress granule (SG) marker. Cellular DNA was stained with DAPI (blue). Cells were analyzed by confocal microscopy. (C) For each transfection, 100 cells were examined to score SG-positive and SG-negative cells. The results are summarized in bar graphs. (D) U2OS cells were cotransfected with a full-length L1 expression plasmid encoding FLAG-tagged ORF1 and either empty vector (pcDNA), SAMHD1 T592A-myc or D207N-myc. Cells were stained with antibodies targeting ORF1-FLAG (green), SAMHD1-myc (red) or endogenous G3BP1 (cyan) as SG marker two days posttransfection. Cellular DNA was stained with DAPI (blue). Cells were analyzed by confocal microscopy.

To analyze the impact of SAMHD1 overexpression on SG formation in the presence of L1, HEK 293T cells were transfected with either a full-length L1 expression plasmid encoding FLAG-tagged ORF1p (Fig. 28A) or an expression plasmid for ORF1-FLAG (Fig. 28B) together with empty vector (pcDNA), SAMHD1 T592A-myc, or D207N-myc. Cells were stained for ORF1-FLAG, SAMHD1-myc, and the SG marker G3BP1 two days posttransfection and were analyzed by confocal microscopy. One hundred cells for each preparation were scored for SG-positive and SG-negative cells, together with the controls mentioned before (Fig. 28C). ORF1p expressed from full-length L1 was exclusively located in the cytoplasm, occasionally forming dense aggregates (Fig. 28A). Interestingly, expression of ORF1p alone yielded the same results (Fig. 28B). In the absence of SAMHD1, only a few SG-positive cells were found. However, in SG-positive cells, aggregates of ORF1p colocalized to SGs as published previously (Doucet et al., 2010). Coexpression of the highly active SAMHD1 T592A mutant did not change the overall number of SG-positive cells (Fig. 28C), indicating that SAMHD1 T592A does not prevent L1 retrotransposition by promoting the accumulation of L1 RNPs in cytoplasmic SGs. In agreement to Hu and colleagues, coexpression of enzymatically inactive SAMHD1 (D207N) did not promote SG formation (Hu et al., 2015). Moreover, no colocalization of ORF1p and SAMHD1 was detected, although SAMHD1 coprecipitates with L1 (Fig. 26). To confirm the findings

88 Results obtained from HEK 293T cells and to exclude cell type-specific artefacts, U2OS cells were transfected with the same approaches for full-length L1 (Fig. 28D). Again, no difference between pcDNA- and SAMHD1-transfected cells was found. In summary, these results demonstrate that SAMHD1 does not inhibit L1 retrotransposition by promoting its accumulation in cytoplasmic SGs.

89 Discussion

VI. DISCUSSION

1. The role of SAMHD1-dependent restriction and sensing in mice

Mammalian cells are constantly confronted with various challenges, including exogenous pathogens like retroviruses, as well as endogenous parasitic nucleic acids such as retrotransposons. Therefore, cells have evolved numerous defense mechanisms to protect themselves and their genome integrity. The innate immune system uses pathogen recognition receptors (PRRs) to recognize danger-associated (DAMPs) or pathogen- associated molecular patterns (PAMPs), which upon activation initiate a cascade of intracellular signaling pathways to cause protection of the host (reviewed in Dempsey et al., 2003). Nucleic acids are such a trigger, and thus can induce immune responses under certain conditions (reviewed in Ahlers and Goodman, 2016; Radoshevich and Dussurget, 2016). Cytosolic DNA sensors trigger the activation of different signaling pathways leading to activation of transcription factors such as the IFN regulatory factor 3 (IRF3) or nuclear factor kappa B (NF-κB). Both factors are responsible for the production of IFN-I as well as pro- inflammatory cytokines and are activated by the central adapter molecule stimulator of interferon genes (STING). In some cases, DNA accumulates within the cytoplasm from either exogenous (viral or bacterial infections) or endogenous sources (cellular stress or DNA damage) and activates the innate immune system. The best-studied cytosolic DNA sensor is cyclic GMP-AMP (cGAMP) synthase (cGAS). Upon DNA binding, cGAS catalyzes the synthesis of the second messenger cGAMP, which in turn leads to STING activation (reviewed in Chen et al., 2016). Moreover, intrinsic antiviral immunity, which is mediated by so-called restriction factors, represents a first-line defense against retroviral infection (reviewed in Harris et al., 2012; Hatziioannou and Bieniasz, 2011). Over the past years, several proteins with anti-HIV-1 activity have been identified. Interestingly, many of them not only inhibit the replication of HIV-1, but were also found to restrict endogenous transposable elements (reviewed in Colomer-Lluch et al., 2016; Goodier, 2016). These proteins include SAMHD1, an important restriction factor in non-cycling human and murine cells, which is believed to impede reverse transcription by lowering the intracellular dNTP pool (reviewed in Herrmann et al., 2016).

Acute HIV-1 infection induces an IFN-I response, which, however, is not sufficient to clear the infection reviewed in (Altfeld and Gale, 2015; Jakobsen et al., 2015). Under certain conditions, lentiviral RNA, reverse-transcribed cDNA, or viral proteins were reported to trigger IFN-I responses (Beignon et al., 2005; Gringhuis et al., 2010; Jakobsen et al., 2013; Manel et al., 2010; Pertel et al., 2011). To elucidate the role of SAMHD1 in a HIV-1-induced IFN-I response, primary BMDCs of wt mice or SAMHD1, IFNAR, and IFNAR/SAMHD1 double KO cells were infected with HIV-GFP. Knockout of SAMHD1 resulted in more HIV-

90 Discussion

GFP-positive cells compared to SAMHD1-competent wt cells (Fig. 6A), thus confirming that murine SAMHD1 acts as a restriction factor for HIV-1 (Behrendt et al., 2013; Rehwinkel et al., 2013; Wittmann et al., 2015). In agreement with previous work from Maelfait and colleagues, loss of IFNAR further increased the susceptibility to HIV-GFP compared to cells lacking only SAMHD1 (Fig. 6A) (Maelfait et al., 2016). This result demonstrates that the presence of IFNAR-mediated signaling augments SAMHD1-dependent HIV-1 restriction in primary murine BMDCs. Furthermore, this finding highlights the role of the positive IFNAR- mediated feedback loop during HIV-1 infection in the absence of SAMHD1.

Recently, cGAS was shown to be critical for mounting an immune response towards HIV-1 through recognizing reverse transcription (RT) products during viral replication in human THP-1 cells (Gao et al., 2013). However, the role of SAMHD1 in cGAS-mediated HIV-1 restriction and sensing is still elusive. In vitro HIV-GFP infection studies in PMA-differentiated SAMHD1-competent human THP-1 cells lacking cGAS conducted within this thesis showed a higher infectivity compared to wt cells (Fig. 5C and D). Notably, the absence of cGAS in primary murine BMDCs had none or only a slight effect on the infection with HIV-GFP (Fig. 6C and D). The same results were obtained by Maelfait and colleagues, who also did not find any differences in HIV-1 infectivity on cGAS KO cells compared to wt cells (Maelfait et al., 2016). The discrepancy between human and murine cells points towards species-specific differences in the sensing of HIV-1-derived RT products or a response of murine myeloid cells to HIV-1 infection at a step downstream of cGAS sensing. Strikingly, both human THP-1 cells and murine BMDCs lacking cGAS and SAMHD1 showed a greatly enhanced infection rate compared to single KO cells (Fig. 5C and D, Fig. 6C and D), demonstrating that SAMHD1 covers cGAS-mediated restriction of HIV-1 infection in vitro. Taken together, the data provided within this thesis indicate that SAMHD1 limits the amount of RT products, thus impeding cGAS-mediated IFN-I and cytokine production in response to HIV-1 infection. In vitro infection experiments performed within this study revealed that the lack of cGAS had a greater impact on HIV-GFP infectivity compared to the loss of STING (Fig. 5 and 6). This difference might be explained by the fact that cGAS-mediated sensing of HIV-1 takes place upstream of STING and that STING is activated by the cGAS-synthesized second messenger cGAMP. In THP-1 cells, the absence of both STING and SAMHD1 resulted in an enhanced infection rate compared to the respective single KO control cells (Fig. 5C and D). This finding demonstrates that SAMHD1 not only covers cGAS-mediated sensing of HIV-1, but also diminishes STING-dependent signaling in human cells. In contrast, infectivity of primary murine STING/SAMHD1 double KO BMDCs was decreased compared to that of SAMHD1 KO cells. Notably, defects such as developmental delays or hydranencephaly were more frequently observed in STING/SAMHD1 double KO mice compared to wt and single KO mice. On a cellular level, such defects might influence the susceptibility towards HIV-1

91 Discussion infection, thus explaining the reduced infectivity and the discrepancy between primary murine BMDCs and the THP-1 cell line. Overall, HIV-GFP infectivity on cGAS/SAMHD1 double KO cells was more enhanced than that of STING/SAMHD1 double KO cells compared to single KO cells in both, human and murine cells, indicating that cGAS might play a more important role in sensing of HIV-1 than STING. This is rather surprising, since cGAS has been reported to only signal through STING so far (Ablasser et al., 2013a; Diner et al., 2013; Sun et al., 2013). Therefore, the data obtained from this study indicate that cGAS has an additional, STING-independent antiviral function, which is normally covered by the restrictive activity of SAMHD1. Several viruses were found to directly counteract cGAS/STING signaling. For example, various herpes- or adenoviruses, inhibit cGAS activity or interfere with the downstream signaling of STING (reviewed in Ma and Damania, 2016). Thus, it is not surprising that HIV-1 has also evolved mechanisms to counteract cGAS/STING-mediated sensing. For example, HIV-1 infection has been shown to upregulate the mitochondrial nucleotide-binding domain and leucine-rich-repeat-containing protein 1 (NLRX1), which binds to STING and prevents downstream signaling (Guo et al., 2016). Moreover, the HIV-1- encoded accessory proteins Vpr and Vif can degrade IRF3 by ubiquitination and proteasomal degradation (Okumura et al., 2008). Furthermore, both proteins can bind TBK1 and block its phosphorylation in myeloid cells, thereby preventing downstream signaling cascades (Harman et al., 2015). In addition to interference with the cGAS/STING pathway, HIV-1 has evolved mechanisms to evade IFN-mediated antiviral activities (reviewed in Lahaye and Manel, 2015). These include interaction of the viral capsid with protective host factors or counteraction of antiviral restriction factors by viral accessory proteins. The restrictive activity of SAMHD1 is counteracted by Vpx (Hrecka et al., 2011; Laguette et al., 2011), which is encoded by HIV-2 and some SIV strains. Since HIV-1 does not encode Vpx or a protein with equal function, it cannot directly circumvent SAMHD1-mediated restriction. The data provided within this thesis support the hypothesis that HIV-1 avoids the counteraction of SAMHD1 to prevent cGAS-mediated induction of a strong immune response. Thus, SAMHD1 joins the ranks of host factors that enable escape from innate immune detection, including Trex1, CPSF6, or CypA.

Loss of SAMHD1 results in the spontaneous induction of IFN-I and ISGs in the absence of viral infection in mice, resembling the autoimmune reaction of AGS patients (Behrendt et al., 2013; Rehwinkel et al., 2013; Rice et al., 2009). To clarify the question whether the same or different genes are induced in autoimmunity and during HIV-1 infection, global gene expression profiles of uninfected and infected wt and SAMHD1 KO BMDCs were analyzed by RNA sequencing. Comparison of the transcriptome of uninfected wt and SAMHD1 KO BMDCs revealed that more ISGs were upregulated in SAMHD1-deficient cells (Fig. 8), resembling the spontaneous IFN-I signature in the absence of SAMHD1. However, these

92 Discussion genes partially differed from the ones previously identified by Behrendt and colleagues (Behrendt et al., 2013). Since the earlier study used peritoneal macrophages and the experiments presented within this thesis were performed with BMDCs, this discrepancy might be explained by cell type-specific expression patterns. This finding demonstrates that SAMHD1 plays an important role in the suppression of a spontaneous activation of the IFN-I system under normal conditions. The majority of the genes that were endogenously upregulated in the absence of SAMHD1 were further enhanced upon HIV-GFP infection (Fig. 8). This result supports the hypothesis that at least in part the same genes are responsible for both, the endogenous autoimmune reaction and the anti-HIV-1 response in SAMHD1-deficient cells. Furthermore, this finding indicates that the same central sensor is responsible for the induction of both immune responses. Since SAMHD1 covers cGAS- mediated restriction of HIV-1 infection in primary murine BMDCs, cGAS represents a promising candidate for this sensor. Interestingly, an involvement of the cGAS/STING pathway has also been reported for the AGS-related proteins Trex1 and RNaseH2 (Ablasser et al., 2014; Gao et al., 2015; Gray et al., 2015; Pokatayev et al., 2016), thus further confirming this hypothesis. The most prominent upregulation in HIV-GFP-infected compared to uninfected SAMHD1 KO cells was observed for immune responsive gene 1 (Irg1) and pro- platelet basic protein (Ppbp). Irg1 acts as an enzyme that produces itaconic acid, which has been reported to exhibit antiviral effects on RSV, HBV, and different neurotropic positive- stranded RNA virus infections (Cho et al., 2013; Liu et al., 2017). Ppbp is a potent chemoattractant and activator of neutrophils (Di Stefano et al., 2009) and exhibits antimicrobial effects in human monocytes (Schaffner et al., 2004). Therefore, both proteins might represent HIV-1 restriction factors that are normally covered by the restrictive activity of SAMHD1. Using the well-established SAMHD1 KO mouse model provides an opportunity to study their role in HIV-1 infection in vitro and in vivo in greater detail.

Previously, SAMHD1-deficiency was reported to be associated with the upregulation of genes related to IFN-I and cytokine signaling in the absence of HIV-1 infection (Oh et al., 2018). To address the question whether infection of SAMHD1-deficient cells results in a similar phenotype, upregulated transcripts of infected SAMHD1 KO and wt cells were analyzed for differences in gene ontology clustering using the DAVID online platform. Infection of SAMHD1-deficient cells is associated with the enhanced expression of genes involved in inflammatory and innate immune signaling as well as defense responses to viral infection (Fig. 7A). These data show that the absence of SAMHD1 has an influence on gene regulation, especially ISGs with potential antiviral activity, thereby affecting the susceptibility towards HIV-1 infection. Moreover, these data further support the hypothesis that the restrictive activity of SAMHD1 impedes the induction of an anti-HIV-1 response in vitro. Although wt BMDCs were not susceptible to HIV-GFP infection (Fig. 6), many genes

93 Discussion associated with innate and antiviral immune responses were also upregulated in these SAMHD1-competent cells (Fig. 7B). This finding supports the hypothesis that abortive RT intermediates in the presence of SAMHD1 are also potent activators of the innate immune system, most likely by activation of the cGAS/STING pathway (Lahaye et al., 2013; Rasaiyaah et al., 2013).

Quantification of GFP-positive cells isolated from IFNAR/SAMHD1 double KO mice revealed a 3-times higher susceptibility to HIV-GFP infection compared to SAMHD1 KO cells (Fig. 6A). Moreover, intravenous infection of IFNAR/SAMHD1 double KO mice resulted in a 5-fold enhanced HIV-GFP infectivity on total splenocytes (Behrendt and Wittmann, unpublished data). Thus, the absence of IFNAR-mediated signaling additonally augments SAMHD1-dependent HIV-1 restriction in vivo. To identify genes that contribute to the inhibitory effect of SAMHD1 compared to IFNAR/SAMHD1 double KO cells, upregulated transcripts in infected SAMHD1 KO compared to IFNAR/SAMHD1 double KO cells were determined (Fig. 10A). Infection of SAMHD1 KO BMDCs results in the upregulation of almost 100 genes, possibly exhibiting antiviral activities (Fig. 10). Interestingly, the majority of these genes corresponds to a list previously published in an AGS-related study (Pokatayev et al., 2016), thus validating the results obtained within the present thesis. Furthermore, this finding supports the hypothesis that similar genes are upregulated during HIV-1 infection, which are also enhanced in autoimmunity. A nearly 2000-fold upregulation was observed for Oas1, which is a member of the oligoadenylate synthase family. Upon binding to long dsRNA, Oas1 catalyzes the synthesis of the second messenger oligoadenlyate, leading to the activation of RNaseL, which in turn degrades of cellular and viral RNAs (reviewed in Hartmann, 2017). Furthermore, transcripts for the IFN-induced protein 44 (ifi44) were nearly 750-fold enhanced. Ifi44 has been shown to inhibit HIV-1 replication in vitro by suppressing the LTR promoter activity (Power et al., 2015). Among other genes that were identified with the help of this transcriptome analysis, these candidate genes might account for the enhanced infectivity of IFNAR/SAMHD1 double KO compared to SAMHD1 KO BMDCs. Although IFNAR/SAMHD1 double KO cells were more susceptible to HIV-GFP infection compared to SAMHD1 KO cells, genes that were upregulated in SAMHD1-deficient cells were reduced in the absence of IFNAR upon infection (Fig. 9B). This result points towards an impaired antiviral response in the absence of the IFN-I receptor and SAMHD1.

To identify genes that are directly induced upon HIV-1 infection, the transcriptome of uninfected and infected IFNAR/SAMHD1 double KO cells was analyzed. Due to the absence of SAMHD1 expression, these cells are more susceptible towards HIV-1 infection compared to SAMHD1-competent cells. Furthermore, due to the absence of IFNAR, these cells lack the

94 Discussion amplifying positive IFN feedback loop. Thus, analysis of induced genes in the absence of IFNAR results in directly induced genes that do not require IFNAR-dependent amplification. Comparison of both gene expression profiles resulted in 98 genes that were significantly upregulated upon HIV-GFP infection (Fig. 11). Most of the genes that were upregulated in double KO cells were also elevated in the presence of SAMHD1, indicating that their induction is SAMHD1-independent. However, a few genes were enhanced only in the absence of SAMHD1, pointing towards a SAMHD1-dependent upregulation. These genes include IFN-inducible protein with tetratricopeptide repeats 1 (ifit1). Ifit1 has been reported to exhibit antiviral activity by recognizing 5’-triphosphate RNA (Pichlmair et al., 2011). Interestingly, Ifit1 has been shown to inhibit HIV production, although HIV-1 RNA does not contain a 5’-triphosphate group (Nasr et al., 2017). Since Ifit1 is upregulated only in the absence of SAMHD1, this finding indicates that the restrictive activity of SAMHD1 covers the anti-HIV-1 activity of Ifit1 in murine BMDCs. Taken together, the analysis of upregulated genes in IFNAR/SAMHD1 double KO BMDCs performed within this thesis identified almost 100 genes that could exhibit antiviral activity.

To validate the results obtained from the transcriptome analysis, the upregulation of selected genes was confirmed by qRT-PCR. In agreement with the RNA sequencing data, upregulation of pyrin domain containing 4 (pydc4), irg1, and the interferon-activated gene 206 (ifi206) was detected in SAMHD1 KO compared to wt cells 16 hpi. A dramatic induction of ISGs 16 h upon HIV-1 infection has already been reported in a similar setting (Johnson et al., 2018), demonstrating that this early time point after infection is most suitable to detect an HIV-1-induced IFN-I response. Interestingly, Ifi206 represents the murine orthologue to the IFN-γ-inducible protein 16 (IFI16), which has already been reported as a DNA sensor for HIV-1 (Jakobsen et al., 2013; Unterholzner et al., 2010). Furthermore, IFI16 is required for sensing of HIV-1 derived cDNA in human macrophages by supporting the production and function of cGAMP, the second messenger that is produced by cGAS in response to cytosolic DNA (Jonsson et al., 2017). Therefore, it would be interesting to further investigate the interplay between IFI16 and cGAS in dendritic cells as well as in vivo using the already established cGAS and cGAS/STING mouse models. Taken together, the data obtained from the transcriptome analysis were confirmed, thus validating the experimental approach used for this study.

Collectively, the data presented within this study highlight the role of SAMHD1 in diminishing the IFNAR- and cGAS/STING-dependent susceptibility of primary murine BMDCs towards HIV-1 infection. Moreover, SAMHD1 plays an important role in dampening IFN-I-dependent immune responses in primary murine BMDCs in autoimmunity as well as upon HIV-1 infection. Targeting SAMHD1 by small molecule inhibitors might represent a way for a

95 Discussion specific induction of an immune response in resting infected cells to elicit better protection of the host. Future studies based on these results will help to better understand the induction of pathogenic autoimmune and HIV-1-induced antiviral reactions in vitro and in vivo.

2. The role of SAMHD1 in the inhibition of endogenous retroelements

All known genes that are linked to AGS play a role in the metabolism of nucleic acids (reviewed in Crow and Manel, 2015). Besides DNA replication or DNA damage-dependent by-products, nucleic acids of replicating transposable elements are discussed as a trigger for the induction of autoimmune responses in the absence of AGS-related proteins (Li et al., 2017; Rehwinkel et al., 2013; Stetson et al., 2008). The idea of transposable elements as causative of autoimmune reactions is supported by the finding that retroelement-derived ssDNA accumulates in cells lacking the three prime repair exonuclease 1 (Trex1) (Stetson et al., 2008). Furthermore, reverse transcriptase inhibitors have been shown to rescue disease symptoms in Trex1 KO mice in the absence of viral infection through inhibition of reverse transcription of endogenous retroelements (Beck-Engeser et al., 2011). In addition, murine cells expressing mutants of the ribonuclease H2 (RNaseH2) also show elevated L1 cDNA levels (Pokatayev et al., 2016). Two initial publications showed that the AGS-related protein SAMHD1 restricts L1 retrotransposition in cell culture (Hu et al., 2015; Zhao et al., 2013). However, both studies postulated different mechanisms of L1 inhibition, which differ from what is known about SAMHD1-mediated HIV-1 restriction. Therefore, the aim of this study was to analyze the inhibitory effect of SAMHD1 on L1 retrotransposition in greater detail and to identify the underlying mechanism of restriction.

In agreement with the previous studies (Hu et al., 2015; Zhao et al., 2013), experiments conducted within this thesis confirmed the SAMHD1-mediated restriction of L1 in a dose- dependent manner in HEK 293T cells (Fig. 13C). Notably, the inhibition of L1 occurs in cycling cells, whereas the block of HIV-1 was limited to non-cycling cells (Fig. 5A and 16), pointing towards a distinct mechanism of restriction or a different regulation of this restrictive activity. In contrast to the previous studies, the presence of wt SAMHD1 reduced retrotransposition at the maximum of 40 %. A possible reason for this discrepancy might be the position of the epitope tag sequence. While the initial studies were performed with N•terminally tagged SAMHD1, C-terminally myc-tagged SAMHD1 was used within this thesis. This study identified N-terminally FLAG-tagged SAMHD1 to be more active against L1 than untagged or C-terminally myc-tagged SAMHD1 (Fig. 14C). Thus, an N-terminal tag might influence the anti-L1 activity of SAMHD1, thereby explaining the enhanced restriction of wt SAMHD1 observed in the previous studies (Hu et al., 2015; Zhao et al., 2013).

The phosphorylation status of SAMHD1 at T592 has been shown to regulate its antiviral activity in a cell cycle-dependent manner (Cribier et al., 2013; Welbourn et al., 2013; White et

96 Discussion al., 2013b). However, the study of Zhao and colleagues did not reveal a difference of the non-phosphorylated T592A or the phosphomimetic T592D mutant of SAMHD1 regarding their anti-L1 activities (Zhao et al., 2013). In contrast, retrotransposition assays conducted within this study clearly demonstrate that unphosphorylated SAMHD1 T592A almost completely abolishes retrotransposition and thus is far more active against L1 than the phosphorylated wt or the T592D variant (Fig. 14A). This finding leads to the assumption that SAMHD1 is only active against L1 in non-cycling cells in vivo, in which CDK-mediated phosphorylation is lost. Analysis of the phosphorylation state of SAMHD1 and its anti-L1 activity in cell types in which L1 was shown to be active will be required to confirm this idea. Although unphosphorylated SAMHD1 restricts HIV-1 in non-cycling cells (Fig. 5A), overexpression of non-phosphorylated SAMHD1 T592A did not block HIV-1 infection in cycling HEK 293T cells (Fig. 16). This discrepancy suggests a similar regulation but different mechanism of L1 inhibition compared to HIV-1 restriction. This finding also indicates that an additional, still unknown cell cycle-dependent mechanism for the SAMHD1-mediated HIV-1 restriction exists. While Zhao and colleagues initially reported that dNTPase-defective SAMHD1 still inhibits retrotransposition (Zhao et al., 2013), Hu and colleagues later found that dNTPase mutants lost their ability to restrict L1 (Hu et al., 2015). In agreement with the findings from Hu et al., this study clearly identified the enzymatically active site to be required for the inhibition of L1 retrotransposition (Fig. 14, 19, and 20). Concludingly, the mechanism of L1 restriction is regulated by T592-phosphorylation and requires enzymatically active SAMHD1, but also a yet unrecognized determinant, which explains the differences between HIV-1 and L1 inhibition.

To further address the role of T592 phosphorylation in L1 restriction, the retrotransposition assay was performed in presence of CDK inhibitors to prevent CDK-mediated phosphorylation of SAMHD1 (Cribier et al., 2013; St Gelais et al., 2014; White et al., 2013b). Treatment with CDK inhibitors drastically reduced L1 retrotransposition (Fig. 15A), indicating that CDK-mediated phosphorylation of L1-encoded proteins might be important for retrotransposition. Indeed, Cook and colleagues recently showed that ORF1p is phosphorylated in a CDK-dependent manner, which is required for efficient retrotransposition (Cook et al., 2015). Given that the treatment with CDK inhibitors interfered with efficient L1 replication, no enhanced anti-L1 activity of wt SAMHD1 in their presence could be detected. To circumvent this problem, the DNA polymerase inhibitor aphidicolin was used to induce cell cycle arrest at the early S phase, in which SAMHD1 exists in its unphosphorylated state. However, aphidicolin treatment during the retrotransposition assay almost completely abolished L1 replication (Fig. 15B), indicating that cell cycle progression is required for efficient retrotransposition. Although some studies detected retrotransposition in non-dividing and terminally differentiated cells like neurons or glioma cells (Kubo et al., 2006; Macia et al.,

97 Discussion

2017), several other studies showed a strongly reduced efficacy of retrotransposition in cell cycle-arrested cells (Shi et al., 2007; Xie et al., 2013). It is not fully understood yet how L1 RNPs enter the nucleus. However, it is supposed that the breakdown of the nuclear membrane during represents an opportunity for L1 to enter the nucleus, as it is the case for some exogenous retroviruses (Goff, 2007; Suzuki and Craigie, 2007). Thus, aphidicolin-induced cell cycle arrest might prevent or diminish the access of L1 RNPs to the genomic DNA, explaining the drastically reduced retrotransposition events (Fig. 15B). Interestingly, Mita et al. recently showed that retrotransposition peaks during S phase (Mita et al., 2018). In this state, the dNTPase activity of SAMHD1 is reduced (Franzolin et al., 2013), resulting in high levels of dNTPs, which are restricted during other cell cycle phases in a SAMHD1-dependent manner. Since high amounts of dNTPs are required for replication, this finding points towards an adaptation of L1 replication to the cell cycle stage, which supports efficient target-primed reverse transcription (TPRT).

In addition to overexpressed SAMHD1, endogenous SAMHD1 was reported to block L1 replication (Hu et al., 2015; Zhao et al., 2013). To confirm this finding, stable HEK 293T shSAMHD1 cells were generated. However, depletion of endogenous SAMHD1 did not enhance the number of retrotransposition events (Fig. 17). As expected for cycling cells, endogenous SAMHD1 was found to be phosphorylated at T592 in HEK 293T cells, explaining the lack of an anti-L1 activity of endogenous SAMHD1. This result corroborates the finding that phosphorylated SAMHD1 is not or only slightly active against L1, highlighting the importance of the T592-dependent regulation of its anti-L1 activity. The absence of any residual activity of endogenous compared to exogenously overexpressed SAMHD1 might result from low expression levels of endogenous SAMHD1 in HEK 293T cells.

In addition to L1, SAMHD1 T592A mutant efficiently blocked the replication of non- autonomous Alu and murine LTR-retroelements such as IAP and MusD (Fig. 18). In contrast, the enzymatically inactive D207N mutant lost the ability to restrict retrotransposition of all elements tested. Since SAMHD1 T592A inhibits replication of both non-LTR and LTR elements from different species, this points towards a broadly acting mechanism of restriction. While phosphorylated wt SAMHD1 did not inhibit L1 or MusD (Fig. 18B and E), it was slightly active against Alu and IAP (Fig. 18C and D), indicating that some transposable elements might be more susceptible to SAMHD1-mediated restriction than others. Collectively, the data obtained within this study suggest a common principle of restriction with element-specific differences.

Mutational analysis of SAMHD1 revealed that restriction of endogenous retroelements like L1 relies on an enzymatically functional HD domain as well as on the allosteric GTP-binding site. In contrast to HIV-1 restriction, a mutation that interferes with the proposed RNase

98 Discussion activity of SAMHD1 (Ryoo, 2014) had only marginal effects on its anti-L1 activity (Fig. 19 and 20), indicating that the postulated RNase activity is not required for L1 restriction. Furthermore, this result again supports the hypothesis that HIV-1 and L1 are inhibited by different SAMHD1 functions. In addition to human SAMHD1, also murine SAMHD1 potently restricted L1 retrotransposition (Fig. 21A), suggesting that L1 inhibition is a conserved feature of mammalian SAMHD1. Like human SAMHD1, the anti-L1 activity of murine SAMHD1 is also regulated by phosphorylation and requires an enzymatically active site, as well as functional allosteric GTP-binding sites (Fig. 21A). Notably, the inhibitory effect of the allosteric site mutation was less pronounced in isoform 2 compared to isoform 1, since the isoform 2 mutant remained some of its anti-L1 activity, the effect. Interestingly, Bloch and colleagues recently showed that isoform 2 of murine SAMHD1 is more active than isoform 1 or human SAMHD1 and allosteric GTP-binding is not required for activation (Bloch et al., 2017). In addition to isoform 2, which lacks the regulatory T603 phosphorylation site, the phosphorylated isoform 1 of murine SAMHD1 was found to be far more active against L1 than phosphorylated human SAMHD1 (Fig. 21B). This discrepancy suggests the presence of species-specific inhibitory or regulatory host factors, which are not present or do not interact with murine SAMHD1 in human cells. Future analysis and comparison of interaction partners of human and murine SAMHD1 will help to identify the illusive SAMHD1 regulatory factors. Since murine and human SAMHD1 exhibit similar rates of dNTP hydrolysis in vitro (Buzovetsky et al., 2018), differences within the dNTPase activity of both species cannot be responsible for the unequal anti-L1 activities. About 3000 active L1 copies exist in mice compared to approximately 100 active copies in humans (Goodier et al., 2001; Penzkofer et al., 2017). Therefore, murine SAMHD1 might be more efficiently adapted to control retrotransposition compared to human SAMHD1, which would explain its enhanced anti-L1 activity.

Since the dNTPase activity of SAMHD1 is thought to be the main antiviral mechanism, the ability of the different SAMHD1 mutants to degrade dNTPs was analyzed. Overexpression of SAMHD1 strongly reduced the intracellular dNTP pool of cycling HEK 293T cells (Fig. 23A and B). However, the decreased dNTP levels were still high enough to support efficient reverse transcription of HIV-1, since the HIV-1 RT has a high affinity for dNTPs with

Km values ranging between 0.5 and 1 µM. This finding could explain the lack of an anti-HIV-1 activity of overexpressed unphosphorylated SAMHD1 T592A in cycling HEK 293T cells

(Fig. 16). Previous in vitro data suggest a Km for L1 ORF2 between 0.38 for dCTP and 0.83 µM for dTTP (Dai et al., 2011). However, dNTP quantifications conducted within this study revealed dCTP levels of approximately 0.5 µM and dTTP levels ranging from 2 to 4 µM in presence of enzymatically active SAMHD1 (Fig. 23). Thus, ORF2-RT is most likely not affected by the lower dNTP level upon overexpression of SAMHD1. Interestingly, no

99 Discussion significant differences in dNTP degradation between phosphorylated SAMHD1 wt, non- phosphorylated T592A, or phosphomimetic T592D were found. This finding demonstrates that in HEK 293T cells phosphorylation of SAMHD1 at T592 regulates its anti-L1, but not its dNTPase activity. Unexpectedly, overexpression of SAMHD1 D137A/T592A reduced intracellular dNTP level almost as efficiently as the wt. In contrast, a previous study of Yan and colleagues showed that recombinant D137A was defective for tetramerization and dNTP degradation in an in vitro assay (Yan et al., 2013). However, recombinant SAMHD1 might differ from eukaryotically expressed SAMHD1 regarding posttranslational modifications, which could account for the observed differences. Failure of SAMHD1 D207N/T592A to degrade cellular dNTPs correlates with its inability to restrict retrotransposition, demonstrating that a functional HD domain, and most likely the dNTPase activity of SAMHD1, is necessary for L1 inhibition. Since the dNTPase activity is not regulated by phosphorylation at T592, these data strongly suggest an additional, phosphorylation- dependent yet uncharacterized activity of SAMHD1 to be responsible for L1 inhibition.

Since TPRT of L1 occurs in the nucleus, the requirement of nuclear localization of SAMHD1 for L1 inhibition was analyzed. Although cytoplasmic SAMHD1 is phosphorylated at T592 and degrades intracellular dNTPs to the same extent as wt SAMHD1, it was far more active against L1 (Fig. 22 and 23). These data support the hypothesis that a cytoplasmic activator or nuclear inhibitor of SAMHD1 exists.

To identify the step of the L1 life cycle at which SAMHD1-mediated restriction occurs, L1 promoter activity, RNA transcription, and protein expression were examined. A L1 promoter assay revealed that SAMHD1 does not restrict L1 retrotransposition by interfering with its promoter activity (Fig. 23A). Furthermore, overexpression of SAMHD1 did not affect L1 mRNA levels (Fig. 23B), suggesting that SAMHD1 does not degrade L1 RNA as it was recently postulated for genomic HIV-1 RNA (Ryoo et al., 2014). This result also confirms the findings obtained from mutational analysis of SAMHD1-mediated L1 restriction, in which the RNase mutant Q548A/T592A still blocked L1 replication (Fig. 20). While Zhao and colleagues reported that SAMHD1 does not alter ORF1p expression, they found reduced ORF2 protein levels upon SAMHD1 expression (Zhao et al., 2013). In contrast, analysis of ORF1p and ORF2p levels in the presence of SAMHD1 within the present study revealed that SAMHD1 does not induce degradation of L1-encoded proteins (Fig. 24C-F). On the contrary, SAMHD1 coexpression resulted in elevated ORF2 protein levels, suggesting that SAMHD1 contributes to ORF2p stabilization.

Initially, it has been postulated that SAMHD1 prevents successful reverse transcription of L1 RNA in vitro (Zhao et al., 2013). Therefore, the effect of SAMHD1 on L1 RT activity was analyzed using the well-established L1 element amplification protocol (LEAP), which

100 Discussion measures the ability of ORF2p to reverse transcribe L1 RNA in vitro (Fig. 25A). L1 RNPs were isolated from transfected cells by ultracentrifugation through a sucrose cushion. ORF2 RT activity was used to reverse transcribe L1 RNA that is enclosed in L1 RNPs. As a control, L1 RNA was isolated from the RNP samples and reverse-transcribed using recombinant MLV-RT. The MLV-RT control revealed no difference in the amplification of cDNA from SAMHD1-containing samples generated by MLV-RT compared to the empty vector control, demonstrating that SAMHD1 does not affect the L1 RNA content in RNPs (Fig. 25C, Fig. 21, Fig. 24B). Furthermore, the ORF2p-mediated RT activity did not differ between SAMHD1 T592A- or D207N-containing samples (Fig. 25C). This result stands in contrast to the findings reported by Zhao and colleagues, who reported a SAMHD1-dependent inhibition of ORF2p-mediated RT activity (Zhao et al., 2013). Notably, the presence of excessive amounts of exogenously added nucleotides during the in vitro RT reaction may cover possible effects of a dNTPase-mediated restriction. Thus, influences of dNTP degradation on successful TPRT in vivo cannot be excluded.

To analyze whether SAMHD1-mediated restriction of L1 occurs through direct interaction, co•immunoprecipitation experiments were performed. Initial experiments revealed that SAMHD1 wt, T592A, and T592D interact with ORF1p expressed from a full-length L1 construct (Fig. 26A). Interestingly, co-precipitation of the non-phosphorylated T592A mutant resulted in a slightly stronger signal compared to wt or the phosphomimetic T592D mutant, indicating that phosphorylation at T592 regulates the interaction of SAMHD1 with L1 RNPs. To answer the question whether SAMHD1 interacts with L1 ORF1p or ORF2p, both proteins were expressed separately in the presence of SAMHD1. Although SAMHD1 was co- precipitated with both L1 proteins, pulldown of ORF2p resulted in a stronger SAMHD1 signal despite a lower expression level (Fig. 25B). The considerably weaker SAMHD1 signal upon precipitation of ORF1p could result from an interaction of ORF1p with endogenously expressed L1 RNPs (Fig. 25B), which points towards a direct interaction of SAMHD1 with ORF2p rather than ORF1p. Although SAMHD1 has been reported to bind to RNA (Beloglazova et al., 2013; Goncalves et al., 2012; Seamon et al., 2015), co-precipitation of SAMHD1 with full-length L1 was not affected by RNase treatment (Fig. 25C). This finding supports a direct protein-protein interaction of SAMHD1 with L1 ORF2p. To answer the question whether SAMHD1 interacts with functional L1 RNPs, the immunoprecipitation protocol was combined with the LEAP assay. ORF2p-encoded RT activity was pulled down together with SAMHD1 wt, T592A, and T592D (Fig. 25D), thereby confirming that SAMHD1 interacts with functional RNPs. In contrast to Zhao et al., this finding also demonstrates that SAMHD1 does not impede ORF2p-mediated RT activity in vitro. Notably, slightly more LEAP products were found after precipitation of the non-phosphorylated T592A mutant compared to wt or the phosphomimetic T592D mutant suggesting that phosphorylation at T592

101 Discussion regulates the interaction with L1 RNPs. Taken together, the data presented within this study support a model in which SAMHD1 interacts with L1 RNPs in a phosphorylation-dependent manner, preferentially by binding to ORF2p.

Although Hu and colleagues previously reported that SAMHD1 promotes the formation of cytoplasmic stress granules (SG) (Hu et al., 2015), expression of SAMHD1 alone (in the absence of L1 RNA or protein) did not induce SGs in HEK 293T, HeLa HA, or U2OS cells (Fig. 26). However, expression of full-length L1 or ORF1p alone resulted in a few SG- positive cells, which has been previously reported by several studies (Doucet et al., 2010; Goodier et al., 2007; Hu et al., 2015). Nevertheless, no differences in number or size of SGs between T592A or D207N transfected cells were detected compared to control cells (Fig. 27). Collectively, the results obtained within this study demonstrate that SAMHD1 does not promote the accumulation of L1 RNPs in cytoplasmic stress granules. Thus, sequestration of RNPs in SGs is not responsible for the block to L1 retrotransposition.

In previous studies, the retrotransposition of L1 has been shown to result in DNA double strand breaks (DSBs) (Gasior et al., 2006). Notably, the number of L1-induced DSBs is higher than the predicted number of successful insertions, which might explain the low efficiency of the retrotransposition process. Interestingly, SAMHD1 deficiency was linked to hypersensitivity towards DSB-inducing agents (Daddacha et al., 2017). Enhanced endogenous RT activity concomitant with impaired DSB repair in the absence of SAMHD1 might be responsible for the generation of immunogenic nucleic acids in the cytoplasm. Indeed, treatment with RT inhibitors to prevent endogenous L1 retrotransposition has been shown to decrease cGAS/STING-mediated pro-inflammatory cytokine production in case of the genetic disorder Fanconi Anemia (Bregnard et al., 2016). Interestingly, the cGAS/STING pathway was shown to be responsible for IFN-I and immune-regulatory cytokine induction in response to DNA damage (Gluck et al., 2017; Mackenzie et al., 2017). Moreover, genetic deficiencies that compromise DNA damage response functions also induce elevated cytokine levels and lead to autoinflammatory diseases through aberrant activation of this pathway, thereby linking DSB-inducing agents to autoimmunity. Besides DSBs resulting from insufficient L1 integration into the host genome, L1 cDNA is a potential trigger of cGAS. Noteworthy, several AGS-related genes that play a role in nucleic acid metabolism have already been connected to cGAS/STING-dependent sensing. For example, Trex1-deficient mice develop life-shortening inflammatory phenotypes, which can be rescued by genetic depletion of either cGAS or STING (Ahn et al., 2014; Gao et al., 2015; Gray et al., 2015; Stetson et al., 2008). Furthermore, cells lacking RNaseH2b show persistent genome instability which promotes the formation of micronuclei, thereby leading to recruitment of cGAS and activation of IFN-I signaling (Bartsch et al., 2017; Mackenzie et al., 2017).

102 Discussion

Removal of cGAS or STING reverses inflammation and autoimmune phenotypes in RNaseH2 KO mice (Mackenzie et al., 2017; Pokatayev et al., 2016). Although SAMHD1- deficient mice do not show an overt autoinflammatory phenotype, their myeloid cells express elevated ISG levels that depend on cGAS and STING (Fig. 8) (Maelfait et al., 2016). Therefore, accumulation of nucleic acids derived from retroelements like L1 might be responsible for the cGAS/STING-dependent immune response in the absence of SAMHD1. However, Zhao and colleagues recently reported that endogenous L1 components trigger IFN-β production independently of the cGAS and STING through RNA-sensing pathways involving RIG-I and MDA5 (Zhao et al., 2018). In agreement with the study from Zhao and colleagues, upregulation of retroelement-derived RNA in human cells deficient for SAMHD1 was recently found to trigger IFN production through the PI3K/AKT/IRF3 pathway (Oh et al., 2018). Taken together, involvement of the cGAS/STING pathway in L1 sensing has mainly been reported for murine cells, whereas studies implicating L1 RNA as a trigger for autoimmunity were performed in human cells. Given the discrepancies between murine and human cells, it is possible that different types of nucleic acids induce distinct sensing pathways in mice and humans. Therefore, further studies are required to clarify the nature of the immunogenic L1 nucleic acids in both humans and mice. To address this issue, the KO mouse models for SAMHD1, cGAS, and STING established within this study provide a suitable tool to resolve this conundrum.

In summary, phosphorylation-dependent regulation of the interaction with L1 RNPs as well as the enzymatically active site are prerequisites for SAMHD1-mediated L1 inhibition. Thus, the data provided within this thesis suggest a model in which SAMHD1 directly interacts with L1 RNPs and impedes L1 replication by a locally-restricted depletion of dNTPs in close proximity to the L1-encoded RT. The results lead to the hypothesis that SAMHD1 contributes to genome stability and prevents autoimmunity by restricting retrotransposition of endogenous retroelements, which could be responsible for the development of autoimmune diseases such as AGS. Moreover, these findings strongly suggest that cGAS represents the cytosolic sensor that is responsible for both, the sensing of HIV-1 infection as well as the autoimmune reaction leading to AGS. Further insights into the molecular basis of SAMHD1- dependent sensing of cytosolic DNA derived from either viral infections or retroelements will improve the understanding of host immunity as well as vaccine development, and allow the design of target-specific treatments for inflammatory diseases.

103 Abbreviations

VII. ABBREVIATIONS

ADAR adenosine deaminase acting on RNA GO gene ontology AGS Aicardi-Goutières syndrome GTP guanosine triphosphate APOBEC3 apolipoprotein B mRNA editing HEK cells human embryonic kidney cells enzyme catalytic polypeptide-like 3 HERV human endogenous ART anti-retroviral therapy HIV-GFP HIV-1 reporter virus encoding GFP instead of Nef As2O3 arsenic trioxide HIV-Luc HIV-1 reporter virus encoding luciferase instead BMDCs bone marrow-derived dendritic cells of Nef bp Hsp90 heat shock protein 90 CA capsid IAP intracisternal A particle CDK cyclin-dependent kinase IFI16 gamma-interferon-inducible protein 16 cDNA complementary DNA Ifi206 interferon-inducible gene 206 cGAMP cyclic guanosine monophosphate- Ifi44 interferon-induced protein 44 adenosine monophosphate IFIH1 interferon-induced helicase C domain-containing 1 cGAS cyclic GMP-AMP synthase Ifit1 interferon-induced protein with tetratricopeptide Co-IP co-immunoprecipitation repeats 1 cps counts per second IFN interferon CPSF6 cleavage and polyadenylation IFNAR interferon-α/β receptor specificity factor subunit 6 IFN-I type I interferon CypA cyclophilin A IN integrase DAMP danger-associated molecular pattern IRF3 interferon regulatory factor 3 DAPI 4',6-diamidino-2-phenylindole Irg1 immune responsive gene 1 dATP deoxyadenosine triphosphate ISG interferon stimulated gene DAVID Database for Annotation, Isg20 interferon-stimulated gene 20 Visualization and Integrated Discovery Iso1 isoform 1 of murine SAMHD1 DCs dendritic cells Iso2 isoform 2 of murine SAMHD1 dCTP deoxycytidine triphosphate JM111 retrotransposition-defective full-legth L1 Ddit4l DNA damage-inducible transcript 4 kb kilo-base pair dGTP deoxyguanosine triphosphate kDa kilo dalton DMSO dimethyl sulfoxide KO knockout dN deoxynucleoside LEAP L1 element amplification protein DNA deoxyribonucleic acid LINE-1; L1 long interspersed element 1 Dnah2 dynein axonemal heavy chain 2 LTR long terminal repeat dNTP deoxynucleoside triphosphate Luc luciferase dNTPase deoxynucleoside triphosphate MA matrix triphospho hydrolase MACs macrophages ds double-stranded MDA5 melanoma differentiation-associated protein 5 dTTP deoxythymidine triphosphate MDDC monocyte-derived dendritic cell EGFP enhanced green fluorescent protein MOI multiplicity of infection EN endonuclease MOV10 moloney leukemia virus 10 Env envelope Ms4a4c membrane-spanning 4 domains subfamily A G3BP1 Ras GTPase-activating protein- member C binding protein 1 NC nucleocapsid G418 geneticin Nef negative regulatory factor Gag group-specific antigen neo neomycin GAPDH glyceraldehyde 3-phosphate NF-κB nuclear factor kappa B dehydrogenase

104 Abbreviations

NLRX1 nucleotide-binding domain and RT reverse transcriptase leucine-rich-repeat containing protein 1 Rtp4 receptor-transporting protein 4 NNRTI non-nucleoside reverse transcriptase SAM sterile alpha motif inhibitor SAMHD1 SAM and HD domain containing protein 1 NRTI nucleoside reverse transcriptase SG stress granule inhibitor shCtrl scrambled shRNA, control-shRNA Oas1g oligoadenylate synthase 1g shSAMHD1 shRNA targeting SAMHD1 Oasl1 oligoadenylate synthase-like 1 SINE short interspersed element ORF open reading frame ss single-stranded ORF1p protein encoded by ORF1 STING stimulator of interferon genes ORF2p protein encoded by ORF2 SVA SINE-VNTRP-Alu elment PAMP pathogen-associated molecular pattern T592 Threonine at position 592 PMA phorbol 12-myristate 13-acetate Tat trans-activator of trascription Pol polymerase TBK1 TANK-binding kinase 1 Ppbp pro-platelet basic protein TE transposable element PR protease TLR toll-like receptor PRR pattern recognition receptor TPRT target-primed reverse transcription Pydc4 pyrin domain-containing 4 Trex1 three prime repair exonuclease 1 qRT-PCR quantitative real-time polymerase TRIM5α tripartite motif-containing protein 5α chain reaction Vif viral infectivity factor RIG-I retinoic acid inducible gene I Vlig1 very large interferon-inducible GTPase RLU relative light units Vpr viral protein R RNA ribonucleic acid Vpu viral protein U RNase ribonuclease Vpx viral protein X RNaseH2 ribonuclease H2 VSV-G gylcoprotein of the vesicular stomatitis virus RNaseL ribonuclease L WHO world health organisation RNaseOUT recombinant RNase inhibitor wt wild-type RNP ribonucleoprotein ZAP zinc finger antiviral protein

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119 Appendix

IX. APPENDIX

1. Supplementary information

Table S1. List of genes that were significantly upregulated in wt BMDCs upon HIV-GFP infection.

Gene WT -HIV WT +HIV log2-fold change p-value Irg1 379,1 26495,8 6,07 0 Gramd1c 36,5 1833,5 5,62 0 Ptgs2 243,2 5617 5,07 0 Ccdc141 16,7 513,5 4,97 0 Serpinc1 10,7 314,5 4,88 0 Ifit1 45,4 1342,3 4,8 0 Trim72 10,5 192,8 4,47 0 Hcar2 36,8 694,5 4,45 0 Six4 11,3 231,3 4,36 0 Ifit2 137,8 2870,4 4,36 0 Fosb 17,4 226,6 4,21 0 Igf2bp1 9,2 134,8 3,84 0 Susd2 12,4 270,1 4,21 0 Cxcl5 17,3 328,5 4,17 0 9230114K14Rik 29,3 516,5 4,09 0 Cxcl10 10,4 197,9 4,02 0 Rtp4 43,7 734,4 4 0 Ccrl2 951,2 14985,4 3,96 0 Rsad2 253 3742,8 3,94 0 Cxcl2 386,3 5238,5 3,93 0 Cxcl3 664,8 9643,8 3,87 0 Ralgds 612,1 8736,5 3,82 0 Tnfaip3 2142,4 30070,1 3,77 0 Epha4 16,4 242 3,74 0 Il1b 149,1 1936,7 3,74 0 Isg15 84 1103,2 3,74 0 Fam71f2 87,5 805,4 3,73 0 Cxcl1 38,9 480,6 3,65 0 Oasl1 63 928 3,65 0 Ifi205 14,7 183,9 3,63 0 Cdo1 22,3 270,3 3,6 0 Tnf 349,6 3993,8 3,51 0 Cp 12,2 150,6 3,47 0 Cd200r2 14,6 149,5 3,44 0 Fam181b 18,4 208,8 3,43 0,000001 B3galt2 21,9 292,7 3,39 0 Gm12689 23,4 239,2 3,33 0 RP23-403P24.5 11,2 112,1 3,29 0 Gem 66,5 659,9 3,25 0 Tlcd2 118 1068,9 3,23 0 RP23-167C5.1 12,2 116,2 3,19 0

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Gene WT -HIV WT +HIV log2-fold change p-value Gm15832 440,4 4086,2 3,17 0 AA986860 29,2 258,8 3,15 0 Tigd3 18 158,3 3,14 0 Oasl2 341,1 3030,8 3,13 0 Gm13571 33,4 307,3 3,12 0 Il20rb 67,6 606,3 3,11 0 Gm37084 18,6 159,4 3,05 0 Lgr4 27,2 226,4 3,03 0 Pygm 21,6 173,1 3,01 0 Cmpk2 74,6 610,5 2,99 0 Clec4e 1500 11930,9 2,95 0 Hist1h2be 14,2 109 2,95 0 Itga7 4,1 137,5 5,09 0 Irf7 315,1 2388,3 2,94 0 H2-M2 732,5 5400 2,93 0 Slpi 492,9 3979,1 2,93 0 RP23-382I10.2 19,3 148,3 2,93 0 Tlr2 1468,1 11258 2,9 0 Gm37498 21,9 165,8 2,89 0 Ednrb 111,8 755,9 2,88 0 Cxxc5 44,7 321 2,88 0 RP24-492O4.8 24,8 184,6 2,88 0 Sv2a 18,9 133,4 2,83 0 Boc 14,6 102,2 2,8 0 Neb 8 100,5 3,63 0 Nfkbiz 289,6 2055,5 2,8 0 Gm37188 30,5 218,4 2,75 0 Il1a 250,4 1606,9 2,73 0 n-R5-8s1 58 375 2,72 0,000064 Nr1d1 308 1972,4 2,66 0 Mnda 74,1 452,4 2,66 0 Rnasek 17,8 114,4 2,66 0 Rasgef1b 531,8 3343,9 2,64 0 Ifi44 8,8 111,9 3,48 0 Bnipl 7,5 119,4 4,05 0 Plet1 42,8 253,6 2,63 0,000933 Mss51 57,4 344,6 2,59 0 Saa3 161 955,1 2,55 0 Ccl3 228,1 1241,7 2,54 0 Ets2 1277,1 7162,1 2,53 0 Usp18 123,7 837,7 2,52 0 Inhba 499,5 2824,5 2,5 0 Lox 62,4 354,6 2,49 0 Slc6a9 77,8 427,2 2,46 0 Lrrc32 527,7 2888,1 2,46 0 Gadd45b 1536,1 8482,5 2,43 0

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Gene WT -HIV WT +HIV log2-fold change p-value Ptpn14 33,9 179,2 2,43 0 Tma16 503,7 2680 2,42 0 Orm1 25,8 139 2,42 0 Gp5 32,3 164,8 2,42 0,000001 Zbp1 103,7 601 2,38 0 Psrc1 318,9 1637,1 2,37 0 Pcx 206,5 1076,4 2,35 0 Sod2 2124,1 10867,9 2,34 0 Clmp 707,6 3637,3 2,33 0 Pabpc1l 23,5 116,6 2,31 0 Dusp4 352,7 1576,8 2,29 0 Slfn2 4642 22631,4 2,28 0 Slfn1 60,1 350,8 2,28 0,000031 Unc5b 52,4 246,2 2,27 0 RP23-441I24.5 79,6 382,5 2,26 0 Mx2 40,1 206,4 2,25 0 G630090E17Rik 42,8 199,1 2,25 0 1700056N10Rik 21,6 106,3 2,25 0 Hmox1 27992 131557,4 2,24 0 Trim69 2,8 465,8 7,42 0 Tnfsf15 40,1 194,8 2,23 0 Pim3 522,4 2472,4 2,22 0 Il1f9 1351,4 6143,7 2,22 0 Spic 56,2 265,6 2,21 0 Fosl1 28,7 125 2,19 0 Xaf1 186 871 2,17 0 9130230L23Rik 164,3 736,5 2,16 0 Plcxd2 212,5 948,6 2,16 0 Msantd1 33,6 151,9 2,15 0 Gm340 309,1 1388,8 2,15 0 Phlda1 167 673,9 2,14 0 Ptx3 94,3 405,4 2,11 0 Vasn 25,2 105,1 2,1 0,000002 Ptges 297,2 1294,7 2,1 0 Plk2 4552,5 19082 2,09 0 Chrnb2 64,1 269,7 2,09 0 Rasd2 62,9 283,7 2,09 0,000012 Oas1g 56,9 245,2 2,09 0 Gm4955 9,3 187,7 4,24 0 Fpr2 545,8 2491,6 2,08 0 BC025920 61,5 258,7 2,08 0 Treml4 138,6 578,8 2,05 0 Rltpr 26,7 108,4 2,02 0 Tchh 170,5 666,8 2,02 0 Gm11427 543,4 2204,5 2,02 0 Fam212b 96,5 383,8 2 0

122 Appendix

Gene WT -HIV WT +HIV log2-fold change p-value Gtse1 93,3 360,4 1,99 0 Rab11fip1 1778,3 7141,3 1,99 0 Gm26667 41,7 168,6 1,99 0 Cish 206,1 800,9 1,98 0 Nfkbie 796 3148,6 1,97 0 Cdkn2b 152,9 613,5 1,97 0 Ly6i 170,1 749,8 1,96 0,000042 Tarm1 479,5 1864,3 1,96 0 BC055308 42,6 166,7 1,96 0 Rab20 431,7 1648,9 1,95 0 Phf11d 61,3 234,1 1,94 0 Ifi47 224,4 868,2 1,94 0 Slc19a2 427,1 1595 1,93 0 Foxq1 32,1 129,6 1,92 0 Hp 747 3261,2 1,9 0,000002 Ppp1r15a 1050,6 3844,6 1,88 0 Gm17230 31,3 112,6 1,88 0 Rgl1 786,7 2864,8 1,86 0,000222 Sqstm1 15919,8 56947,5 1,85 0 Tmem178 37,5 137,6 1,85 0 A530099J19Rik 8,7 140,4 3,97 0 Gbp5 136,7 520,8 1,85 0 Ppap2b 673,7 2401,7 1,84 0 Gpr84 36,9 135,9 1,84 0 Gbp2 464,6 1663,4 1,83 0 Htr2a 98,5 346,7 1,83 0 Zfp503 344,2 1145,7 1,83 0 H2-T24 345,4 1245,7 1,83 0,000022 Ccno 31,6 105,6 1,82 0,000002 Sstr5 6,4 175,9 4,81 0 Hilpda 1772,9 5101,9 1,82 0,000128 Bcl2a1a 263,7 936,5 1,82 0 Ampd3 1601,2 5445,7 1,81 0 Gm23935 2295,2 8061,5 1,81 0,000054 Gm15708 65,3 227,7 1,81 0 Serpinb2 41,7 114,5 1,8 0,021029 Aoc2 136,5 471,7 1,8 0 Csf1 837,6 2896,5 1,79 0,000014 Fah 65,6 227,6 1,79 0 Cd14 6531,7 16745,5 1,79 0,019425 Gm37607 30,6 106,5 1,79 0,000001 Tiparp 1896,8 6400 1,78 0 Mcoln2 793,6 2717,4 1,77 0 Hmcn2 69,1 241 1,77 0 Gm12226 95,7 325,9 1,77 0 Rasgrp1 192,9 680,5 1,76 0

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Gene WT -HIV WT +HIV log2-fold change p-value Msantd3 192,1 628,9 1,76 0 Ankrd24 225,4 775,5 1,75 0 Tmem236 0,7 227,1 8,53 0 Mdm2 5669,9 18624 1,74 0 Filip1l 853 2953,9 1,74 0 Ifit3b 5,6 173 4,86 0 Oas3 1033,6 3531,6 1,73 0,00002 Ptgr1 5059,9 16548,8 1,72 0 Gbp7 83,9 274,3 1,72 0,000025 Cdc42ep2 201,3 663,1 1,72 0 Nfkbia 2740,2 8977,6 1,71 0 Ccdc86 664,4 2187,5 1,71 0 Nxt1 259,1 832,5 1,71 0 Arhgef10 5,7 132,8 4,59 0 Gm12250 76,8 267 1,71 0 9930022D16Rik 36,9 116,4 1,7 0,000008 Lsmem1 2,2 104,7 5,46 0 Mir6240 51,4 165,2 1,7 0,00079 Slfn3 117,6 383 1,69 0 Ccnb2 41,8 132,5 1,69 0 Cd40 123,1 444,3 1,66 0,00153 Ddias 48,9 153,9 1,66 0 Slfn8 557,1 1830,8 1,66 0 Lhfpl2 9319,4 28807,4 1,66 0 Six1 75,1 243,5 1,66 0 Per1 1545,9 4939,3 1,65 0 Procr 5030,5 14277,7 1,65 0 Casp4 325,4 1019,9 1,65 0 Aspm 53 164,1 1,65 0,00001 Gm14335 9,7 185 4,15 0 Gss 1331,1 3913,4 1,62 0 Tnfsf9 52,3 161,1 1,62 0,000001 Fam13a 52,6 162,7 1,62 0 Fpr1 514,2 1636 1,62 0 Tubb4a 50,2 156,8 1,62 0 Nr4a1 275,7 789,9 1,61 0,000007 Olr1 730,3 2234,9 1,61 0 Slfn5 2103,1 6275,6 1,61 0,000003 Ccl4 61,1 184,8 1,6 0,000008 Icam1 1681,3 5164,7 1,6 0 Rrs1 494,3 1476,1 1,6 0 Tgif1 3167,1 9576,1 1,59 0 Timm23 44,9 134,9 1,58 0 Mtfr2 211,7 627,5 1,58 0 Adora2a 393,1 1282 1,58 0 Slc7a11 14875,2 44799,2 1,58 0

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Gene WT -HIV WT +HIV log2-fold change p-value Met 4379,3 13010,9 1,57 0 Casz1 234,3 689,2 1,57 0 Gdf15 393,2 1140,9 1,57 0,000054 Gm37123 56,1 166,5 1,57 0 Socs3 135,9 399,9 1,56 0 Gm19325 8,9 226,1 4,66 0 Esam 56,8 161,9 1,55 0,000008 Tnfrsf10b 109,1 318,9 1,55 0 Wnk2 443,4 1302,1 1,55 0,000397 Gm5424 2470 7241,6 1,55 0 Gm37755 2,9 139,8 5,63 0 Gm37621 7 159,7 4,59 0 Prmt6 321,5 934,4 1,55 0 Dok7 108,9 311,8 1,54 0,000066 Gbp3 179,9 533,1 1,53 0 Dusp10 102,5 284,1 1,53 0 RP23-250D22.2 1,8 127,1 6,06 0 Oser1 180,1 511,4 1,52 0 H2-Q4 1365,2 3990,7 1,52 0 Ppfibp2 2046,2 5889,2 1,52 0 Acsl1 2471,8 7304,1 1,51 0 Zfp142 709,5 2045,2 1,51 0 Ppp1r10 1505,8 4307,4 1,51 0 Ass1 560,9 1594,5 1,51 0 Vcam1 42,7 117,4 1,5 0,000065

Table S2. List of genes that were significantly upregulated in SAMHD1 KO BMDCs upon HIV-GFP infection.

Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Cxcl10 13,5 552,8 5,35 0 Ifit1 70,4 2778,8 5,3 0 Irg1 924,2 33849,9 5,19 0 Gramd1c 40,8 1232 4,91 0 Gm4955 15 359,7 4,59 0 Oasl1 156 3730,8 4,58 0 Rsad2 294 6765,5 4,52 0 Ifit2 217,7 4874 4,48 0 Cp 17,4 385,3 4,47 0 Susd2 39,7 880 4,47 0 Rtp4 72,3 1266,4 4,13 0 Ifit3b 12,3 212,7 4,12 0 Isg15 110,1 1884 4,1 0 Ccdc141 23,8 404 4,09 0 Ifi44 13,5 210,8 3,97 0 Gm19325 11,3 167,1 3,88 0

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Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Ifi205 17 244 3,84 0 Fam71f2 26,9 371,8 3,79 0 Oasl2 437,1 5484,5 3,65 0 Irf7 349 4184,6 3,58 0 Cmpk2 83,5 928,2 3,48 0 Mnda 58,2 644,9 3,47 0 Cxcl5 70,9 775,8 3,45 0 Cfb 13 130,3 3,32 0 Serpinc1 15,1 145,7 3,27 0 Ptgs2 154,3 1469,1 3,25 0,000446 Cxcl3 1541,7 14524,5 3,24 0 Slpi 914 7626,3 3,06 0 Gm14446 15,2 118,6 2,97 0 Ccrl2 797,6 6070,9 2,93 0 Gm15832 353,2 2664,7 2,92 0 Adhfe1 14,8 107,5 2,86 0 Fam181b 38,3 275,7 2,85 0,000015 AA986860 20,9 148,8 2,82 0 Gm12689 31,8 224,4 2,82 0 Ms4a4c 8,9 199,9 4,49 0 Six4 20,2 137,7 2,77 0 Tnfsf15 48,8 329,4 2,76 0 Slfn1 115,3 769,3 2,74 0 Tlcd2 84 558 2,73 0 Zbp1 186,6 1219,8 2,71 0 Cdo1 17,3 113 2,71 0 Slc6a9 96,7 620,7 2,68 0 Cst7 35 223,6 2,68 0 Fam212b 109,2 677,7 2,63 0 Usp18 253,3 1510,3 2,58 0 Clec4e 2608,8 15641,1 2,58 0 Cxcl2 670,2 4002,7 2,58 0,000003 Mx2 53,2 316,5 2,57 0 Ednrb 144,6 852,4 2,56 0 Saa3 251,8 1475,1 2,55 0 Epha4 27,3 153,9 2,49 0,00004 Tnf 611,2 3420 2,48 0 Lgr4 26,5 145,4 2,45 0 Ralgds 912,1 4718,9 2,37 0 Oas1g 98,8 509,7 2,37 0 Phf11d 50,2 258,7 2,37 0 Artn 7,8 142,1 4,2 0 H2-M2 1701,5 8691,8 2,35 0 RP24-492O4.8 21,3 109,4 2,35 0 Gm13571 33,8 169,5 2,33 0 Xaf1 215,2 1074,9 2,32 0

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Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Psrc1 355,1 1688,3 2,25 0 Ccl4 75,8 358,6 2,24 0 Ptges 366,7 1704,5 2,22 0 9230114K14Rik 34 159,6 2,22 0 Hcar2 75,9 350,8 2,21 0,006081 Ddit4l 23,2 108,4 2,21 0 Gtse1 99,5 447,9 2,17 0 Ankrd24 318,1 1434 2,17 0 Sod2 2738,7 12165,5 2,15 0 Fpr2 1118,8 4970,4 2,15 0 Oas3 1154,1 5042,1 2,13 0 Unc5b 54,7 236,8 2,11 0 Trim69 4,5 190,9 5,42 0 Cxcl1 80 346,6 2,11 0 Pcx 434,5 1835,9 2,08 0 C1s1 37,8 159,6 2,08 0 Ly6i 443,4 1862,7 2,07 0,000004 Tnfaip3 2782 11633,4 2,06 0 Gbp5 260,8 1086,4 2,06 0 Gm12250 90,5 370,9 2,04 0 Il20rb 109,3 446,5 2,03 0 G630090E17Rik 27,3 108,8 1,99 0 Cd38 699,7 2722,2 1,96 0 H2-T24 435 1695,7 1,96 0,000002 Ifi47 324,3 1262,6 1,96 0 Gm11427 438,2 1703,8 1,96 0 Acsl1 2746 10613,6 1,95 0 Isg20 51,3 198,4 1,95 0 Mss51 65,9 252,7 1,94 0 Tma16 729,2 2794,3 1,94 0 Slfn5 2024,1 7656,4 1,92 0 Il1f9 1702 6363,1 1,9 0 Ptpn14 61,6 228,1 1,89 0 Clmp 918,2 3414,3 1,89 0 Slfn8 570 2108,9 1,89 0 Gbp2 895,8 3275,9 1,87 0 Hmcn2 97,7 358,5 1,87 0 Orm1 50,4 183,4 1,86 0 Slc19a2 412,6 1483,6 1,85 0 RP23-441I24.5 69,2 245,7 1,83 0 Ddias 63,4 221,1 1,8 0 Gm37498 32,8 113,9 1,79 0 Slfn3 117,9 403,9 1,78 0 Ms4a6b 168,9 581,4 1,78 0,000272 Procr 2847,2 9751,7 1,78 0 Hp 2050,5 7022,6 1,78 0,000007

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Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value A530099J19Rik 6,5 155,4 4,58 0 Cdkn1a 6321,2 21375,2 1,76 0 Il6 88,1 296,5 1,75 0,000079 Sstr5 6,7 111 4,03 0 Ddx60 547,2 1843,7 1,75 0 Treml4 236,7 795 1,75 0 Oas1a 458,5 1518,3 1,73 0 Gem 130,6 431,7 1,72 0 Nfkbiz 417,4 1372,5 1,72 0 Phf11a 45 147,5 1,71 0,000469 Nr1d1 281,6 918,4 1,7 0 Pyhin1 562,8 1827,8 1,7 0 Tarm1 913,4 2973,3 1,7 0 Ampd3 1251,8 4025,7 1,69 0 Tmem236 2,4 125,8 5,73 0 Il1a 350 1126,9 1,69 0 Rasd2 109,8 354,7 1,69 0,000461 Ifi204 1090,6 3508,9 1,69 0 Hmox1 31650,7 100637,6 1,67 0,000032 Rasgrp1 338,1 1074,6 1,67 0 Ptgr1 4767,4 15176,9 1,67 0 Mcoln2 881,9 2788,7 1,66 0 Pydc4 9,4 239,5 4,66 0 Dhx58 540,3 1712,5 1,66 0 Rasgef1b 496,7 1566,9 1,66 0 Hsh2d 36,5 115,2 1,66 0,00009 Gbp7 129,6 407,6 1,65 0,000025 Gm4070 32,5 102 1,65 0,002075 Chrnb2 53,6 166,8 1,64 0 Ankrd66 235,4 734,4 1,64 0 Eda2r 42,7 132,2 1,63 0,000008 Gm7204 560 1727,8 1,63 0 Cd40 398,2 1221,7 1,62 0,000746 Ccne2 34,5 106,5 1,62 0,000425 Dck 970,7 2992,4 1,62 0 Trim30d 725,2 2215,7 1,61 0 Ddx58 1102,2 3351,3 1,6 0 Kctd1 73,7 222,4 1,59 0 Tnfrsf10b 166,3 498,2 1,58 0 Gss 1112,2 3326,8 1,58 0 Fosl1 38,6 115,4 1,57 0,000028 Gbp3 286,6 853,3 1,57 0 Prdx1 74543,8 220982,6 1,57 0 Irgm1 873,9 2603,1 1,57 0 Gm21399 16154,2 47903,2 1,57 0 Il1b 540,5 1590,2 1,56 0,006506

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Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Phf11b 184,1 538,2 1,55 0 Phlda3 659 1921,7 1,54 0 Mdm2 4761,1 13719,6 1,53 0 Cxxc5 44,2 128,3 1,53 0,000024 Lrrc32 520,5 1501,6 1,53 0,000037 Gm37249 65,9 190,2 1,53 0 Cdkn2b 225,6 649 1,52 0 Msantd1 37,5 107,3 1,51 0,000185

Table S3. List of genes that were significantly downregulated in wt BMDCs upon HIV-GFP infection.

Gene WT -HIV WT +HIV log2-fold change p-value Hba-a1 162 12,5 -4,78 0,000024 Ccr2 4683,5 185,7 -4,26 0 Abca9 176,4 11,9 -3,75 0 Slco2b1 932,8 71,3 -3,71 0 Slc9a9 496,3 32,8 -3,68 0 Pcp4l1 114,2 15,1 -3,09 0 Rnf150 1892,9 245,4 -2,94 0 Nxpe5 481,5 63,2 -2,89 0 Nlrp1c-ps 485,5 63,1 -2,82 0 Skint3 86,4 12 -2,79 0,000008 2900026A02Rik 277,9 46,7 -2,63 0 Hdac9 71 11,7 -2,59 0,000002 Iqgap3 288,5 47,4 -2,57 0 Mef2c 749 129,9 -2,55 0 Nlrp1b 1258,6 212,5 -2,51 0 Hpgds 2605,3 501,6 -2,48 0 Krt80 76,2 13,3 -2,48 0,000005 Kif26b 139 22,1 -2,45 0,000003 Kcnj10 1157,3 194,1 -2,43 0 Arhgap19 3786,2 735,7 -2,39 0 5031425F14Rik 119,5 22,6 -2,39 0 Rnase6 98,7 18,1 -2,36 0,000001 Dmpk 88 18,3 -2,36 0 Gatm 489,7 109,2 -2,35 0 Reps2 941,2 192,3 -2,35 0 Notch4 118,8 24,2 -2,31 0 Usp2 180,7 36,1 -2,31 0 Rab3il1 1516,9 313,6 -2,29 0 Arhgap15 601 117,1 -2,29 0,000017 Gm5150 318,4 65,2 -2,29 0 Zdhhc14 805,9 164,7 -2,28 0 Arg1 377,7 64,2 -2,26 0,015592 Plxnb3 134,7 27,5 -2,24 0 Gm38248 90,2 19,1 -2,24 0

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Gene WT -HIV WT +HIV log2-fold change p-value Kif19a 142,3 30,5 -2,2 0 Plscr4 260,5 59,8 -2,19 0 Plxna4os1 91,7 19,7 -2,19 0 C5ar2 1411 319 -2,17 0 Prkar1b 70,5 15,6 -2,16 0 Sirpb1a 817,9 178,8 -2,14 0 Sirpb1c 2133,4 481,4 -2,11 0 Gm15513 466 113,3 -2,1 0 Cd300lg 86,3 18,8 -2,08 0,000071 Hacd4 7326,1 1731,8 -2,08 0 Slc46a3 796,6 201,6 -2,08 0 Oscp1 67,8 15,8 -2,08 0 Plxdc2 7980,8 1852,5 -2,05 0 Ppp1r9a 576,1 134,9 -2,05 0,00007 Sirpb1b 519,8 122,9 -2,04 0 Ypel4 55,9 14,5 -2,03 0,000002 Gpr35 597,4 155,1 -2,01 0 Vipr1 132,4 31,4 -2,01 0 Mxd4 2835,3 687,1 -2,01 0 Enpp1 236 58,2 -2,01 0 Cd300c 40,9 10,2 -2 0,000036 Adcy3 1120,5 284,3 -1,99 0 Ace 172,5 42,1 -1,98 0,000021 Iqgap2 1128,8 282,6 -1,97 0,000005 Frmd4b 1525,3 385,1 -1,97 0 Sult1a1 60 15 -1,97 0,000034 Ksr2 1154,4 288,1 -1,97 0 Gm8113 42 10,7 -1,97 0,000004 Htra3 60,9 16 -1,96 0,000034 Sat2 56,9 14,7 -1,96 0,000001 Sh2d1b1 672,2 169,5 -1,96 0 C5ar1 9185 2448,2 -1,95 0 Ophn1 444,8 114,9 -1,94 0 Ttc30b 77,7 20,5 -1,94 0 Deptor 1249,9 310,7 -1,93 0 Card11 983 256,6 -1,93 0,000268 Pald1 1741,6 443,9 -1,92 0 Nat8l 613,2 159,4 -1,92 0 Fn1 35777,5 8207,4 -1,91 0,000459 RP23-418O21.3 55 15,2 -1,91 0,000068 Slc7a4 106,6 28,4 -1,9 0,000004 Maf 1640 456,2 -1,89 0,000006 Itga6 3532,4 945,8 -1,88 0 Ap1s2 3409,1 921,8 -1,88 0 Gm29397 111,6 30,2 -1,88 0 D3Ertd751e 144,5 39,5 -1,87 0

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Gene WT -HIV WT +HIV log2-fold change p-value Ramp1 1032 277,1 -1,87 0 Lair1 2473,7 676,3 -1,87 0 Pkd1l2 85,8 23,5 -1,86 0,000001 Neat1 42874,5 11796,4 -1,86 0 Sesn1 1392,8 386,8 -1,85 0 Arhgap18 2117,1 596,8 -1,85 0 Havcr2 857,9 260 -1,84 0 Zfp395 634,2 184,6 -1,84 0 Pdk1 1909,6 531 -1,83 0 Mertk 4264,2 1207,6 -1,82 0 Slc16a7 395,3 108,4 -1,82 0 Slc18a1 88,1 23,9 -1,82 0,000011 Gng7 101,4 28,6 -1,82 0 Ccr5 11059,7 3107,7 -1,82 0 Gm1673 85,8 24,4 -1,81 0,000015 Mrc1 5527,5 1601,9 -1,8 0 Gng2 1529,8 438,7 -1,8 0 Hhat 33,8 10 -1,79 0,003499 Mafb 7567,1 2301,7 -1,79 0 Tlr5 60 18,2 -1,79 0,000011 Adora3 67,6 20,4 -1,78 0,00134 Nes 554,4 163,7 -1,78 0 Tmem37 1076,6 323 -1,78 0 A430078G23Rik 130,7 35,5 -1,78 0,000041 Enc1 4400,4 1245,1 -1,76 0 Cd72 1671,4 503,3 -1,75 0 Prune2 903,7 266,1 -1,75 0 Camk2a 90,6 27 -1,74 0,000023 Cfh 236,1 80,1 -1,74 0,000047 Daglb 5487,8 1689,7 -1,74 0 Espn 71,8 21,5 -1,73 0,000008 Zfyve28 782,5 248 -1,73 0 1810011H11Rik 982,4 290,2 -1,73 0 E230016K23Rik 37,7 11,2 -1,73 0,015632 Klhl3 114,2 34,7 -1,72 0 Fam110b 92,8 28,1 -1,72 0,000488 Alg6 300,8 90,9 -1,72 0 Syn1 196,4 60,3 -1,71 0 Nlrp10 145,5 45,9 -1,71 0 Nav2 1148,9 348,1 -1,71 0,00001 Lpar5 215,7 68 -1,7 0 Gpr183 522,1 161,8 -1,69 0 Smim13 1137,7 356,9 -1,67 0 Lrrc14b 190,2 64,3 -1,66 0,000477 4933431E20Rik 42,9 13,8 -1,66 0,001045 Slc37a2 14265,3 4812,5 -1,65 0

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Gene WT -HIV WT +HIV log2-fold change p-value Ctla2b 164,1 50,9 -1,65 0,000001 Gm26778 154 48,9 -1,65 0,000008 Lipn 109 36,1 -1,64 0 Clec12a 5202 1649,9 -1,64 0 3300005D01Rik 39,6 13,6 -1,64 0,003598 Hfe 1477,2 479,7 -1,63 0 Tspan8 32,5 10,5 -1,63 0,003764 Fcgr4 901,7 302,6 -1,63 0 Mamdc2 5104,1 1709,3 -1,62 0 Ldlrad3 503 157,8 -1,62 0,000002 Wdpcp 76,3 25 -1,61 0,000014 Abcg2 248,7 81,7 -1,61 0 Plcb1 74,2 23,4 -1,61 0,000829 Gm4980 126,9 42,3 -1,61 0,000001 Etv5 343,7 114 -1,6 0 RP24-80F7.5 70,1 24,1 -1,6 0,000604 Chn2 47,9 16 -1,59 0,019262 Gm28417 32,2 10,7 -1,59 0,002082 Sgsh 1009 333,4 -1,58 0 Abcc3 5375,9 1960,7 -1,57 0 Porcn 64,5 21,7 -1,57 0,000008 Kcnq3 44,1 13,5 -1,57 0,04246 Kif9 134,5 44,1 -1,56 0,000001 C3ar1 14927,8 5386,4 -1,56 0,000005 Clspn 54,5 18,5 -1,56 0,000462 Clec4a3 2881,8 936,5 -1,56 0 Gm11545 43,6 15,1 -1,56 0,000117 Gm13091 33 11,2 -1,56 0,007148 Slc36a2 3411,2 1105,1 -1,55 0 Fgd2 136,5 50,8 -1,55 0,00001 Dchs1 170,5 57,4 -1,55 0 Camk1d 1610,4 510,2 -1,55 0,002678 Gm21975 259,1 89,3 -1,55 0 Itgb5 12942 4365,2 -1,54 0 Cand2 49,5 15,6 -1,54 0,005367 Klrb1a 80,9 27,6 -1,54 0,000067 Siglecf 419,1 156,7 -1,54 0,000046 Gpx3 1263,6 455,7 -1,53 0,000001 Cela1 54,6 19,4 -1,53 0,000219 Slamf9 390,8 135,9 -1,53 0 Pitpnc1 1641,1 570,7 -1,53 0 Cysltr1 715,3 242,2 -1,53 0 Svip 222,3 75,7 -1,53 0,000001 4930480K23Rik 34,3 11,8 -1,53 0,001244 Gpd1 58,4 22,2 -1,52 0,000105 Hgf 1158,5 405,2 -1,52 0

132 Appendix

Gene WT -HIV WT +HIV log2-fold change p-value Rhoj 296 106 -1,52 0 B3glct 159,1 55,4 -1,52 0 Acot1 282,1 103,4 -1,52 0 Gm20707 140,2 48,8 -1,52 0 Stxbp4 82,4 28,4 -1,51 0,000001 Kctd12b 2010,6 706 -1,51 0 Gapt 248,6 88,1 -1,51 0 Zfp791 37,1 14,7 -1,51 0,012447 Eya4 112,6 40,5 -1,5 0,000088 Pdgfc 216,1 76,6 -1,5 0 Tlr8 6585,6 2288,6 -1,5 0

Table S4. List of genes that were significantly downregulated in SAMHD1 KO BMDCs upon HIV-GFP infection.

Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Slc9a9 703,5 49,7 -3,82 0 Ccr2 8936,8 686,2 -3,7 0 Abca9 181,7 16,7 -3,44 0 Cd300lg 181,4 23,3 -2,95 0 Arg1 470 62,2 -2,92 0,002606 Slco2b1 671,9 91,6 -2,88 0 Rnf150 1666,9 246,1 -2,76 0 Dclk3 224 34,5 -2,69 0 Gpr34 234,2 38 -2,62 0,00029 Nxpe5 617,1 105,8 -2,54 0 Gm13710 57,8 10,5 -2,44 0,000122 Dmpk 96,3 19 -2,35 0 Hpgds 1759,4 359,9 -2,29 0 C5ar2 1283,5 262 -2,29 0 Mrc1 6861,7 1446,5 -2,25 0 Kcnj10 1284,2 270,3 -2,25 0 2900026A02Rik 169,3 35,4 -2,25 0 Gpx3 1349,8 284,8 -2,24 0 Fcrls 487,7 104,2 -2,23 0 Pcyt1b 44,3 10 -2,15 0,000289 Fn1 52607,3 11915,5 -2,14 0,000083 Plxnb3 160,7 37,6 -2,09 0,000001 Havcr2 618,1 146,3 -2,08 0 Fzd4 64,2 15,2 -2,07 0,000141 Arhgap19 3279,1 793,9 -2,05 0 F13a1 134,6 32,7 -2,04 0,000024 Plscr4 224,8 55,4 -2,01 0 Maf 1496,8 373,9 -2 0,000001 Notch4 42,2 10,6 -1,99 0,000664 Pkd1l2 92,3 23,2 -1,99 0

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Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Zfp395 474,7 119,1 -1,99 0 Usp2 218 55,3 -1,97 0 Sepp1 13420,4 3426,4 -1,97 0 Reps2 671,9 173,2 -1,95 0 A430078G23Rik 195,7 51 -1,93 0,000002 Iqgap3 470,7 124,2 -1,92 0 Nat8l 721 189,8 -1,92 0 Gm15513 297,7 78,9 -1,92 0 Plxna4os1 80,5 21,3 -1,92 0,000001 Mef2c 500,1 132,3 -1,91 0 Zfyve28 477,7 127,2 -1,91 0 Slc16a7 416,8 112,9 -1,88 0 Cfh 89,5 24,3 -1,88 0,000141 Enpp1 264,3 71,3 -1,88 0 RP23-297M4.7 215,5 58 -1,88 0 Adam22 86,2 23,5 -1,87 0,005468 Hdac9 96,4 26,4 -1,86 0,00021 Rab3il1 1309,1 363,6 -1,85 0 Zdhhc14 806,7 223,3 -1,85 0 C5ar1 6977,3 1939,5 -1,85 0 Ksr2 1240,1 344,4 -1,85 0 Gatm 356,5 99,7 -1,84 0 Mamdc2 3404,5 949,5 -1,84 0 Itga6 3246,8 911,2 -1,83 0 Ophn1 348 97,7 -1,83 0 Krt80 103,3 29 -1,83 0,0003 Nlrp10 141,7 40 -1,83 0 Scel 37,3 10,5 -1,82 0,003315 Vipr1 164,5 46,3 -1,82 0 Rhoj 221,6 63 -1,81 0 Gng2 1647,4 474,3 -1,79 0 Nlrp1c-ps 512 148,3 -1,79 0,000002 Ms4a8a 39,6 11,6 -1,78 0,01159 Slc46a3 617,1 179,6 -1,78 0 Ace 277 81 -1,77 0,000066 Arhgap15 888,7 260,4 -1,77 0,000809 Kif26b 160,3 47,3 -1,76 0,000708 Nes 270,1 80,1 -1,75 0 Daglb 4357,4 1298,2 -1,75 0 Gm1673 94,1 27,8 -1,75 0,00002 Espn 69,3 20,7 -1,74 0,00001 Sult1a1 83 24,6 -1,74 0,000074 Adcy3 1185 358,4 -1,73 0 Lyzl4 67,7 20,4 -1,73 0,000005 Lair1 1893,7 571 -1,73 0 Camk2a 112 33,7 -1,72 0,000013

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Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Gm29397 86,9 26,3 -1,72 0,000001 Lmln 189,6 57,8 -1,71 0 Itga8 46,7 14,3 -1,71 0,002857 Ramp1 1470,9 450,5 -1,71 0 Lpar5 198,3 60,7 -1,71 0 Sirpb1c 2249,8 686,3 -1,71 0,000002 Zfp541 39,1 11,6 -1,71 0,00035 Rnase6 253,3 77,7 -1,7 0,000051 Deptor 1563,9 482,2 -1,7 0 Kazald1 711,7 218,8 -1,7 0 Hacd4 7467,4 2297,9 -1,7 0 Slc18a1 67,1 20,6 -1,69 0,000111 Clec12a 3662 1132,3 -1,69 0 Sirpb1b 559,1 173,3 -1,69 0 Mxd4 3058,5 964,8 -1,66 0 Ear2 941,5 297,4 -1,66 0,003397 Gm8113 54,1 17,1 -1,66 0,000023 Sirpb1a 921,4 291,7 -1,66 0 Hfe 1439,2 457,5 -1,65 0 Gm28417 39,2 12,5 -1,65 0,000632 Rnase4 679,1 217,6 -1,64 0 Pald1 2353,1 762,1 -1,63 0 Fam110b 72,8 23,6 -1,63 0,00193 Nynrin 71,2 22,9 -1,63 0 Kif19a 140,3 45,8 -1,62 0,000011 Sgsh 862,3 281,7 -1,61 0 Efr3b 524,6 171,9 -1,61 0 Cela1 44,4 14,5 -1,61 0,000255 C3ar1 8452,5 2767,7 -1,61 0,000002 Gm38248 75,8 24,6 -1,61 0,000015 Plxdc2 9139,7 3042,5 -1,59 0,000179 Card11 1147,4 380,5 -1,59 0,003691 Syn1 205,2 67,8 -1,59 0 Rhobtb1 87,8 29,4 -1,58 0,000001 Ppfia4 451,7 151 -1,58 0 Mafb 7989,1 2664,8 -1,58 0,000001 Clec1a 141,7 47,7 -1,57 0,000006 Prune2 907,2 306,2 -1,57 0 Sesn1 1115,5 377 -1,56 0 Tmem37 995,2 338,4 -1,56 0 Ccr5 11437,5 3881,5 -1,56 0 Itgb5 12464,8 4258,2 -1,55 0 Prkar1b 53,4 18,1 -1,55 0,000494 Cd28 420,3 142,9 -1,55 0,000001 Enc1 3971,8 1352,5 -1,55 0,000003 Gm5150 234,9 80,1 -1,55 0,000005

135 Appendix

Gene SAMHD1 KO -HIV SAMHD1 KO +HIV log2-fold change p-value Gm9733 79,5 26,7 -1,55 0 5031425F14Rik 117,1 39,9 -1,55 0,000001 Zranb3 745,5 257 -1,53 0 Plcb1 112,6 39 -1,53 0,00053 Zfp791 35,5 12,4 -1,53 0,010385 Id1 175,4 61,6 -1,52 0 Slc6a19 32,2 11,4 -1,51 0,010514 Htra3 78 27,5 -1,51 0,000757 Siglecf 385,9 134,9 -1,51 0,000053 Oscp1 63,1 21,9 -1,51 0,000162 Nlrp1b 1325,6 464,3 -1,51 0,000195 Spink2 51,5 18,3 -1,5 0,032351 Sh2d1b1 802,4 282,4 -1,5 0

Table S5. List of genes that were endogenously upregulated in SAMHD1 KO compared to wt BMDCs. Genes that were further enhanced upon HIV-GFP infection are highlighted in dark red.

log2-fold SAMHD1 SAMHD1 log2-fold Gene WT -HIV WT +HIV change KO -HIV KO +HIV change Irg1 379,1 924,2 1,13 924,2 33849,9 5,19 Ppbp 955,2 4717,5 1,92 4717,5 13048,9 1,47 H2-M2 732,5 1701,5 1,08 1701,5 8691,8 2,35 Apol7c 549,4 3384,1 1,99 3384,1 6732,3 0,99 Clu 840,8 2849,7 1,28 2849,7 4517,4 0,66 Dusp5 1261,8 2813,8 1,02 2813,8 3368,8 0,26 Ifitm1 629 2264,8 1,31 2264,8 2423,7 0,1 Slc2a6 412,6 1248,2 1,3 1248,2 1899,9 0,61 Adam23 377,2 1144,8 1,01 1144,8 1592,2 0,48 Il1b 149,1 540,5 1,97 540,5 1590,2 1,56 Dnah2 349,9 1409,4 1,58 1409,4 1386,4 -0,02 Spib 279,3 1166,4 1,5 1166,4 1277,2 0,13 Cd40 123,1 398,2 1,18 398,2 1221,7 1,62 Vcan 253 719,3 1,29 719,3 1085 0,59 Atrnl1 291 1050,3 1,42 1050,3 974,4 -0,11 Zmynd15 234 640,3 1,07 640,3 892,5 0,48 Mab21l3 113,6 439,5 1,37 439,5 685,2 0,64 Eps8l2 119,6 417,9 1,34 417,9 648,5 0,63 H2-Eb2 38,8 320,3 2,64 320,3 614,9 0,94 Egr1 569,2 1565,3 1,56 1565,3 612,6 -1,35 Gpr55 133,9 450,9 1,27 450,9 600,6 0,41 Ifi27l2a 75,4 229,3 1,2 229,3 582,3 1,34 Amica1 372,4 1044,4 1,21 1044,4 579,8 -0,85 Pdcd1lg2 132,9 466,4 1,46 466,4 571,8 0,29 Zfp366 103,4 384,5 1,29 384,5 563,9 0,55 Dpysl5 104,3 442,4 1,44 442,4 525 0,25 Il2ra 96,9 367,3 1,28 367,3 511,7 0,48 Spn 266 607,9 1,02 607,9 503,3 -0,27

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log2-fold SAMHD1 SAMHD1 log2-fold Gene WT -HIV WT +HIV change KO -HIV KO +HIV change Plet1 42,8 339,3 3,23 339,3 437,5 0,37 Ccl17 43,3 191,8 1,61 191,8 437 1,19 Mtss1l 88,4 301,6 1,33 301,6 418,2 0,47 Gm8221 10,8 155,5 3,4 155,5 414,4 1,41 Six1 75,1 190,2 1,16 190,2 397 1,06 Mgl2 79,1 655,9 2,91 655,9 392 -0,74 Gm7676 77,8 277,4 1,3 277,4 385,4 0,47 Crispld2 166,9 350,5 1,05 350,5 374,4 0,1 Mylk 43,7 231,9 2,1 231,9 356,8 0,62 Nrg1 102,5 291,8 1,23 291,8 356 0,29 Cxcl1 38,9 80 1,14 80 346,6 2,11 Scube1 89,7 318,9 1,25 318,9 343 0,1 2610528A11Rik 75,7 314,8 1,63 314,8 339,3 0,11 Dpp4 81,1 314,8 1,42 314,8 331,4 0,07 Lad1 19,3 125,6 2,29 125,6 296,8 1,24 Sod3 71,9 216,2 1,1 216,2 294,5 0,44 H2-Oa 72,8 212,2 1,12 212,2 265,3 0,32 Kcnh3 55,8 184 1,2 184 248,7 0,43 Vcam1 42,7 135,3 1,44 135,3 245,9 0,86 Gpr84 36,9 92,3 1,11 92,3 236 1,35 Cdh23 54,5 188 1,25 188 233,3 0,31 Col5a1 64,6 160,7 1,06 160,7 219,1 0,45 Kazald1 306,7 711,7 1,07 711,7 218,8 -1,7 Klk1b11 78,8 433,5 2,11 433,5 211,3 -1,03 Cacng8 43,2 122,5 1,19 122,5 201,7 0,72 Asgr2 20 182,4 2,76 182,4 193,3 0,08 Ptk6 49,7 154,7 1,19 154,7 192,8 0,32 Ccdc80 111,7 289,5 1,14 289,5 185,2 -0,64 Tnfrsf18 62,6 163,1 1,03 163,1 183 0,16 Gm12164 83,2 169,8 1,12 169,8 170,5 0,01 Aff3 35,4 159,8 1,68 159,8 169,3 0,08 Mmp23 44,5 140,2 1,27 140,2 166 0,24 Aqp9 66,8 148,5 1,08 148,5 165,4 0,16 Samd5 20,7 80 1,58 80 155,1 0,95 Apol10b 13,3 89,6 2 89,6 149,7 0,74 Hmmr 63,6 114,5 1,15 114,5 141,4 0,3 Siglece 31,5 97,2 1,3 97,2 136,7 0,49 Nid1 39,4 127 1,23 127 125,6 -0,01 Rrad 35,1 85,2 1,01 85,2 123,5 0,53 Tubb2b 42,9 99,4 1,02 99,4 122,5 0,3 Cdh2 36,5 103,7 1,13 103,7 116,7 0,17 Abcg4 25,8 72,6 1,11 72,6 115,4 0,67 Stxbp6 23,9 94,5 1,37 94,5 110,6 0,23 Ccnb2 41,8 98,8 1,38 98,8 110 0,15 Aif1l 14,6 84 2 84 108,8 0,37

137 Appendix

log2-fold SAMHD1 SAMHD1 log2-fold Gene WT -HIV WT +HIV change KO -HIV KO +HIV change Hepacam2 37,5 115,4 1,25 115,4 107,7 -0,1 Ltbp2 42 109,1 1,01 109,1 106,2 -0,04 2010005H15Rik 28,3 81,4 1,23 81,4 99,2 0,28 Fndc5 20,6 70,3 1,43 70,3 95,9 0,45 Ocln 18,2 55,1 1,27 55,1 83,9 0,6 BC035044 9,3 40,3 1,67 40,3 80,8 1 Gm37422 13 57,5 1,66 57,5 80,8 0,49 Gal3st2 9,9 78,1 2,51 78,1 80,7 0,05 Myo5b 9,1 59 2,25 59 79,6 0,43 Rnase6 98,7 253,3 1,15 253,3 77,7 -1,7 Epx 13,9 45,7 1,61 45,7 74,1 0,7 Dscam 17,3 75,7 1,66 75,7 73,6 -0,04 Kcnq3 44,1 129,8 1,14 129,8 70,9 -0,87 Acvr1c 6,9 35,9 1,91 35,9 68,8 0,94 St8sia1 17,1 56 1,42 56 65,9 0,23 Gm266 13,8 48,9 1,39 48,9 64,4 0,4 Ccl2 18,3 112 2,59 112 62,9 -0,83 Dpf3 18,4 58,2 1,31 58,2 62,7 0,1 Prr11 38,8 79,3 1,07 79,3 59,9 -0,41 Plekha6 36,8 82,3 1,24 82,3 58,6 -0,49 Cenpf 49,4 99 1,21 99 58,3 -0,76 Ncam1 24,2 59 1,07 59 57,7 -0,03 Mfsd6l 18 43,6 1,2 43,6 54,1 0,3 Gli3 15,1 52,5 1,46 52,5 53,5 0,02 Egr3 25,9 120,6 2 120,6 51 -1,23 Fgd5 16,5 56,6 1,27 56,6 50,1 -0,18 Phf19 26,3 54,6 1,01 54,6 47,9 -0,19 Gfpt2 9,5 30 1,42 30 47,7 0,67 1700024P16Rik 9,7 53,8 2,15 53,8 47 -0,19 Vat1l 5,6 32,1 2,25 32,1 46,3 0,53 Gm5833 3,5 42,9 3,25 42,9 45,6 0,09 Tcaf2 8,1 26,8 1,41 26,8 44,6 0,73 Cdhr1 10,8 37,3 1,37 37,3 43,6 0,22 Gfi1b 15,3 37,2 1,37 37,2 43 0,21 Kctd14 5,8 19,5 1,47 19,5 41,6 1,1 Kmo 22,4 81,1 1,37 81,1 41,6 -0,96 Atp8b3 8,5 35,2 1,64 35,2 40,6 0,2 Gm10851 8,7 37,3 1,59 37,3 40,4 0,11 Klrd1 10,1 36,7 1,43 36,7 39,6 0,11 Ntn4 14,5 45 1,33 45 39,2 -0,2 Ccr4 9,5 34,5 1,38 34,5 37,4 0,11 Gm26535 5,8 24,7 1,53 24,7 35,4 0,51 Npr2 10,5 41,3 1,65 41,3 33,8 -0,29 Chrm3 4,5 20,5 1,83 20,5 32,5 0,67 Dao 7,6 35,5 1,74 35,5 32 -0,16

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log2-fold SAMHD1 SAMHD1 log2-fold Gene WT -HIV WT +HIV change KO -HIV KO +HIV change Htr7 6,1 24,7 1,72 24,7 31,4 0,35 Gm2065 4 28,5 2,48 28,5 31,3 0,13 Ear1 9,5 26,8 1,13 26,8 31,3 0,23 Ccl24 2,7 15,9 2,11 15,9 29,3 0,88 Cmah 26,4 58,5 1,21 58,5 29,3 -0,99 Gcnt4 7,4 23,1 1,22 23,1 27,7 0,26 Susd4 6,6 39,5 1,98 39,5 27,4 -0,53 Cdkn2a 6,6 24,6 1,77 24,6 27,1 0,14 Rasl10b 7,9 28,4 1,47 28,4 25,8 -0,13 Kif4 16,1 43,3 1,42 43,3 25,2 -0,78 Camk4 3,3 16,1 1,96 16,1 24,6 0,62 Cd1d2 4,1 18,8 2,08 18,8 24,4 0,37 Ces2c 2,3 14,9 2,69 14,9 24,1 0,69 Dkkl1 2,5 19,8 2,61 19,8 23,9 0,27 Dll1 3,8 19,7 2,01 19,7 23,7 0,27 Ttc36 10 31,7 1,48 31,7 22,4 -0,5 Nek2 21 41,2 1,25 41,2 19,8 -1,06 Gm9994 0,9 16,7 3,89 16,7 19,5 0,22 Irgc1 13,8 43,2 1,53 43,2 19,5 -1,13 D630039A03Rik 5,4 19,4 1,57 19,4 19,3 -0,01 Adgrg5 2,3 17,9 2,64 17,9 19,2 0,11 Fam178b 3,6 21,2 2,23 21,2 18,3 -0,22 Klk1b27 17,1 49,8 1,08 49,8 17,6 -1,48 Kif2c 14,1 39,9 1,62 39,9 17,1 -1,21 Gm6537 2,8 17,8 2,23 17,8 17 -0,07 Cadm3 4,7 25 2,05 25 16,4 -0,62 Vwa3b 4 18,2 1,82 18,2 16,2 -0,17 Fam160a1 1,7 14,8 2,47 14,8 15,9 0,11 Stk19-ps1 7,6 22,4 1,42 22,4 15,2 -0,56 Dcn 1,7 13,9 2,84 13,9 13,4 -0,05 Grin2c 1 10 2,57 10 13,2 0,4 Lmo1 2,9 12,2 1,84 12,2 12,8 0,09 Kif14 15,9 30,3 1,25 30,3 12,7 -1,25 Klk1b9 1,6 16,6 3,14 16,6 12,4 -0,41 Tgfb1i1 9,9 32 1,5 32 12,2 -1,39 Adgrl3 4,9 32,1 2,29 32,1 12,1 -1,4 Dnajc22 1,1 9,2 2,52 9,2 11,3 0,3 Cemip 3 23,6 2,6 23,6 10,3 -1,21 Gad1-ps 4,6 19,3 1,57 19,3 8 -1,28 Plet1os 5 15,2 2,06 15,2 6,1 -1,32 Gprc5d 0,9 7,5 2,85 7,5 5,9 -0,34 Aox3 9,1 34 1,34 34 4,8 -2,8 Klhl32 0 5,9 5,03 5,9 4,1 -0,55 RP24-465E5.2 0,6 5,9 3,21 5,9 3,2 -0,87 C8a 0,3 7,9 3,9 7,9 3 -1,35

139 Appendix

log2-fold SAMHD1 SAMHD1 log2-fold Gene WT -HIV WT +HIV change KO -HIV KO +HIV change Grb14 2 10,2 2,03 10,2 2,9 -1,81 Gm3272 0,3 4,7 4,04 4,7 2,6 -0,84 Aplnr 1,5 8,4 2,32 8,4 2,1 -2,05

Table S6. List of genes that were significantly upregulated in SAMHD1 KO compared to IFNAR/SAMHD1 double KO BMDCs upon HIV-GFP infection.

Gene SAMHD1 KO +HIV IFNAR/SAMHD1 KO +HIV log2-fold change p-value Oas1g 509,7 0 10,95 0 Ifi44 210,8 0 9,54 0 Oas1a 1518,3 6,3 7,93 0 Rtp4 1266,4 4,7 7,92 0 Gm14446 118,6 0,4 7,86 0 Gm4955 359,7 1,5 7,55 0 Ms4a4c 199,9 1,2 7,25 0 Ifit3b 212,7 1,2 7,12 0 Oasl2 5484,5 44,6 6,9 0 Ifit2 4874 59,8 6,32 0 Gm4070 102 1,2 6,32 0 Ifi27l2a 582,3 5,8 6,24 0 Oas3 5042,1 67,5 6,13 0 Pydc4 239,5 2,3 6,11 0 Cmpk2 928,2 29,3 4,9 0 Gm12250 370,9 11,6 4,8 0 Irf7 4184,6 157,1 4,77 0 Usp18 1510,3 49,6 4,62 0 Zbp1 1219,8 47,8 4,41 0 Oasl1 3730,8 187,5 4,12 0 Mnda 644,9 45,2 4 0 Ifi205 244 15,3 3,96 0 Xaf1 1074,9 61,5 3,94 0 Slfn5 7656,4 586,1 3,85 0 Slfn8 2108,9 147,9 3,7 0 Rsad2 6765,5 561,1 3,69 0 Slfn1 769,3 47,6 3,69 0 Mx2 316,5 20,4 3,63 0 Ly6a 891,5 60,6 3,56 0 Ifi47 1262,6 116,2 3,41 0 Cxcl10 552,8 48,4 3,4 0 Ifi203 1254,6 121 3,27 0 Ifit1 2778,8 278,6 3,25 0 Phf11d 258,7 28 3,21 0 H2-T24 1695,7 196,3 3,03 0 Ifitm3 3219,7 301,5 3,02 0 Phf11a 147,5 15,6 2,99 0 Ddx60 1843,7 221 2,89 0

140 Appendix

Gene SAMHD1 KO +HIV IFNAR/SAMHD1 KO +HIV log2-fold change p-value Ifi27 1580,2 201,4 2,87 0 Isg15 1884 265,1 2,85 0 Oas1b 259,2 37,3 2,68 0 Phf11b 538,2 72 2,67 0 Mgl2 392 55 2,62 0,000101 Ppbp 13048,9 1846 2,57 0,000002 Ddx58 3351,3 566,9 2,53 0 Trim30d 2215,7 345,4 2,52 0 Siglec1 963,3 151,6 2,51 0,000003 Gbp7 407,6 80,7 2,41 0 Pyhin1 1827,8 309 2,36 0 Isg20 198,4 37,6 2,34 0 Ifi204 3508,9 825,4 2,33 0 Fcgr1 623,8 132,2 2,31 0 Ms4a6b 581,4 130,8 2,25 0,000007 Trim30a 4883 978,4 2,21 0 Rnf213 15217,9 3042,8 2,2 0 Plet1 437,5 114,3 2,17 0,005623 Nlrc5 2560,8 515,5 2,17 0 Ly6i 1862,7 373,3 2,08 0,000002 Mndal 1948,7 430 2,06 0 Asgr2 193,3 38,4 2,03 0,000191 Hsh2d 115,2 22,7 2,03 0,000002 Slfn9 259 63,8 1,99 0 Dhx58 1712,5 423 1,98 0 H2-Eb2 614,9 120,4 1,97 0,000096 Gm7676 385,4 81,1 1,95 0,000001 Gbp5 1086,4 242,8 1,95 0 Irf9 2770,7 690,8 1,93 0 Stat2 5293,8 1302,1 1,93 0 Lad1 296,8 66,2 1,89 0,000064 Parp14 8726 2282,9 1,88 0 Gbp3 853,3 203 1,87 0 Cxcl5 775,8 178,8 1,83 0,000646 Gm1966 772,8 182,4 1,82 0 Stat1 2922,6 759,2 1,8 0 Unc13b 113,9 34,4 1,8 0 Oas1c 192,7 50 1,77 0 Sp100 3979,1 1129,8 1,77 0 Irgm2 1114,7 320,1 1,75 0 Gm5431 896,2 242,9 1,74 0 Igtp 535,2 167,2 1,72 0 Ccnd1 348,5 104,5 1,71 0,000911 Slfn3 403,9 122,1 1,7 0 Eif2ak2 2827 888,2 1,7 0 Gbp2 3275,9 898,3 1,7 0

141 Appendix

Gene SAMHD1 KO +HIV IFNAR/SAMHD1 KO +HIV log2-fold change p-value Ms4a6c 1218,3 314,7 1,7 0 Tnfsf15 329,4 90,6 1,68 0 Trim30b 153,4 39,8 1,67 0,000155 9230114K14Rik 159,6 47,7 1,67 0,000203 Kcnn3 602,3 169 1,65 0,000009 Irgm1 2603,1 879,3 1,62 0 Bst2 3707,3 1179,4 1,59 0 Lgals3bp 12740,1 4148 1,58 0 Vcan 1085 342 1,53 0,006558 Cfb 130,3 32,6 1,53 0,000026 Ccl17 437 121,6 1,52 0,001428 Fgl2 2677,6 920,8 1,52 0 Batf2 120,9 40,1 1,51 0 Il1b 1590,2 597,8 1,5 0,018099

Table S7. List of genes that were significantly upregulated in IFNAR/SAMHD1 double KO BMDCs upon HIV-GFP infection. Log2-fold change of IFNAR/SAMHD1 double KO cells was compared to fold change of infected IFNAR KO BMDCs.

log2-fold change log2-fold change Gene IFNAR/SAMHD1 KO +HIV IFNAR KO +HIV Tmem236 5,96 6 Gramd1c 5,6 5,73 Irg1 5,17 5,39 Ifit1 4,83 2,6 A530099J19Rik 4,4 4,35 Ccdc141 4,16 4,42 Susd2 4,01 4,24 Gm19325 3,7 3,91 Cxcl3 3,66 4,23 Cxcl2 3,51 3,24 Oasl1 3,4 1,46 Cxcl5 3,35 3,46 Cxcl1 3,32 3,08 Ptgs2 3,27 3,72 Ednrb 3,2 3,67 Ddit4l 3,12 2,09 Serpinc1 3,09 3,86 Cp 3,05 3,19 Tnf 3,03 2,62 Slpi 2,94 3,05 Fam181b 2,86 3,85 Fam71f2 2,78 4,31 Tlcd2 2,7 3,58 Gm15832 2,69 3,55 Fam212b 2,67 1,45 Gm12689 2,66 3,04

142 Appendix

log2-fold change log2-fold change Gene IFNAR/SAMHD1 KO +HIV IFNAR KO +HIV Clec4e 2,58 2,62 Slc6a9 2,42 2,83 Cdo1 2,4 3,29 Rsad2 2,38 1,45 Isg15 2,37 1,79 Gtse1 2,31 1,63 RP24-492O4.8 2,29 2,55 H2-M2 2,24 2,38 AA986860 2,2 2,68 Il20rb 2,15 2,33 Pcx 2,14 2,47 Psrc1 2,14 1,64 Sod2 2,1 2,23 Tnfrsf10b 2,09 1,22 Lgr4 2,08 2,53 Il1b 2,05 1,83 Six4 2,05 3,64 Mss51 2,01 3,18 Fosl1 2,01 1,95 Hmcn2 1,97 1,84 Orm1 1,94 2,18 Saa3 1,94 2,33 Slc19a2 1,94 1,54 Treml4 1,94 2,16 Spic 1,93 1,42 Il1f9 1,91 2,54 Ccrl2 1,89 2,48 Tarm1 1,89 2,41 Ralgds 1,88 2,49 Unc5b 1,86 2,06 Ankrd24 1,86 1,97 Hmox1 1,84 2,56 Tnfaip3 1,84 1,99 Ddias 1,84 0,95 Ccl4 1,83 0,94 Eda2r 1,8 0,92 Fpr2 1,74 1,87 RP23-441I24.5 1,74 2,32 Gdf15 1,73 2,07 Cdkn2b 1,73 1,51 Gnal 1,72 1,98 Gm340 1,72 2,31 Fam13a 1,69 1,72 G630090E17Rik 1,68 1,92 Cdkn1a 1,67 1,25

143 Appendix

log2-fold change log2-fold change Gene IFNAR/SAMHD1 KO +HIV IFNAR KO +HIV Ptpn14 1,66 2,3 Ptgr1 1,65 2,09 Gem 1,64 2,05 Nfkbie 1,63 1,53 Hp 1,63 1,76 Cd38 1,62 1 Ampd3 1,61 1,91 Tma16 1,61 1,82 Rasgef1b 1,61 1,54 Gm13571 1,61 2,18 Gm11427 1,59 2,17 Phlda3 1,58 0,82 Lox 1,56 1,91 Ptges 1,55 1,46 Tmem178 1,54 2,01 Ankrd66 1,54 1,9 Clmp 1,53 1,97 Nfkbiz 1,53 1,72 Cdc42ep2 1,53 1,61 9130230L23Rik 1,51 1,7 Il1a 1,5 1,73

144 Appendix

2. Publications

Herrmann A., Wittmann S., Thomas D., Shepard C., Kim B. Ferreiros N., and Gramberg T. (2018). The SAMHD1-mediated block of LINE-1 retroelements is regulated by phosphorylation. Mobile DNA (2018) 9:11.

Herrmann A., Happel A. and Gramberg T. (2016) SAMHD1 in Retroviral Restriction and Innate Immune Sensing – Should We Leash the Hound? Current HIV Research, 2016, 14, 225-234.

Kahle T., Volkmann B., Eissmann K., Herrmann A., Schmitt S., Wittmann S., Merkel L., Reuter N., Stamminger T. and Gramberg T. (2015) TRIM19/PML Restricts HIV Infection in a Cell Type-Dependent Manner. Viruses 2016, 8, 2; doi:10.3390/v8010002.

3. Contributions to national and international conferences

3.1. Oral presentations

Herrmann A., SAMHD1 inhibits endogenous retroelements in a phospho-dependent manner. 28th Annual Meeting of the Society for Virology, Würzburg.

Herrmann A., Ross J. and Gramberg T. (2014), The Role of SAMHD1 in LINE-1 Retrotransposition. 13. Workshop des GfV-Arbeitskreises “Immunologie von Virusinfektionen”, Bad Dürkheim.

3.2. Poster presentations

Herrmann A., Behrendt R., Wittmann S. and Gramberg T. (2018) SAMHD1-dependent retroviral restriction and sensing in mice. 27th Annual Meeting of the Society for Virology, Würzburg.

Herrmann A., Behrendt R., Wittmann S. and Gramberg T. (2017) SAMHD1-dependent retroviral restriction and sensing in mice. 27th Annual Meeting of the Society for Virology, Marburg.

Herrmann A., Wittmann S., Shepard C., Thomas D., Ferreiros Bouzas N., Kim B. and Gramberg T. (2016) The SAMHD1-mediated inhibition of LINE-1 retroelements is regulated by phosphorylation. Frontiers of retrovirology, Erlangen.

Herrmann A., Wittmann S., Shepard C., Thomas D., Ferreiros Bouzas N., Kim B. and Gramberg T. (2016) The SAMHD1-mediated inhibition of LINE-1 retroelements is regulated by phosphorylation. International Congress on Transposable Elements. Saint Malo, France.

145 Appendix

Herrmann A., Wittmann S., Shepard C., Thomas D., Ferreiros Bouzas N., Kim B. and Gramberg T. (2016) The SAMHD1-mediated inhibition of LINE-1 retroelements is regulated by phosphorylation. 26th Annual Meeting of the Society for Virology, Münster.

Herrmann A. and Thomas Gramberg (2015) The role of SAMHD1 in LINE-1 retrotransposition. 25th Annual Meeting of the Society for Virology, Bochum.

Herrmann A. and Thomas Gramberg (2015) The role of SAMHD1 in LINE-1 retrotransposition. 24th Annual Meeting of the Society for Virology, Alpbach, Austria.

146 Appendix

4. Acknowledgement

An dieser Stelle möchte ich mich bei allen Menschen bedanken, die mich auf meinem Promotionsweg begleitet haben:

Vielen Dank an Prof. Überla für die Möglichkeit, meine Doktorarbeit am Virologischen Institut anfertigen zu können.

Mein Dank gilt besonders Prof. Nitschke und Prof. Marschall für die schriftliche Begutachtung meiner Arbeit. Des Weiteren danke ich Prof. Slany und Prof. Eichler sowie Prof. Gramberg für die Teilnahme an meiner mündlichen Prüfung.

Ein besonderes Dankeschön geht an meinen Betreuer Prof. Gramberg dafür, dass ich meine Doktorarbeit in seiner Arbeitsgruppe durchführen durfte. Ich danke ihm für sein Vertrauen, seine Geduld und dafür, dass er mir stets mit Rat und Tat zur Seite stand.

Bedanken möchte ich mich auch bei allen ehemaligen und aktuellen Mitgliedern der AG Gramberg. Besonders danken möchte ich Bine für ihre experimentelle Unterstützung sowie Franziska Kühner für ihren Beitrag während ihrer Bachelorarbeit. Außerdem möchte ich Bianca und Janina für die lustige und lockere Laboratmosphäre danken.

Vielen Dank an alle Mitarbeiter der Virologie, besonders der AGs Marschall, Stamminger und Marschall für die vielen Ratschläge und Materialien. Besonders bedanken möchte ich mich außerdem bei Kirsten, Pia, Pierre, Stephan, Dennis und allen anderen für die stets unterhaltsamen Mittagspausen sowie Anna, Theresa, Sigrun, Eric, Eva und Myri für die tolle Zeit im Schreibraum sowie für eure Geduld und Unterstützung.

Mein Dank gilt auch unseren Kooperationspartnern Dominique Thomas, Nerea Ferreirós, Caitlin Shepard, Baek Kim und Rayk Behrendt für die experimentelle Unterstützung, von denen meine Publikation sehr profitiert hat bzw. welche eine wichtige Grundlage für eine zukünftige Publikation geliefert hat.

Ich möchte mich außerdem sehr bei meinen Freunden bedanken, besonders bei Tanja, Anna und Herta, die immer ein offenes Ohr für mich hatten und mich auch in schwierigen Zeiten stets aufgemuntert haben.

Außerdem möchte ich mich auch bei meiner Familie bedanken, die immer an mich geglaubt und mich tatkräftig unterstützt hat.

Mein größter Dank gilt meinem Partner Friedrich, der während der letzten Jahre immer für mich da war und mich mit viel Geduld und Verständnis unterstützt hat. Ohne dich hätte ich es wahrscheinlich nie so weit geschafft, danke!

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