Identification of Anti-Poxviral Agents by High-Throughput Image-Based Screening

Home , Orthopoxvirus

Identification of anti-poxviral agents by high-throughput image-based screening

Jerzy Samolej

A dissertation submitted in partial fulfilment

of the requirements for the degree of

Doctor of Philosophy

University College London

MRC Laboratory for Molecular Cell Biology

Supervised by

Dr Jason Mercer and Dr Ricardo Henriques

September 2019

Declaration

I, Jerzy Samolej, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis.

Signed

Date 2019-09-21

Abstract

Poxviruses are a family of large, double-stranded DNA viruses that infect a wide range of organisms, from insects to humans. They possess a complex structure and an intricate replication cycle that takes place entirely in the cytoplasm. Additionally, their genome is remarkably large for viruses, encoding over 200 proteins. Historically, one representative of the orthopox genera – variola virus, the causative agent of smallpox – has claimed hundreds of millions of human lives. Despite its eradication, smallpox, and some zoonotic poxviruses, are still considered as potential threats, and new means of combating them are in demand. To this end research has continued on vaccinia virus (VACV), the prototypic orthopox virus which was used as the smallpox vaccine. In this project a library of 1,280 FDA approved molecules was screened for cell-based VACV inhibitors using a high-throughput image-based screen, following the principles of drug repurposing. After confirmation of initial hits using secondary screens, 9 Early inhibitors, 3 Late inhibitors, and 7 Spread inhibitors were identified.

Additionally, 14 cardiac glycosides were identified as Early inhibitors. Using focused assays, the specific phases of the virus replication cycle at which inhibition occurs were also identified. This project led to the discovery of new compounds that block

VACV infection at different stages in cell culture. Additionally, the initial hits were tested on African swine fever virus and 5 potential inhibitors were identified.

Collectively, these results have furthered our insights into host – pathogen interaction, while providing potential hits for testing in animal models and against other viruses.

Impact statement

In the 20th century smallpox, the most infamous member of the poxvirus family killed between 300 and 500 million people. Although eradicated, smallpox, is still considered a potential bioterrorism threat, while zoonotic poxviruses represent a continued threat to individuals and populations. TPOXX, the first anti-smallpox drug approved by the FDA, and its subsequent stockpiling by the US government demonstrates the need for development of novel anti-poxvirus agents. Vaccinia virus,

VACV, the prototypic poxvirus is 96% identical to smallpox, and was used as live vaccine for its eradication. The research presented in this thesis has made the first steps in identifying potential FDA-approved VACV inhibitors for drug repurposing.

A high-throughput microscopy-based screen followed by targeted assays has successfully identified inhibitors of various stages of VACV replication, paving the way for additional insights into poxviruses host-pathogen interaction through determining the mechanism of action of the identified inhibitors. In addition, the identified hits were used in an analogous, small-scale screen directed against African swine fever virus (ASFV), which causes 100% lethality in infected herds resulting in significant economic losses. Collectively, the research presented here confirms the utility of the high-throughput screening methodology, providing a blueprint for future drug screens on VACV and similar pathogens.

The true delight is in the finding out rather than in the knowing. - Isaac Asimov

Table of Contents

Declaration ...... 2

Abstract ...... 3

Impact statement ...... 4

Acknowledgments ...... 10

List of Tables ...... 11

List of Figures ...... 12

Abbreviations ...... 14

1 Introduction ...... 16

1.1 Poxviruses ...... 16

1.1.1 Smallpox ...... 17

1.1.2 Other poxviruses and African swine fever virus ...... 19

1.2 Vaccinia virus ...... 21

1.2.1 Modulation of the immune response ...... 22

1.2.2 VACV structure(s) ...... 26

1.2.3 VACV replication cycle ...... 27

1.3 Molecular tools ...... 37

1.3.1 Recombinant viruses ...... 37

1.3.2 VACV inhibitors ...... 38

1.4 Poxvirus prevention and treatment ...... 39

1.4.1 Existing anti-poxvirals ...... 40

1.4.2 Drug repositioning and screening for antiviral drugs ...... 41

1.5 Vaccinia virus screens ...... 42

1.5.1 VACV drug screens ...... 43

1.5.2 LOPAC screen ...... 43

1.6 Thesis aims ...... 46

2 Materials and methods ...... 47

2.1 Virus list ...... 47

2.2 Prestwick Chemical Library ...... 48

2.3 General methods ...... 48

2.3.1 Cell culture ...... 48

2.3.2 Virus preparation ...... 49

2.3.3 Plaque assay ...... 50

2.4 Primary screen ...... 50

2.4.1 Imaging ...... 51

2.4.2 Image analysis ...... 51

2.5 Secondary screen ...... 52

2.5.1 Flow cytometry ...... 52

2.6 Cytotoxicity ...... 53

2.7 Early assays ...... 53

2.7.1 Pre-incubation ...... 53

2.7.2 Binding ...... 54

2.7.3 Acid bypass ...... 54

2.7.4 AraC release assay...... 54

2.8 Microscopy assays...... 54

2.8.1 Entry assay ...... 54

2.8.2 Pre-replication and replication site formation ...... 55

2.8.3 Edu incorporation ...... 55

2.8.4 Spread hits assay ...... 56

2.9 24h EEV and IMV yields ...... 56

2.9.1 Extracellular enveloped virions ...... 57

2.9.2 Intracellular mature virions...... 57

2.10 Transmission electron microscopy ...... 57

2.11 African swine fever virus ...... 58

2.12 STAT3 experiments ...... 58

2.12.1 Flow cytometry ...... 58

2.12.2 Pre-incubation ...... 59

2.12.3 Addition timepoints ...... 59

2.12.4 STAT3 siRNA knockdown...... 59

2.12.5 Western blots ...... 59

3 Results ...... 61

3.1 Microscopy-based high-throughput drug library screen ...... 61

3.2 Flow-cytometry based confirmation screen ...... 64

3.3 Cytotoxicity ...... 69

3.4 Early hits ...... 70

3.4.1 Cardenolides ...... 72

3.4.2 Direct effect on virus particle...... 77

3.4.3 Binding to cell surface ...... 78

3.4.4 Acid bypass of internalization and fusion ...... 79

3.4.5 AraC release – secondary, Late inhibition? ...... 82

3.4.6 Is inhibition by Cardenolides ATPase dependent?...... 84

3.4.7 Microscopy of VACV binding, internalization, and fusion...... 87

3.4.8 Early hits – conclusions ...... 90

3.5 Late hits ...... 92

3.5.1 Intermediate gene expression ...... 93

3.5.2 Pre-replication sites and replication sites formation ...... 94

3.5.3 Edu incorporation ...... 95

3.5.4 Late hits – conclusions ...... 97

3.6 Spread hits ...... 98

3.6.1 24h IMV and EEV yield ...... 100

3.6.2 Fluorescent phenotyping of infected cells ...... 101

3.6.3 EM investigation of VACV morphogenesis ...... 104

3.6.4 Spread hits – conclusions ...... 115

3.7 African swine fever virus screen ...... 116

3.8 Signal transducer and activator of transcription 3 ...... 119

3.8.1 Vaccinia virus inhibition ...... 119

3.8.2 Pre-incubation ...... 119

3.8.3 Addition timepoints ...... 121

3.8.4 Phosphomimetic cell line ...... 122

3.8.5 STAT3 siRNA knockdown...... 123

4 Discussion ...... 124

4.1 Vaccinia virus high-throughput screens ...... 126

4.2 Prestwick Chemical Library screen...... 126

4.2.1 Limitations ...... 127

4.2.2 Early hits ...... 130

4.2.3 Late hits ...... 132

4.2.4 Spread hits ...... 132

4.3 African swine fever virus ...... 134

4.4 Signal transducer and activator of transcription 3 ...... 134

4.5 Summary ...... 135

5 References ...... 136

6 Supplementary ...... 160

Acknowledgments

Above all else, I want to thank my advisor, Dr Jason Mercer, for being the best mentor anyone could ask for, not only in science but in life. He has been a close friend and an outstanding role model and will continue to be throughout my life.

Victoria, for keeping chaos at bay. Artur, A FELLOW HUMAN, for ZedMate and help with data analysis. David, for being fabulous. Jen, for tolerating my distractions in the office. Moona, Nooo Niiii! Susanna, for free hearing tests.

Becky, for asking all the questions. Caroline, for fixing the cytometer, checking my spelling, and repeating my mistakes. Corina, for being a trailblazer and doing this first. Hattie, for her help with my British accent and fashion advice. Laura, for not getting my references and making me feel old. Melanie, for her help with my German accent. Noelia, for her help with my Spanish accent. Rob, for being the smart one in the lab.

Rosalie and Sam, for their work on the LOPAC screen.

Joana and Janos, for their help with the primary screen. Ian, for the EM imaging and entertaining tweets.

Pippa and Chris at The Pirbright Institute, for their help with the ASFV work.

Paul, for keeping the building in one piece, and the kitchen staff, for the daily PBS and trypsin.

The Breakfast Club, for picking me up and putting me down every morning.

And finally, my family, without whom all this would not be possible.

List of Tables

Table 2.1. VACV and VACV recombinants used in this thesis...... 48 Table 3.1. Confirmed hits...... 68 Table 3.2. Cardenolides in the literature...... 75 Table 3.3. Cardenolides confirmed by flow cytometry...... 76 Table 3.4. Early hits summary table...... 91 Table 6.1. Hits identified in the primary screen...... 161

List of Figures

Figure 1.1. Virus Taxonomy: 2017 Release...... 16 Figure 1.2. Smallpox was a major human pathogen...... 18 Figure 1.3. ASFV global distribution (2016-2018)...... 20 Figure 1.4. ASFV structure...... 21 Figure 1.5 Viral detection and IFN activation and JAK1/STAT pathway...... 24 Figure 1.6. VACV structure(s) ...... 26 Figure 1.7. VACV replication cycle...... 28 Figure 1.8. VACV entry...... 29 Figure 1.9. Genome replication...... 32 Figure 1.10. IMV formation...... 34 Figure 1.11. EM micrograph of VACV particle formation...... 35 Figure 1.12. Wrapping and egress...... 36 Figure 1.13. Primer designed to insert E-EGFP and L-mCherry into VACV TK locus...... 38 Figure 1.14. Intravenous Cidofovir and TPOXX capsules...... 40 Figure 1.15. Global map of significant and new emerging infections: spread to new areas since 1997...... 42 Figure 1.16. STAT3 pathways...... 44 Figure 3.1. VACV replication cycle and its inhibitors ...... 61 Figure 3.2. E-EGFP L-mCherry virus plaque formation and gene expression over time...... 62 Figure 3.3. Primary screen controls...... 63 Figure 3.4. Prestwick Chemical Library screen results...... 65 Figure 3.5. Cytotoxicity of confirmed hits...... 69 Figure 3.6. Early gene expression block by CHX...... 70 Figure 3.7. Cardenolides general structure...... 72 Figure 3.8. Direct effect of Early hits on virus particle...... 77 Figure 3.9. Binding assay test...... 78 Figure 3.10. VACV binding to cells...... 79 Figure 3.11. VACV acid bypass...... 80 Figure 3.12. Acid bypass...... 81 Figure 3.13. AraC release: independent Late inhibition...... 82 Figure 3.14. AraC release, 24h yield...... 84 Figure 3.15. VACV Cardenolide-dependent inhibition in HEK cells expressing mouse Na+/K+ ATPase...... 86

Figure 3.16. Microscopy-based entry assay...... 89 Figure 3.17. Late gene expression block by AraC...... 92 Figure 3.18. Late gene inhibition...... 93 Figure 3.19. Intermediate gene inhibition...... 94 Figure 3.20. Initiation site and replication site formation inhibition...... 95 Figure 3.21. EdU incorporation...... 96 Figure 3.22. Spread block by BFA...... 98 Figure 3.23. 24h IMV and EEV yield...... 101 Figure 3.24. Immunofluorescence imaging of Spread hits inhibition...... 103 Figure 3.25. EM imaging of VACV particle formation...... 105 Figure 3.26. EM - DMSO ...... 107 Figure 3.27. EM - Amethopterin ...... 108 Figure 3.28. EM - Colchicine ...... 109 Figure 3.29. EM - Pinaverium bromide ...... 110 Figure 3.30. EM - Zardaverine ...... 111 Figure 3.31. EM - Progesterone ...... 112 Figure 3.32. EM - Norgestimate ...... 113 Figure 3.33. EM - Testosterone ...... 114 Figure 3.34. ASFV screen controls...... 117 Figure 3.35. ASFV screen hits...... 118 Figure 3.36. VACV inhibition by STAT3 phosphorylation inhibitors and their toxicity...... 120 Figure 3.37. Stattic preincubation and addition timepoints...... 121 Figure 3.38. VACV inhibition in Y705F/Y705F cell line...... 122 Figure 3.39. siRNA knockdown of STAT3...... 123 Figure 4.1. VACV inhibitors identified in the Prestwick Chemical Library drug screen...... 125 Figure 4.2. Experimental workflow from hit identification to in vivo testing...... 129 Figure 6.1 Early, Cardenolides, and Spread hits titres...... 163 Figure 6.2 VACV Cardenolide-dependent inhibition in HEK cells ...... 164

Abbreviations

Cytarabine, cytosine Ethylenediaminetetraacetic AraC EDTA arabinoside Acid

ASFV African Swine Fever Virus Extracellular Enveloped EEV Virion BFA Brefeldin A EFC Entry Fusion Complex BSA Bovine Serum Albumin Enhanced Green Fluorescent EGFP Centers for Disease Control Proteins CDC and Prevention Epidermal Growth Factor EGFR Cell-associated Enveloped Receptor CEV Virion 5-(N-Ethyl-N- EIPA CHIKV Chikungunya Virus isopropyl)amiloride

(Transmission) Electron CHX Cycloheximide EM Microscopy

CMPV Camelpox Virus ER Endoplasmic Reticulum

CMV Cytomegalovirus EV Enveloped Virion

CPXV Cowpox Virus EV71 Enterovirus 71

Crypto Cryptotanshinone FA Formaldehyde

CVB3 Coxsackievirus B3 FBS Foetal Bovine Serum

DENV Dengue Virus Food and Drug FDA Administration DHFR Dihydrofolate Reductase GAG Glycosaminglycans Dulbecco's Modified Eagle DMEM Medium HBV Hepatitis B Virus

DMSO Dimethyl Sulfoxide HCMV Human Cytomegalovirus

EBOV Ebola Virus HCV Hepatitis C Virus

ECTV Ectromelia Virus

Human Immunodeficiency MuLV Murine Leukemia Virus HIV Virus Nucleocytoplasmic Large NCLDVs Hpi Hours post infection DNA Viruses

HRV Human Rhinovirus NTPs Nucleoside Triphosphates

HSV Herpes Simplex Virus PBS Phosphate-Buffered Saline

Intracellular Enveloped PCL Prestwick Chemical Library IEV Virion Pfu Plaque forming units IMV Intracellular Mature Virion RT Room Temperature IV Immature Virion Severe Acute Respiratory SARS-CoV Immature Virion with Syndrome Coronavirus IVN Nucleoid SINV Sindbis Virus KRAB-associated protein-1, KAP1 TRIM28 SNS Strategic National Stockpile

LB Lateral Bodies Signal Transducer and STAT Activator of Transcription LDH Lactate Dehydrogenase TK Thymidine Kinase Laboratory for Molecular LMCB Cell Biology TNF Tumor Necrosis Factor

The Library of VACV Vaccinia Virus LOPAC Pharmacologically Active Compounds VARV Variola Virus Mouse Embryonic MEF Venezuelan Equine Fibroblasts VEEV Encephalitis Virus Middle East Respiratory MERS-CoV Vaccinia Immune Globulin, Syndrome Coronavirus VIGIV Intravenous Molluscum Contagiosum MOCV Virus VZV Varicella Zoster Virus

MOI Multiplicity of Infection WR Western Reserve

MPXV Monkeypox Virus WT Wild Type

1 Introduction

1.1 Poxviruses

The virus family Poxviridae includes a total of 71 species in 16 genera and 2 subfamilies (ICTV Virus Taxonomy 2014; Fig. 1.1). The two subfamilies are Entomopoxviridae, infecting insects, and Chordopoxviridae, infecting vertebrates. All members of Poxviridae are large, double stranded DNA viruses, with genomes ranging in size from 130 kbp to 300 kbp, which carry their own transcription machinery (Moss 2007). The genomes of poxviruses are highly conserved, with at least 66 genes conserved in both subfamilies, and an additional 53 conserved within the Chordopoxviridae subfamily (Iyer et al. 2006).

Figure 1.1. Virus Taxonomy: 2017 Release. Poxviridae are divided into two subfamilies and 16 genera. The family includes a total of 71 viruses, as of 2018.

Source: International Committee on Taxonomy of Viruses (ICTV)

The host range of poxviruses is highly dependent on the species. Some are highly specific, like Variola virus (VARV), Camelpox virus (CMPV) or Ectromelia (mousepox virus, ECTV).

Others, like Cowpox virus (CPXV), may infect a broad range of hosts (humans, cows, cats, foxes, zoo animals) (McFadden 2005). And while there are only two human-specific poxviruses, VARV (causative agent of smallpox) and Molluscum contagiosium virus (MOCV, a generally benign skin condition potentially dangerous to the immunocompromised), at least 13 poxviruses are known to cause zoonotic infections in humans (Haller et al. 2014, Essbauer, Pfeffer, and Meyer 2010). Among the poxviruses most relevant to researchers are the abovementioned VARV and MOCV, as well as Vaccinia virus (VACV, the prototypic poxvirus, used as the smallpox vaccine), Monkeypox virus (MPXV, endemic in Africa), and CPXV (endemic in Brazil).

1.1.1 Smallpox

Evidence of smallpox has been found on mummies more than 3,000 years old, though it is certain that the disease is much older than that (Fenner et al. 1988). Originally, smallpox was called just “pox”, as the name comes from the characteristic skin lesions caused by the infection (Fig. 1.2A). However, when syphilis started spreading in Europe in the early 16th century, because the pustules it caused were larger than those of pox it was called the “great pox”, and the name “smallpox” was used to differentiate the two diseases (Farhi and Dupin 2010). It is estimated that in the 18th century smallpox killed ~400,000 people every year (Behbehani 1983). Smallpox was used as a biological weapon by the British who purposefully employed it against the French and Native Americans during the French and Indian Wars (1754–1763) (Fenn 2000).

In the 1950s, almost 150 years after Edward Jenner published his vaccine research, there were still approx. 50 million cases of smallpox every year, with a death rate of up to 20% (Fenner et al. 1988). It wasn’t until 1979 when the World Health Organization made the announcement that changed the world: smallpox, one of the worst infectious diseases in human history, had been eradicated (Fig. 1.2B). The Smallpox Eradication Program, started in 1966, involved an intensive programme of vaccinations, surveillance, and prevention on an international scale (Fenner et al. 1988). It involved ring vaccination, a process whereby after identifying an outbreak all infected individuals were isolated, and everyone living nearby was vaccinated. Apart from the fact that an effective vaccine existed, the elimination of the disease was possible in major part due to the lack of zoonotic reservoirs of VARV. The last natural infection occurred in Somalia in 1977. The last known infection ever occurred in a lab in 1978 and resulted in three people dying: the victim died of smallpox, her father of a heart attack, and the scientist responsible for the research committed suicide (Shooter 1980).

Figure 1.2. Smallpox was a major human pathogen. (A) A person suffering from smallpox, showing characteristic lesions. (B) Cover of the WHO magazine celebrating the eradication in 1980. Smallpox was eliminated from the environment thanks to a WHO-lead immunization programme.

Source: (A) CDC Public Health Library (B) WHO

While VARV has been eliminated from the natural environment, USA and Russia are still in possession of samples of the virus. This has caused controversies in the past, when both sides refused to destroy the samples in case the other would not; others have cited research needs as the reason to maintain stocks (Hammond 2007). Recently, breakthroughs in synthetic biology have created the possibility of an orthopox virus being re-created from known genomic sequences (Noyce, Lederman, and Evans 2018). Since the vaccination programme has been discontinued following the eradication, the population is now vulnerable to a potential spread of the virus. This is why smallpox is still in the Category A of the CDC Bioterrorism Agents/Diseases. Furthermore, there have been incidents where samples were lost from official storage, or found, previously lost and forgotten (CDC 2014). While the threat of smallpox being used as a bioweapon may seem unlikely, steps must be taken to prepare for any eventuality. Currently, there are three main options to control a potential smallpox outbreak: immunization, antibody administration, and antiviral drugs. These will be discussed in more detail in section 1.4.

However, the potential bioterrorist threat of smallpox is not the only reason for the search for poxvirus antivirals. As mentioned earlier, some zoonotic viruses already possess a wide host range, while other may adapt to human hosts. This means potentially non-threatening poxviruses could become a major threat to human life. Additionally, basic research on model

poxviruses can lead to better understanding of other nucleocytoplasmic large DNA viruses (NCLDVs).

1.1.2 Other poxviruses and African swine fever virus

Monkeypox

Monkeypox, despite the name, is primarily a rodent disease. However, it was first discovered in laboratory monkeys and the name remained. While monkeypox virus is a zoonotic virus usually restricted to tropical forests of West Africa, there have been cases where it has spread to other parts of the world, most notably the USA in 2003, where an outbreak in humans occurred in six states – spread by prairie dogs (Reynolds and Damon 2012). In Africa monkeypox virus infection mortality is between 1% and 10% (Hutin et al. 2001).

Cowpox

Cowpox virus is another zoonotic poxvirus that can infect humans. Cowpox is a historically significant disease, as it led to Edward Jenner creating the first smallpox vaccine. Though whether he used CPXV specifically is now contested (Esparza et al. 2017). Currently the virus can still be found in Europe, and while it is primarily a rodent virus, it can also infect other animals, including cats, which can in turn infect humans (Switaj et al. 2015).

Molluscum contagiosum

Molluscum contagiosum virus is, like VARV, a human virus with no non-human reservoirs. Unlike zoonotic poxviruses, where person to person spread is extremely rare, MOCV is spread by contact with infected skin or surfaces and clothes that have been in contact with the infected person. Prevalence among children in the UK is estimated at between 5.1% and 11.5% (Olsen et al. 2015). In a healthy individual infection will be limited, resulting in localized skin lesions that will clear on their own. However there are growing concerns that the disease may have a greater impact on quality of life than commonly assumed (Olsen et al. 2014). More importantly, immunodeficient individuals are at a greater risk due to potential complications, especially children (Braue et al. 2005).

African Swine Fever Virus

African swine fever virus (ASFV) is the solitary member of the Asfarviridae family, and a member of the NCLDV order alongside poxviruses (Dixon et al. 2013). ASFV infects domestic and wild pigs and has been identified in over 50 countries, mostly in Africa and Eastern Europe, but is also present in China (Fig. 1.3). Importantly, it has a high potential socio- economic impact due to the lack of available vaccine or treatment (Arias et al. 2018, Gallardo

Figure 1.3. ASFV global distribution (2016-2018). Source: World Organization for Animal Health (OIE) (2018) et al. 2015). Animals must be destroyed to prevent spread, and in conjunction with trade restrictions that occur, an outbreak in countries with high pork production can be devastating.

ASFV is an icosahedral enveloped virus that shares numerous similarities with poxviruses: temporal sequence of gene expression, packaging of transcription machinery, and cytoplasmic replication site formation (Netherton and Wileman 2013) (Fig. 1.4). Evidence suggests many similarities to VACV viral entry as well, including macropinocytosis, late endosome disassembly, and fusion to release the core into the cytosol (Alonso et al. 2013, Hernaez et al. 2016, Sanchez, Perez-Nunez, and Revilla 2017).

For ASFV, a vaccine would be the preferred solution to prevent viral infection in the first place; however, antivirals could be an efficacious alternative. First, they could be used as molecular tools to better understand ASFV and its replication cycle. Second, they could potentially increase survival time, allowing infected animals to generate an immune response and resistance. Third, they could be used in combination with future vaccines to improve the clinical outcomes of infection (Arias et al. 2018, Alonso et al. 2013). As ASFV is on the Specified Animal Pathogen Order Schedule 1, any research on the virus is highly restricted. As such, it is possible that preliminary screens of potential drugs could be done in a similar virus with less stringent safety requirements, i.e. VACV.

Figure 1.4. ASFV structure. (A) Electron microscope image of ASFV particle (B) ASFV model. The lipoprotein envelope surrounds the proteinaceous core containing the double-stranded DNA genome.

Source: (A) Institute for Animal Health (B) Philippe Le Mercier

1.2 Vaccinia virus

Vaccinia virus is perhaps the most interesting poxvirus. It is used as the vaccine against Variola virus, with which it shares more than 90% sequence identity (Massung et al. 1994). Historically it was assumed that the vaccine developed by Jenner came from the virus isolated from cows, hence the term vaccine and vaccination – vaccinus is Latin for “from cow”. However, a different form of vaccination existed: horsepox virus was often used for equination, (equus – horse) (Esparza et al. 2017). Presently, evidence is mounting that VACV is more closely related to the horsepox virus than the cowpox virus. These claims are supported by genomic analysis of the various poxviruses and historical vaccines (Schrick et al. 2017, Medaglia et al. 2015, Noyce, Lederman, and Evans 2018, Esparza et al. 2017). In the end, VACV is neither a cow- nor a horse-pox virus, but a distinct laboratory and vaccine species – and its true origins may never be known.

VACV infections can occasionally occur as a result of vaccination, as seen in the cases related to military personnel or laboratory staff who were vaccinated (CDC 2007, 2008, 2009, 2012, 2013). However, there is also evidence of natural reservoirs of VACV in Brazil, with rodents spreading the virus to farm animals, which in turn can infect humans (Oliveira et al. 2017). While VACV is best known for its role as the vaccine for smallpox, its use in molecular biology cannot go unmentioned. It holds a special place on the list of “first animal virus”: first seen under an electron microscope; first cultured in cells; first to be purified and characterized (Moss 2007). It also inspired research into transcription and translation after it was

discovered that VACV packages transcriptional machinery into the virion, challenging the contemporary ideas about viruses (Moss 2007). Currently, it is still used for basic molecular cell biology involving infection and immunity. It is also an important potential oncological agent (Thorne, Bartlett, and Kirn 2005, Jefferson, Cadet, and Hielscher 2015) and can be used as a protein expression system (Bleckwenn, Bentley, and Shiloach 2003, Hebben et al. 2007). Finally, it still serves as the smallpox vaccine, while also being used as a backbone in vaccines against other viruses, e.g. HIV (Jacobs et al. 2009, Walsh and Dolin 2011, Verardi, Titong, and Hagen 2012).

In the Mercer lab the Western Reserve (WR) strain is used most commonly, while other strains used in research include Modified Virus Ankara (MVA), International Health Department-J (IHD-J), Copenhagen, and Lister.

1.2.1 Modulation of the immune response

Like all viruses, VACV encounters and needs to overcome the host immune response during infection. The host innate immune system, which is the first line of defence, depends on sensing of VACV via pattern-recognition receptors (PRR) and subsequent cytoplasmic sensing of viral DNA and RNA once viral replication is initiated. Within an organism, this triggers a systemic response, involving interferons (IFNs) and other cytokines, the complement system, as well as natural killer cells, macrophages, neutrophils, and dendritic cells. Activation of the innate immune response shapes and is followed by the adaptive immune response which relies largely on antibody and cell-mediated response. This of course is the basis of vaccine- based immunity. Poxviruses devote between 30 to 50% of their genome to proteins that modulate the immune response. Not surprisingly, given this large repertoire, VACV has evolved mechanisms at multiple stages of infection to suppress the host immune system. Given its relevance to the hits discovered in the LOPAC screen, described in 1.5.2, here I will describe the interferon signalling and how VACV interferes with it.

Nucleic acid sensors

VACV double stranded RNA (dsRNA) and double stranded DNA (dsDNA) can be detected by the cell as pathogen associated molecular patterns (PAMPs). Retinoic acid inducible gene I (RIG-1) and melanoma differentiation-associated protein 5 (MDA5) are two RIG-I-like receptor dsRNA helicase enzymes that have been shown to detect viral RNA, and both trigger anti-viral response by activating mitochondrial antiviral-signalling protein (MAVS). This, in turn, results in activation of the IRF3 and NF-κB pathways (Yoneyama et al. 2004, Andrejeva et al. 2004). To prevent detection, VACV E3 protein sequesters dsRNA (Chang, Watson, and

Jacobs 1992) and reduces dsRNA formation by avoiding transcription of overlapping sequences (Smith et al. 1998).

There are at more than 10 proposed dsDNA sensors (Paludan and Bowie 2013), with DNA protein kinase (DNA-PK) (Ferguson et al. 2012, Scutts et al. 2018), absent in melanoma 2 (AIM2) (Rathinam et al. 2010) and cyclic GMP-AMP synthase (cGAS) (Sun et al. 2013, Georgana et al. 2018) identified as VACV PRRs. DNA-PK and cGAS pathways induce IFN expression via IRF3, while AIM2 works by stimulating inflammasome activation. TLR9 is so far the only described endosomal-based DNA sensor and is a strong inducer of IFN-α via MyD88 adaptor (Samuelsson et al. 2008). As an evasion strategy, VACV prevents dsDNA detection by initially shielding the genome within the core and delaying DNA replication until many of the early genes encoding immune-modulatory proteins have been expressed.

Toll-like receptors

Toll-like receptors (TLRs) can recognize a variety of virus-derived ligands, both inside the cell and on the cell surface. These, in turn, via signalling cascades involving IRF3, NF-κB, and AP- 1 (Wathelet et al. 1998), regulate transcription of type I interferons (e.g. interferon β - IFNβ) as well as other cytokines and chemokines. INFβ is secreted from the cell and via autocrine or paracrine signalling initiates the Janus kinase / signal transducer and activator of transcription (JAK/STAT) pathway. As a result, an anti-viral response is mounted in the already infected cell, as well as in the surrounding uninfected cells (Fig. 1.5) There are multiple viral proteins that can inhibit the signalling cascades described above. However, STAT3 (section 1.5.2) is part of the JAK1/STAT signalling pathway, and while inhibiting IFN at any earlier stage would affect it, STAT proteins only play a role after the pathway has been activated, therefore viral proteins blocking IFN response will not be further discussed.

JAK/STAT

The JAK1/STAT signalling pathway is activated by IFNs type I, II, and III binding to their receptors. STAT1/2 heterodimers are formed when JAK1/ tyrosine kinase 2 (TYK2) are activated by IFN type I or III, while formation of STAT1 homodimers is controlled by IFN type II. Phosphorylation of the heterodimers leads to their association with interferon regulatory factor 9 (IRF9) and formation of IFN-stimulated gene factor 3 (ISGF3) complex. Phosphorylation of STAT1 homodimers leads to direct translocation into the nucleus and binding to γ-activated sequences (GAS). The ISGF3 complex is also translocated to the nucleus, where it binds to the IFN-stimulated response elements (ISREs). Both result in production of hundreds of interferon stimulated genes (Stark and Darnell 2012). VACV

Figure 1.5 Viral detection and IFN activation and JAK1/STAT pathway. Pathogen associated molecular patterns are detected by pattern-recognition receptors (PRRs) or Toll-like receptors (TLRs). The NF-κB, IRF3, and AP1 pathways thus activated all lead to production of interferons (IFN). Via autocrine and/or paracrine signalling the IFN is bound by the interferon-α/β receptor (IFNAR), activating JAK-STAT signalling pathways. STAT dimers translocate to the nucleus and induce interferon-stimulated gene (ISG) expression. This results in an anti-viral response.

ISGF3 – IFN-stimulated gene factor 3. ISRE – IFN-stimulated response elements. VACV interference with signalling indicated by pink blobs.

Adapted from Smith et al. 2013. proteins B8 and B18 have been identified to bind to IFN receptors and neutralize them (Symons, Alcami, and Smith 1995, Alcami and Smith 1995), thereby blocking the antiviral response. VH1 works downstream of the pathway, where it dephosphorylates STAT1 and STAT2 (Najarro, Traktman, and Lewis 2001). STAT3, a relative of STAT1 which it can dimerise with, is further discussed in section 1.5.2.

NK Cells

Natural killer cells (NK) are large granular leukocytes that are critical to the innate immune system. The cell surface of NK cells displays receptors that can activate or inhibit their function, which recognize different molecules on the surfaces of infected cells. They preferentially kill cells with low levels of major histocompatibility complex (MHC), the so

called “altered self” phenotype. The NKG2A receptor binds MHC, preventing NK cells from activating. NKG2D and natural cytotoxicity receptors (NCRs), on the other hand, stimulate NK mediated cytolysis (Vivier and Ugolini 2011). During VACV infection, despite MHC downregulation (Kirwan et al. 2006) and enhanced recruitment of NK cells at the site of infection (Jacobs et al. 2006), it appears that the recognition of infected cells is dependent on changes in the NCRs (Chisholm and Reyburn 2006). VACV is able to somewhat modulate this response via protein A56, present on infected cell surface and EEVs, which binds NCRs and lowers the susceptibility of infected cells to recognition by NK cells (Jarahian et al. 2011).

Compliment system

Complement system triggers inflammation, phagocytosis, and membrane attack of infected cells. The classical component pathway is used to complement the adaptive immune system, while the alternative pathway is part of the innate immune system and is activated by a complex cascade starting with cleavage of component C3. VACV VCP protein, which is secreted from infected cells, can block the activation of the complement by binding to proteins C3b and C4b, causing their cleavage, disrupting the classical and alternative pathways (Kotwal and Moss 1988)(McKenzie et al. 1992). VCP can also bind to A56, which places it at the correct location (cell surface) to act against complement (Dehaven, Gupta, and Isaacs 2011). Additionally, VACV EEVs acquire a set of hosts complement control proteins (CD46, CD55 and CD59) in their envelope, hiding the virus from the complement (Vanderplasschen et al. 1998).

Adaptive immunity

Adaptive immune system has a delayed onset and is the basis of vaccination. The immunological memory acquired after an initial exposure to a pathogen in the form of antibodies, CD4+ and CD8+ T-lymphocytes and B-memory cells protects the host from reinfection (Crotty et al. 2003). Neutralizing antibodies generated against MV proteins are in the majority (A27, H3, D8, A28, A13, A17) (Davies et al. 2005), with only a few antibodies against EEV proteins (A33 and B5) known. And, as we know, immunization with VACV confers protection from VARV.

1.2.2 VACV structure(s)

VACV is a large, enveloped virus that produces two forms of infectious particles: Intracellular Mature Virions (IMVs; also known as Mature Virions, MVs) and Extracellular Enveloped Virions (EEVs; also known as Enveloped Virions, EVs) (Fig. 1.6A). The IMV and EEV forms of VACV have no common proteins on their surfaces, and their membranes have different cellular origins (Moss 2007). The IMVs have a single lipid bilayer surrounding the core and lateral bodies, while EEVs have an additional outer membrane (Moss 2007). EEV particles differ from IMVs in a few key features. The EEV membrane contains a number of unique proteins: A33, A34, A36, A56, B5, and F13 (Payne 1978). Additionally, they lack A25 and A26 proteins normally found in the IMV membrane (Ulaeto, Grosenbach, and Hruby 1996). IMVs and EEVs have been shown to bind to different cellular receptors (Vanderplasschen and Smith 1997), however the specifics of EEV binding are as of yet unclear.

Figure 1.6. VACV structure(s) (A) Model of VACV structure and structures (B) Imaged with Electron Microscopy: Lateral bodies, Core, and Membrane. (C) Atomic Force Microscopy, with arrows pointing out LBs, and (D) Super-resolution microscopy, where a LB protein F17 is tagged with EGFP and a core protein A5 is tagged with mCherry.

Sources: (B) Mercer (unpublished) (C) (Kuznetsov, Gershon, and McPherson 2008) (D) (Gray et al. 2016)

Another structure unique to poxviruses are the lateral bodies (LBs): proteinaceous structures located bilaterally to the concaved core (Cyrklaff et al. 2005, Cyrklaff et al. 2007, Ichihashi, Oie, and Tsuruhara 1984). Their existence has been verified by electron microscopy (EM) (Fig. 1.6B) (Condit, Moussatche, and Traktman 2006), and atomic force microscopy (AFM) (Fig. 1.6C) (Kuznetsov, Gershon, and McPherson 2008) as well as super resolution microscopy (SR) (Fig. 1.6 D) (Gray et al. 2016). As the core is deposited in the host cytoplasm, the lateral bodies detach from the core and undergo degradation in the cytoplasm in a proteasome-dependent manner (Schmidt et al. 2013). Very little is known about the composition and function of LBs, though it is likely that the proteins located in them may act as immunomodulators (Bidgood and Mercer 2015). The phosphoprotein F17, the oxidoreductase G4, and dual-specificity phosphatase VH1, have been identified as LB proteins, with the latter dephosphorylating STAT1 to counteract antiviral responses (Schmidt et al. 2013). F17 is also the most abundant protein in the LB, and third most abundant in the entire virus particle (Chung et al. 2006). As its degradation was necessary for the activity of VH1, it is likely that F17 is a LB structural protein, with as of now hypothetical immunomodulatory function (Schmidt et al. 2013).

1.2.3 VACV replication cycle

The VACV replication cycle is a complex process with multiple stages, depicted in Fig. 1.8. Briefly, infection begins with the viral particle attaching to the cell and its internalization by macropinocytosis or direct fusion, followed by core activation and early gene expression. Only then the genome is uncoated, and replication can begin. Next, intermediate and late genes are expressed, and structural proteins are translated. The process of virus morphogenesis results in the formation of Intracellular Mature Virions (IMVs). A small number of IMVs is transported to the wrapping site where they are enveloped in two additional membranes, resulting in formation of a triple membrane particle, the Intracellular Enveloped Virions (IEVs). These are in turn transported to the cell membrane on microtubules where they exit the cell by fusing their outermost membrane. These particles can either remain Cell-associated (CEVs) or they can separate from the cell (Extracellular Enveloped Virions - EEVs). The remaining IMVs are released when the cell lyses approx. three days post infection.

Figure 1.7. VACV replication cycle. VACV infection begins with the macropinocytic uptake of the viral particle, followed by core deposition in host cytoplasm. Early gene expression occurs while the genome is still within the core and is required for DNA uncoating. DNA replication and intermediate and late gene expression are followed by morphogenesis, and the resultant mature particle may become wrapped and exit the cell as early as 8h post infection.

IV – immature virion; IVN – immature virion with nucleoid; IMV – intracellular mature virion; IEV – intracellular enveloped virion; CEV – cell-associated extracellular virion; EEV – extracellular enveloped virion.

Mature Virion entry

Since viruses need a host to reproduce, they evolved mechanisms to attach to and enter cells. Following the binding to cellular receptors of their intended host, there are various paths that a virus can take. One is very direct, via fusion of the viral membrane with the plasma membrane, or some other mechanism (membrane lysis, genome injection) by which the DNA is moved through the membrane into the cytoplasm (Marsh and Helenius 2006). Alternatively, the virus particle can take advantage of cellular endocytosis, which in the case of VACV is induced by the particle (Mercer and Helenius 2008) (Fig. 1.8).

Figure 1.8. VACV entry. VACV binds to the cell membrane, and then is internalized via macropinocytosis. Fusion with the membrane releases the core into the cytoplasm. Early gene expression is required for genome uncoating.

Binding

VACV uses multiple attachment strategies, as demonstrated by its ability to bind to a wide range of hosts and cell types (Oliveira et al. 2017). The IMV-associated proteins involved in attachment include D8, A27, H3, and A26. Each target glycosaminoglycans (GAGs) – linear sulfated polysaccharides containing repeating disaccharide units located at the cell surface (Matho et al. 2018). D8 binds chondroitin sulfate (Hsiao, Chung, and Chang 1999), while A27 and H3 bind heparan sulfate (Chung et al. 1998, Lin et al. 2000, Hsiao, Chung, and Chang 1998). However, VACV GAG reliance is cell line dependant (Bengali, Townsley, and Moss 2009). Another attachment mechanism depends on filopodia, suggesting indirect interaction with the actin cytoskeleton via an unknown receptor (Huang et al. 2008, Mercer and Helenius 2008).

Internalization

As previously mentioned, enveloped viruses can either fuse with the plasma membrane directly or from endosome after virus particles are internalized via endocytosis. On one hand, direct fusion is simpler: a merging of membranes, and release of the viral core directly into the cytoplasm. This, however, may have some disadvantages. Firstly, the newly deposited viral core still needs to be transported to the site of transcription and replication. Secondly, the viral envelope proteins left behind may be detected by the host immune system. These issues are circumvented by the endocytic entry route.

Studies of VACV internalization by electron microscopy were inconclusive, as examples for both possibilities were documented (Chang and Metz 1976, Dales 1963, Townsley et al. 2006). Direct fusion at the plasma membrane observed by EM might be over-represented due to the very high multiplicity of infection required for the experiments, as some more recent work has demonstrated strong evidence for the endocytic pathway (Mercer and

Helenius 2008, Huang et al. 2008, Sandgren et al. 2010, Townsley et al. 2006, Mercer et al. 2010a). These studies used biochemical, fluorescence microscopy-based internalization and gene expression assays where specific inhibitors of endosomal process have resulted in the direct inhibition of virus entry and infection. Importantly, this inhibition could be overcome by brief acidification, which forces fusion at the plasma membrane, bypassing endocytosis and thereby demonstrating that endocytic uptake was a step required for successful infection (Mercer and Helenius 2008). Additionally, when endocytic factors required for endocytic trafficking and maturation were blocked, viral early gene expression was inhibited (Earp et al. 2005, Marsh and Helenius 2006, Rizopoulos et al. 2015).

It was determined that macropinocytosis is the specific endocytic pathway triggered by VACV (Mercer and Helenius 2008). Macropinocytosis is signal-induced and can be triggered by many different receptors. It requires significant rearrangement of the actin cytoskeleton, resulting in formation of large blebs, leading to internalization of the viral particles. VACV is taking advantage of phosphatidylserine (PS) present in the MV membrane, and it is proposed that this mechanism is a form of apoptotic mimicry (Mercer and Helenius 2010, Hoffmann et al. 2001, Amara and Mercer 2015), as PS is usually exposed in apoptotic bodies (van Meer, Voelker, and Feigenson 2008). The process also requires epidermal growth factor receptor (EGFR) signalling, and a whole set of cellular factors, including GTPase Rac1, its effector kinase p21-activated kinase 1 (Pak1), cholesterol, Na+/H+ exchangers, tyrosine kinases, protein kinase C (PKC), and myosins (Huang et al. 2008, Locker et al. 2000, Mercer et al. 2010b, Mercer, Schelhaas, and Helenius 2010).

Fusion

After the virus particle has been successfully internalised, it needs to exit the endosome and deposit its core in the cytoplasm. This step involves endosome maturation and low pH- dependent endosomal fusion (Townsley et al. 2006, Laliberte, Weisberg, and Moss 2011, Mercer and Helenius 2008). This explains why brief acidification can allow the virus to bypass the endosomal pathway. The Entry Fusion Complex (EFC) plays a central part in the fusion process. The EFC is a complex of 9 transmembrane proteins, which are highly conserved in all poxviruses (Iyer et al. 2006). It is the largest and most complex fusion machinery ever described in a virus. All 9 proteins are essential for viral fusion (Senkevich et al. 2005, Moss 2012). The EFC contains stoichiometric amounts of the proteins A16, A21, A28, G3, G9, H2, J5, L5, and O3 (Izmailyan et al. 2006, Ojeda, Domi, and Moss 2006, Ojeda, Senkevich, and Moss 2006, Senkevich and Moss 2005, Senkevich, Ward, and Moss 2004a, b, Townsley, Senkevich, and Moss 2005a, b, Turner et al. 2007, Nelson, Wagenaar, and Moss 2008, Wolfe, Ojeda, and Moss 2012, Satheshkumar and Moss 2012, 2009). Proteins F9 and L1, the so-

called EFC-associated proteins, interact with the complex and are required for entry but are not required for its assembly or stability (Brown, Senkevich, and Moss 2006, Bisht, Weisberg, and Moss 2008). L1 has been shown to not require GAGs for binding (Foo et al. 2009), and L1 antibodies have been shown to have a potent neutralizing effect on virus entry of IMVs, demonstrating the importance of this protein (Foo et al. 2012, Kaever et al. 2014, Walper et al. 2014, Vanderplasschen and Smith 1997). Another two proteins, A25 and A26, prevent the EFC from activating before the pH drop (Chang et al. 2012, Chang et al. 2010). The exact structure of the complex has not yet been described. It is not known how many copies of the complex are required for fusion. The number of copies in a single virion is also unknown. The EFC proteins have no known homologues outside of the Poxviridae. So far, EFC proteins have only been shown to interact which each other. H2 interacts with A28 (Nelson, Wagenaar, and Moss 2008); A56/K2 interacts with A16 and G9 (Wagenaar, Ojeda, and Moss 2008); and G3 interacts with L5 (Wolfe and Moss 2011).

Extracellular Enveloped Virion entry

Studying EEVs is more complex than studying IMVs. First, the outer membrane is unstable and easily disturbed by traditional extraction and purification methods (Schmidt 2011). Likewise not all released EEVs have an unbroken outer membrane (Ichihashi and Oie 1983), with only 10-40% of infectivity being insensitive to antibody neutralization (Schmidt et al. 2011, Bisht, Weisberg, and Moss 2008, Wolffe, Vijaya, and Moss 1995).

Despite these differences, EEVs are also internalized via the macropinocytotic pathway after inducing blebbing, however the mechanism has not yet been fully elucidated. Furthermore, the disruption of the outer membrane occurs after internalization by endocytosis – the A34 protein has been identified as a pH sensitive “switch” that serves to disrupt the outer membrane (Husain, Weisberg, and Moss 2007, Schmidt et al. 2011, McIntosh and Smith 1996). After the outer membrane has been disrupted fusion can occur the same as it does for an IMV, and the core is released into the cytoplasm.

Genome release, viral transcription and translation, and DNA replication

Upon core deposition the LBs dissociate, and the core expands in volume (Schmidt et al. 2013). This expansion of the core coincides with early gene transcription which takes place within the viral core, with viral mRNA being transported out into the cytoplasm and further trafficked on the microtubule system (Mallardo, Schleich, and Locker 2001). VACV contains all the transcription machinery required for early gene expression within the core, as evidenced by the demonstration that forcing core expansion in vitro in the presence of NTPs allows for early gene expression (Shuman and Moss 1989). Although poorly understood,

Figure 1.9. Genome replication. Following genome uncoating, viral DNA is replicated in distinct replication sites, where the intermediate and late proteins are also synthesised. early mRNAs have been proposed to exit from the core via pores located in its surface (Kates and McAuslan 1967).

All VACV genes: early, intermediate, and late are transcribed by the virus encoded RNA- polymerase, which is made up of nine subunits: J6, A24, H4, A29, E4, J4, A5, D7, G5 (Moss 1994). Analysis of multiple early promoters has identified shared sequence that is variable but highly A–T rich, with a conserved G residue (Davison and Moss 1989). The early transcription factor (ETF), a heterodimer of viral proteins A7 and D6 (Broyles and Fesler 1990), has been identified to bind to all early promoters (Broyles et al. 1988) and the complex then recruits the RNA polymerase (Li and Broyles 1993). ATP is required as an energy source to dissociate ETF from the DNA, to make room for the transcription to proceed (Broyles 1991). As mentioned, the remaining components of the transcription machinery: capping enzyme, poly(A) polymerase, and the transcription termination factor, all also present in the core, allowing for full transcription (Shuman and Moss 1989).

Following early gene expression, the AAA+ ATPase/helicase D5 was identified as the viral genome uncoating factor required for core breakdown whereas the host ubiquitin proteasome system was found to be required on the cellular side (Kilcher et al. 2014, Joklik 1964). While the exact mechanism of uncoating remains undefined, EM images of genome uncoating events suggest that a portion of the core wall is degraded, presumably by the proteasome which would then allow the helicase activity of D5 to facilitate genome unpacking. As the genome is uncoated, so called pre-replication sites are formed (Domi and Beaud 2000). These pre-replication sites can be identified by labelling for I3, a viral single strand DNA binding protein that appears to be important for accumulation of viral genomes (Welsch et al. 2003, Greseth et al. 2012). Once the genome has been released, infection proceeds to DNA replication.

Viral DNA replication takes place in replication sites, or viral factories. These are endoplasmic reticulum compartments within the cytoplasm (Tolonen et al. 2001). By inducing cellular contractility, VACV gathers smaller replication sites near the nucleus (Schramm et al. 2006). The replication of the DNA and transcription and translation of intermediate and late protein takes place in the replication sites, in a sequential manner (Katsafanas and Moss 2007). The replicated DNA is used as template for intermediate and late gene expression, encoding proteins for DNA organization and packaging, as well as core structural proteins, and late transcription factors (Yang et al. 2011). Late proteins include all structural proteins, as well as the early transcription system RNA polymerase complex that will be packaged into new virions (Moss 2007).

Intermediate promoters have an initiator element at the transcriptional start site and an A– T-rich upstream element, containing a TAAAT/A or /GG sequence (Baldick, Keck, and Moss 1992). It is likely that intermediate transcription, unlike early, requires RNA polymerase that has been expressed after viral DNA replication (Hooda-Dhingra, Thompson, and Condit 1989). It is also likely that it lacks the H4 subunit, which has been associated with early transcription (Wright and Coroneos 1995). Viral intermediate transcription factors (VITF) required include: E4 (VITF-1), A8 and A23 (VITF-3) (Rosales et al. 1994, Sanz and Moss 1999). Two host factors also appear to be required, VITF-2 (Rosales, Sutter, and Moss 1994) and the nuclear transcription factor YinYang1 (YY1) (Broyles et al. 1999). Like intermediate transcription, late transcription requires RNA polymerase that has been synthesised after viral DNA replication. The promoters have an initiator-like element (TAAAT) at the start site for transcription and an A–T-rich upstream element (Davison and Moss 1989), they are also targeted by TATA-binding protein (TBP) (Knutson et al. 2006) The transcription factors identified are G8, A1, A2, A2/B1, RBM3 (Keck, Baldick, and Moss 1990, Wright, Oswald, and Dellis 2001), with possible host factor (VLTF-X) involved (Wright, Oswald, and Dellis 2001). Termination of transcription for intermediate and late genes appear to be dependent on viral proteins A18, G2, and J3 (Xiang et al. 1998, Black and Condit 1996, Latner et al. 2000, Xiang et al. 2000). These temporal characteristics of VACV gene expression, combined with the wealth of knowledge on the promoter sequence allow for construction of recombinant viruses expressing non-native proteins at specified stages points during infection. This is discussed in section 1.3.1.

Mature Virion formation

The formation of IMVs is a highly coordinated complex process (Fig. 1.10) that proceeds through four morphologically distinct intermediates which can be distinguished by EM (Fig. 1.11). It begins with the formation of viral crescents. Crescents are an open membrane

Figure 1.10. IMV formation. Viral proteins manipulate cellular membranes to produce new Intracellular Mature Virions. Three preceding distinct stages can be identified: crescents, immature virions, and immature virions with nucleotide. structure whose origin and nature has long been debated, with suggestions ranging from the membranes being formed de novo to them being sourced from multiple different cellular compartments (Dales and Mosbach 1968, Hollinshead et al. 1999, Sodeik and Krijnse-Locker 2002). Recent evidence supports the idea that the membranes are ER-derived, with a host of viral proteins taking part in the process: three structural components A14, A17, and D13 and additional six regulatory proteins, A6, A11, A30.5, F10, H7, and L2 (Chlanda et al. 2009, Husain, Weisberg, and Moss 2006, Maruri-Avidal, Weisberg, and Moss 2011, Maruri-Avidal et al. 2013, Suarez et al. 2013).

The growth of crescents continues until they form a closed structure – the Immature Virion (IV) (Condit, Moussatche, and Traktman 2006). During their formation IVs enclose a portion of viroplasm, that contains core proteins (Szajner et al. 2001, Szajner et al. 2003, Morgan 1976, Cepeda and Esteban 2014). The specific mechanism remains to be elucidated, however, several required proteins have been identified: A15, A30, G7, D2, D3, J1 (Szajner et al. 2004). The protein I6 has been found to bind to viral telomeres and to be necessary for DNA encapsidation - I6 temperature sensitive mutants form abnormal IVs containing the correct protein composition, but no DNA (Grubisha and Traktman 2003). IVs containing DNA are called IVNs (IV with Nucleoid). The viral redox proteins E10, A2.5 and G4 (Locker and Griffiths 1999, Senkevich, White, Koonin, et al. 2002, Senkevich, Weisberg, and Moss 2000, Senkevich, White, Weisberg, et al. 2002, White, Senkevich, and Moss 2002) play a part in the IV – IVN transition at the stage of the formation of disulphide bonds in the reducing cytosol (Hakim and Fass 2010).

The transition of IVNs to Intracellular Mature Virions (IMVs) requires the disassembly of the D13 lattice; reorganization of the proteins within to form the core and the LBs and change of shape from spherical to “brick-like”. The shape change is largely driven by I7 proteolytic

Figure 1.11. EM micrograph of VACV particle formation. Red – crescents. Blue – Immature Virions. Yellow – Immature Virions with Nucleoid. Green – Intracellular Mature Virions. cleavage, and is a major hallmark of the IVN to IMV transition (Byrd, Bolken, and Hruby 2002, Kane and Shuman 1993, Ansarah-Sobrinho and Moss 2004, Hedengren-Olcott et al. 2004, Honeychurch, Byrd, and Hruby 2006). It has also been discovered that I7 phosphorylation, required for its proteolytic activity, is controlled by F10 and H1 (Novy et al. 2018).

The correct packaging of the transcription machinery is important for core activity during infection (Kato, Condit, and Moussatche 2007). Once formed IMVs exit the replication sites and accumulate in the cell cytoplasm awaiting one of two possible fates. They can be released as mature virions when the cell lyses, or they can undergo wrapping and become Intracellular Enveloped Virions (IEVs).

Virus wrapping and egress

A fraction of IMVs is transported on microtubules to the so-called wrapping sites. This is facilitated by viral protein A27, which is required for microtubule dependent transport (Sanderson, Hollinshead, and Smith 2000, Hollinshead et al. 2001). Use of nocodazole, a drug that interferes with polymerization of microtubules, disrupts the transport of IMVs to wrapping sites (Ward 2005). In the wrapping sites IMVs gain two additional membranes derived from the trans-Golgi network (Schmelz et al. 1994) or endosomes (Tooze et al. 1993); the resulting IEVs therefore have three membranes (Roberts and Smith 2008). These additional membranes have been modified by and with the viral proteins A33, A34, A56, B5, F13, A36, F12 (Smith, Vanderplasschen, and Law 2002). The mechanism of wrapping is not

Figure 1.12. Wrapping and egress. IMVs are transported to the wrapping sites via microtubules, where they gain two extra membranes. The IEVs are transported to the cell membrane, where they lose their outermost membrane during exocytosis. Infection initially spreads by CEVs and EEVs. yet fully understood, however certain proteins of importance have been identified. F13 and B5 are essential for wrapping to take place (Blasco and Moss 1991, Wolffe, Isaacs, and Moss 1993) and both proteins localise to the TGN (Schmelz et al. 1994, Lorenzo et al. 2000). The wrapping process is also dependent on E2-F12 protein complex, as their loss results in a wrapping defect (Dodding et al. 2009).

Once wrapped IEVs are transported to the cell surface on microtubules. GFP fused to F13 or B5 has been used to visualise the transport of wrapped particles to cell periphery, and the process, similarly to the IMVs transport, has also been disrupted by nocodazole (Geada et al. 2001, Hollinshead et al. 2001, Ward and Moss 2001a). The IEV specific proteins E2 and F12 have been shown to interact with kinesin light chains and were essential for the movement of virions to the cell membrane (van Eijl et al. 2002, Carpentier et al. 2015, Gao et al. 2017). Deletion of A36 reduced or blocked movement of IEVs (Ward and Moss 2001b, Rietdorf et al. 2001), and interaction between A36 and kinesin-1 has also been observed (Ward and Moss 2004). Additionally, F11 has been shown to play a role in the final step of egress. By inhibiting RhoA signalling F11 facilitates IEV passage through the cortical actin cytoskeleton underlying the plasma membrane, by (Arakawa, Cordeiro, and Way 2007, Irwin and Evans 2012).

IEVs that reach the cell periphery and plasma membrane and fuse while maintain their cellular association, are called Cell-associated Enveloped Virions (CEVs). Those that are released are termed Extracellular Enveloped Virions (EEVs) (Zhang, Wilcock, and Smith 2000).

During exocytosis from the cell the outermost IEV membrane is lost (Smith and Law 2004, Smith, Vanderplasschen, and Law 2002). Actin tail formation under CEVs seems to be particularly important for the spread of infection from cell to cell, as shown by A36 deletion mutants which produce much smaller plaques (Sanderson et al. 1998, van Eijl, Hollinshead,

and Smith 2000, Hollinshead et al. 2001). It has been suggested that A33 is required for A36 localising to the IEV during wrapping (Wolffe, Weisberg, and Moss 2001). During the fusion A36 is translocated underneath the virion, followed by phosphorylation by Src and Abl kinases, which causes the virion to dissociate from kinesin and induces actin polymerization (Newsome, Scaplehorn, and Way 2004, Newsome et al. 2006, Frischknecht et al. 1999). This leads to acting tail formation. As mentioned, the deletion of A36 does not prevent CEV and EEV formation, but greatly reduces plaque size, indicating a spread block consistent with actin tail formation inhibition, hence EEVs appear to be more important in spread from tissue to tissue within the host than spread between hosts (Smith, Murphy, and Law 2003).

EEVs which have been released from the cell surface contain: A33, A34, A36, A56, B5, and F13 (Roper, Payne, and Moss 199, (Duncan and Smith 1992, Parkinson and Smith 1994, Shida 1986, Engelstad and Smith 1993).

To summarize, the replication cycle of VACV is complex, and takes place exclusively in the cytoplasm. Following entry, genes are expressed in a temporal manner. Formation of viral particles requires the breakdown of cellular membranes and their modification by viral proteins. A multi-step process with morphologically distinct phases results in the formation of IMVs. A fraction of this form of infectious particles is then wrapped in additional membranes and exits the cell by exocytosis to form the second infectious form, the EEV, while IMVs are released during cell lysis. Once the viral progeny is released, they may bind to uninfected cells and repeat the entire process of viral replication.

1.3 Molecular tools

As a model orthopoxvirus, VACV has been studied extensively. Consequently, many techniques and tools have been developed to facilitate VACV research. Here I will briefly focus on two: recombinant viruses, and small compound inhibitors.

1.3.1 Recombinant viruses

From very early on it was known that VACV was capable of homologous recombination. (Fenner and Comben 1958, Fenner 1959, Woodroofe and Fenner 1960, Gemmell and Fenner 1960). Researchers eventually discovered they can utilize this mechanism to insert up to 25 kbp fragments within the thymidine kinase (TK) locus, which is non-essential in cell culture (Weir, Bajszar, and Moss 1982, Smith and Moss 1983). To do so, plasmids containing a target protein sequence flanked by sequences homologous to the desired genomic insertion site are transfected into infected cells, allowing for the inherent homologous recombination to

Figure 1.13. Primer designed to insert E-EGFP and L-mCherry into VACV TK locus. An EGFP gene under an early VACV promoter was inserted back-to-back with a mCherry gene under a late VACV promoter into the TK locus. take place. This is used to generate VACV mutants with viral proteins tagged with fluorescent proteins, allowing for visualization of virus particle during various stages of infection (Lorenzo and Blasco 1998). For example, a core protein can be tagged to visualize cell entry; or an EEV protein, to study exit from the cell. Multiple proteins can be tagged at the same time, for more complex analysis.

Another problem to solve was how to control the expression of inserted genes. This was eventually achieved using native VACV promoters, which control the sequential expression of early, intermediate, and late genes (Mackett, Smith, and Moss 1982, 1984). The desired promoter sequence is inserted into the plasmid upstream of the target gene. Using this method, one can generate a VACV that expresses a fluorescent protein with the same temporal expression as a native early viral gene product. As with the tagged proteins, it is possible to insert two fluorescent proteins, for example EGFP and mCherry, into the same recombinant virus, under different promoters (Fig. 1.13). This dual colour expression system allows for visualization of the infection progression by fluorescence microscopy. A combination of tagged and promoter-controlled proteins is also possible, resulting in a virus that, for example, has a mCherry core and makes the cell express EGFP at the same time as VACV late genes (Stiefel et al. 2012).

1.3.2 VACV inhibitors

As the understanding of the VACV replication cycle grows, more compounds that can inhibit the progress of infection are identified. These compounds, while not applicable as anti- poxvirals can nevertheless be extremely useful for studying the basic biology of virus-cell interactions. The most commonly used compounds relevant to this thesis are described below.

Cycloheximide (CHX) is a glutarimide antibiotic, blocking the translocation step in translation elongation, thus blocking protein synthesis (Obrig et al. 1971, Schneider-Poetsch et al. 2010). CHX was shown to block VACV infection during the early stages as early as the 70’s (Moss and Filler 1970). Since CHX blocks protein synthesis, it prevents VACV early genes from being expressed and blocks viral genome uncoating. CHX is easy to use, and extremely stable (private observation). Having been used in VACV research for over 40 years, this is one of the most commonly used inhibitors with well-established characteristics.

Cytarabine (cytosine arabinoside, AraC) is a nucleotide analogue mainly used for treatment of acute myeloid leukemia (Briot et al. 2018). It was shown to inhibit VACV plaque formation as early as the 60’s (Renis and Johnson 1962), and, due to high toxicity at effective doses, was unsuccessfully tested as a potential treatment for smallpox and herpes in the 70’s (Lauter, Bailey, and Lerner 1974, Dennis et al. 1974). AraC inhibits VACV DNA synthesis when it is incorporated into replicated genomes instead of cytosine, stopping infection at the pre- replication stage and as such is an intermediate and late inhibitor.

Brefeldin A (BFA) is a fungal metabolite and has been shown to affect the cell vacuolar system, being particularly effective at disrupting the Golgi (Lippincott-Schwartz et al. 1991). Since VACV uses the trans-Golgi membrane for IMV wrapping, the production of IMVs is not affected; only IEVs, CEVs, and EEVs production is affected (Ulaeto, Grosenbach, and Hruby 1995, Sivan et al. 2016). As such, BFA a good control that allows for early and late gene expression but blocks non-lytic spread of the virus.

1.4 Poxvirus prevention and treatment

In the case of smallpox or any other poxvirus outbreak, there are three possible countermeasures: vaccination, passive antibody administration, and antiviral drugs (CDC 2018).

Vaccination is currently unavailable to the public, as only certain military and health personnel are given the smallpox vaccine. As the vaccine is a live vaccinia virus, a VACV systemic infection is a potential severe complication of vaccination. In such cases another treatment option is needed. Use of Vaccinia Immune Globulin Intravenous (Human) (VIGIV) is the first, FDA approved, option. However, it’s only approved as a treatment for vaccination complication, and not actual smallpox. VIGIV is a gamma globulin (IgG) purified from healthy vaccinated individuals. VIGIV has been used since the 1950’s, when it was first used by the US Army (Wittek 2006, Kempe, Berge, and England 1956, Feery 1976). If VIGIV is unavailable, or treatment with it alone is insufficient, antivirals are the last remaining option.

1.4.1 Existing anti-poxvirals

In cases of a VACV infection or a smallpox outbreak, the FDA allows the use of certain drugs under the Animal Rule. The rule is in place because testing of the drugs in humans would be unethical. Therefore, they have been tested on animal models and there are sufficient indications of their efficacy in humans to advise their use. Currently there are three such drugs: cidofovir, brincidofovir, and TPOXX®.

Cidofovir is a cytidine nucleotide analogue and was approved for treatment of cytomegalovirus (CMV) retinitis in AIDS patients in 1996 (Fig. 1.14 A) (Safrin, Cherrington, and Jaffe 1999). It inhibits the viral polymerase upon incorporation into viral DNA, inhibiting further replication, and as such has been found effective against multiple poxviruses (Safrin, Cherrington, and Jaffe 1999, De Clercq 2002). Bricidofovir has been developed as an orally available alternative to cidofovir which has been shown to be less toxic and more potent (Olson et al. 2014, Trost et al. 2015). Technically neither of the two drugs are FDA-approved for treatment of smallpox and can only be used in an emergency.

There is now a single FDA approved drug with an indication for treatment of smallpox – TPOXX (also known as Tecovirimat and ST-246. Fig. 1.14 B). TPOXX was approved in July 2018 (FDA 2018, Grosenbach et al. 2018) and two million doses are stockpiled by the Strategic National Stockpile (SNS) (SIGA, 2018). TPOXX was discovered in a library drug screen of 356,240 compounds and was demonstrated to protect mice from lethal VACV and ECTV challenge. It was shown that TPOXX targets the IEV protein F13 thereby blocking wrapping and subsequently, virus spread (Yang et al. 2005, Chen et al. 2006, Duraffour et al. 2015). It was then successfully tested against VACV, camelpox virus, and cowpox virus (Duraffour et

Figure 1.14. Intravenous Cidofovir and TPOXX capsules. Source: Mylan and SIGA via AP

al. 2007). When mice (Grosenbach et al. 2008) or macaques (Berhanu et al. 2015) were given the ACAM2000 smallpox vaccine in combination with TPOXX the immune response was not affected, while side effects were reduced. Human single-dose (Jordan et al. 2008, Chinsangaram, Honeychurch, Tyavanagimatt, Bolken, et al. 2012) and multi-dose (Jordan et al. 2010, Chinsangaram, Honeychurch, Tyavanagimatt, Leeds, et al. 2012) studies have demonstrated that TPOXX is safe at doses that should be clinically relevant. Indeed, it has been already used successfully, in conjunction with VIGIV and cidofovir, in a case of a severe eczema vaccinatum in a 28-month-old child (Vora et al. 2008).

More importantly, TPOXX has been shown to inhibit monkeypox and variola virus in cells (Smith et al. 2009) and nonhuman primates (Jordan et al. 2009, Huggins et al. 2009, Mucker et al. 2013), as well as monkeypox in prairie dogs (Smith et al. 2011). However, because the drug targets a viral protein, mutations providing resistance have been identified in the process of drug development, (Duraffour et al. 2015). Since such a mutation may arise naturally, other anti-poxviral drugs are still required.

1.4.2 Drug repositioning and screening for antiviral drugs

Drug discovery is a long, costly, and immensely difficult process. Recent estimates put the price tag on successfully bringing a drug to market at over $2.5 billion (DiMasi, Grabowski, and Hansen 2016), and time to completion of more than a decade (Reichert 2003). This may be one of the reasons why there seems to be a trend of fewer drugs being approved (Scannell et al. 2012). This high cost, in both research time and money, includes compound screens and early stages of discovery that include all the potential hits that are eventually found unfeasible. One way of avoiding the costs of early drug discovery is drug repurposing/repositioning. Simply put, an already approved drug is found to have a different, novel application (Ashburn and Thor 2004, Papapetropoulos and Szabo 2018). The most well- known example of such repurposing is perhaps Viagra, which was originally intended as a hypertension drug (Terrett et al. 1996). A repurposed drug may significantly reduce the time from discovery to approval, and the cost of early studies, as much of the work, e.g. human safety studies, has already been done (Ashburn and Thor 2004, Papapetropoulos and Szabo 2018). Many vendors offer FDA-approved drug libraries. E.g. Prestwick Chemical Library used in this project, which has been cited in 17 publications as reported on Prestwick Chemical publication list.

This approach is even more welcomed when it comes to infectious diseases (Zheng, Sun, and Simeonov 2018). Recent examples of sudden outbreaks include Ebola virus (EBOV) and Zika

Figure 1.15. Global map of significant and new emerging infections: spread to new areas since 1997. The (re)emergence of many diseases around the world explains the need for new antiviral compounds.

Source: (Public Health England 2018) virus (ZIKV) (Fig. 1.15). The EBOV outbreak in 2014 has claimed the lives of 11,325 people mostly in Guinea, Liberia, and Sierra Leone (CDC 2017, Kaner and Schaack 2016). On the other hand, ZIKV infection usually doesn’t cause more than a fever. However, in 2016 in Brazil over 1,000 children born to women infected with ZIKV presented with serious neurological disorders (Costello et al. 2016, Hazin et al. 2016, Mlakar et al. 2016). At the time of the outbreak of both diseases there were no approved vaccines or antiviral drugs.

Since the approval path for repurposed drugs is much shorter, drug repurposing for emerging or re-emerging infectious diseases (Fig. 1.16) is an attractive alternative to the “traditional” drug discovery approach. There are now several publications attempting to address the lack of antiviral drugs against EBOV (Madrid et al. 2013, Johansen et al. 2013, Kouznetsova et al. 2014, Johansen et al. 2015, Zhao et al. 2016) and ZIKV (Barrows et al. 2016, Xu et al. 2016, Mumtaz et al. 2017) via drug repositioning.

1.5 Vaccinia virus screens

VACV has previously been a target for various (high throughput) screens. The temporal organization of the replication cycle and viral spread, as well as the relative ease of creating recombinant VACV, has led to the design of various screening methodologies.

1.5.1 VACV drug screens

The classic procedure measures the ability of compounds to inhibit viral plaque formation. ST-246 (TPOXX®) was identified using this method in a screen of 356,240 compounds (Yang et al. 2005). The screen identified additional 758 compounds that inhibited the cytopathic effect by >50%, however no additional information about those hits was provided in the publication. Other screening techniques may include recombinant viruses expressing various reporter genes, e.g. β-galactosidase, β-glucuronidase, or a fluorescent protein. The latter method was used in conjunction with flow cytometry as a proof of concept of infection quantification (Dominguez, Lorenzo, and Blasco 1998). This approach has been adapted for a high throughput neutralization assay as a method of evaluating smallpox vaccines (Earl, Americo, and Moss 2003). The first microscopy-based library screen using VACV expressing EGFP identified mitoxantrone, as well as 12 other compounds, as VACV inhibitors (Deng et al. 2007). Using EM, mitoxantrone was shown to block the IV/IVN to IMV transition. Mitoxantrone-resistant viruses were generated, and the drug target was identified as a VACV DNA ligase. This screen of 2,880 compounds in BSC40 cells utilised an EGFP under VACV an early/late promoter, with secondary screens using plaque assays. As a result, neither the primary not secondary screen provided any information regarding the stage at which infection was blocked. A tertiary screen using virus yield as a readout was needed to differentiate between a block in replication versus a block in viral spread. To overcome this limitation and further expand the available methods of fluorescent screening, a number of recombinant viruses expressing multiple fluorescent proteins under early, intermediate, and/or late promoters have been developed to be used in screens for inhibitory compounds using a fluorescent plate reader (Dower et al. 2011, Rozelle et al. 2014). Both publications were focused on the design and verification of the screening methodology. The first one reported on one hit, “compound F11”, and the latter did not screen for new compounds.

At the time of writing this thesis, there were only two publications dealing with high- throughput screens of large drug libraries, and only one of them used VACV recombinants expressing fluorescent proteins (Yang et al. 2005, Deng et al. 2007).

1.5.2 LOPAC screen

A screen of the Library of Pharmacologically Active Compounds (LOPAC) done previously in the Mercer lab identified stattic, a Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylation inhibitor, as a VACV infection inhibitor. Stattic inhibits early gene expression

in VACV infection. The JAK1/STAT pathway has been described in section 1.2.1, and information relating to STAT3 is presented here.

STAT3 is a transcription factor that forms homodimers when activated, translocates to the nucleus, and binds to cellular DNA (Akira et al. 1994) (Fig 1.17). It regulates the transcription of genes involved in a variety of important functions, including cell differentiation and proliferation, cell death, angiogenesis, metastasis, and immune responses (Hirano, Ishihara, and Hibi 2000, Johnston and Grandis 2011). While other STATs can be deleted in mice, STAT3 knock-out causes embryonic lethality (Takeda et al. 1997). STAT3 is activated by the IL-6 family of cytokines, epidermal growth factor, granulocyte colony-stimulating factor, and leptin (Levy and Lee 2002). Phosphorylation at tyrosine 705 is normally mediated by JAK1 (Guschin et al. 1995), while phosphorylation at serine 727 is mediated by several other kinases (Decker and Kovarik 2000). Phosphorylation at Y705 leads to STAT3 activation, while phosphorylation at S727 is believed to increase the activity of the dimerized complex (Decker

Figure 1.16. STAT3 pathways. In the classical pathway, STAT3 is phosphorylated at Y705 by the JAK, which leads to dimerization and translocation into the nucleus, where STAT3 acts as a transcription factor. In the non-classical pathway, STAT3 is phosphorylated at S727 by MAPK (or other kinases), and translocates to the mitochondria where it supresses apoptosis and/or the ER, to modulate Ca2+ release.

Source: (Yang and Rincon 2016).

and Kovarik 2000). Phosphorylation at S727 has also been shown to lead to STAT3 accumulation in the mitochondria, where it can supress apoptosis (Cheng, Peuckert, and Wolfl 2017), and in the ER, where it modulates ER-mitochondria Ca2+ release (Avalle et al. 2018). It is evident that STAT3 has varied functions within multiple compartments of the cell.

Contrary to the finding that stattic inhibits VACV, it has been shown that mice treated topically with stattic develop larger lesions and other indications of increased infection, compared to untreated mice, and that keratinocytes use a STAT3 pathways for cell death (He et al. 2014, He et al. 2017). However, both studies used the ACAM-2000 vaccine strain of VACV, which lacks the genes encoding a tumour necrosis factor (TNF) receptor, an INF-α/β binding protein, an elongated thymidylate kinase A, and an ankyrin repeat ortholog (Osborne et al. 2007). Another study suggested that STAT3 is required for the inhibition of VACV and influenza infection. In STAT3-/- Murine Embryonic Fibroblasts (MEFs) VACV infection was increased compared to wild type cells (Mahony et al. 2017). However, the increase in VACV titre was only 2-fold; the drop seen in the LOPAC screen caused by stattic was 2-log, or a virtual block of early gene expression. Considering that VACV inhibits the STAT1/1 and STAT1/2 dimers during infection, it is counterintuitive that inhibition of STAT3 by a drug would lead to inhibition of infection. This result suggests that either STAT3 plays a different function than other members of its family during infection, or stattic’s ability to block viral replication is not by means of STAT3 phosphorylation inhibition. However, a phosphoproteomics screen screen (Novy and Mercer, unpublished) has revealed that KRAB- associated protein-1 (KAP1), also known as TRIM28, is phosphorylated in VACV infected HeLa cells. KAP1 when not phosphorylated inhibits phosphorylation of STAT3, which would suggest that VACV needs STAT3 phosphorylation for successful infection, consistent with the results of the LOPAC screen.

1.6 Thesis aims

The main aim of this PhD project was to identify novel, cell-targeting FDA-approved compounds capable of inhibiting VACV spread in cell culture. To that end, I have screened the Prestwick Chemical Library for VACV spread inhibitors, as well as inhibitors of early and late gene expression. In order to accomplish that, I have combined an image-based screening strategy and EGFP-Early, mCherry-Late expressing recombinant VACV. Taking advantage of the highly coordinated sequential progression of poxvirus replication (Moss 2007), in which each stage of the virus lifecycle depends upon successful completion of the proceeding step, allowed me to differentiate between inhibition at early, late, or spread stage of infection in a single screen. The goal was to then classify identified hits according to the specific stage of viral replication was inhibited, e.g. entry, replication, infectious particle formation, or cell egress. Such compounds could potentially be used as novel molecular tools or, with further development, as antipoxviral agents.

There are two additional topics explored in this thesis. First is the testing of hits identified in the primary screen as potential African swine fever virus inhibitors, in collaboration with The Pirbright Institute. The second covers the expansion of previous work done in the Mercer lab, in which a screen of the LOPAC drug library identified, among other hits, stattic, a STAT3 phosphorylation inhibitor. My contribution to this project includes confirmation of the results and attempts at further elucidating of the mechanism of action of stattic.

2 Materials and methods

2.1 Virus list

All viruses used in this thesis are VACV Western Reserve (WR) strain and are part of the Mercer Lab collection. Apart from the WT virus, they were generated by Florian Schmidt and Jason Mercer (Mercer et al. 2010a, Schmidt et al. 2011, Schmidt 2011) by homologues recombination with a plasmid containing the gene of interest surrounded by flanking genomic sequences of 300bp. Linearized plasmids were transfected and VACV endogenous recombination system relied on to generate the recombinants. All viruses were screened for fluorescence by plaque formation. Resultant fluorescent plaques were purified to homogenity through multiple rounds of plaque purification. Where fluorescent proteins under VACV promoters were added they were inserted into the thymidine kinase (TK) locus, disrupting this gene which is non-essential in vitro. All viruses were validated by sequencing of the resultant virus genomes, flow cytometry for temporal fluorescence expression, and western blots protein expression (Schmidt 2011). All viruses were comparable with parental WT WR VACV with regard to plaque size and 24h yield, with the exception of A5-mCherry F13-EGFP virus, which was 1 log down and had a slight defect in plaque size.

As a result of adding EGFP under a VACV promoter, green fluorescence was detectable in the cell and coincided with expression of early, intermediate, or late viral genes. Tagging A5 protein resulted in green labelling of virion cores. Similarly, tagging the envelope protein F13 resulted in green-labelled enveloped virions (or both green and red in case of A5-mCherry F13-EGFP recombinant), and tagging F17 resulted in green-labelled lateral bodies.

Virus name Description

WT Western Reserve Wild Type

E-EGFP EGFP under VACV early gene promoter, inserted in the TK locus

I-EGFP EGFP under VACV intermediate gene promoter, inserted in the TK locus

L-EGFP EGFP under VACV late gene promoter, inserted in the TK locus

E-EGFP L- EGFP under VACV early gene promoter and mCherry under mCherry VACV late gene promoter, inserted in the TK locus

A5-EGFP EGFP-tagged core protein A5 (C-terminus), endogenous

A5-mCherry mCherry-tagged core protein A5 (N-terminus) and EGFP-tagged F17-EGFP lateral body protein F17 (C-terminus), both endogenous

A5-mCherry mCherry-tagged core protein A5 (C-terminus) and EGFP-tagged F13-EGFP EEV protein F13 (C-terminus), both endogenous

Table 2.1. VACV and VACV recombinants used in this thesis. Apart from the WT virus, all viruses were generated by Florian Schmidt and Jason Mercer (Mercer et al. 2010a, Schmidt et al. 2011, Schmidt 2011).

2.2 Prestwick Chemical Library

The Prestwick Chemical Library (PCL) was purchased from Prestwick Chemical in DMSO at a stock concentration of 10 mM and maintained by the LMCB High Throughput Facility. For the primary screen, compounds were provided as a 1:500 dilution (20 µM) in DMEM in 96-well plates. Dilutions for the secondary screens were made at the requested concentrations using a Tecan Freedom Evo liquid handler, in DMEM in 96-well plates, by Joana Da Costa.

Following the secondary screens, 25 confirmed hits were ordered individually from Prestwick Chemical as powder. Additional cardiac glycosides were ordered from Sigma as powder. The compounds were then dissolved in 98% DMSO, 2% dH2O as 50 µM stock solutions and working aliquots were made in 96-well plates and stored at -80˚C.

2.3 General methods

2.3.1 Cell culture

HeLa (ATCC-CCL2), BSC40 cell lines (from Dr. Paula Traktman), and HEK 293T (expressing empty vector or mouse Na+/K+ ATPase) (from Prof Ariberto Fassati) were maintained in DMEM (Life technologies) supplemented with 10% Foetal Bovine Serum (FBS, Life technologies) and 1% Pen-Strep (Life technologies), under standard conditions: 37°C, 5% CO2. DLD-1 and DLD-1 STAT3 Y705F/Y705F (Horizon; HD PAR-111 and HD 115-016) cell lines were maintained in RMPI (Life technologies) supplemented with 10% Foetal Bovine Serum and 1%

Pen-Strep under standard conditions. Upon reaching confluency cells were washed once with PBS, detached by Trypsin/EDTA (2.5g TRP/litre, 0.2g EDTA/litre), diluted with appropriate media, and split. Cell count was determined using Cellometer (Nexelcom) when needed.

2.3.2 Virus preparation

Virus propagation

Confluent BSC40 cells in a 60 mm dish were infected with multiplicity of infection (MOI) of 1 using the virus to be propagated and incubated for two days. Afterwards, medium was aspirated, and cells were scraped in PBS, followed by centrifugation for 5 min at 300 x g. PBS was aspirated, and the cell pellet was resuspended in 100 µL of 1 mM Tris pH 9.0. Cells were then lysed by freeze-thawing, and the resulting cell lysate was used to infect two 15 cm dishes of confluent BSC40 cells (50 µL per dish).

The procedure was repeated and cell lysate (freeze-thawed in 1 ml of 1 mM Tris pH 9.0) from two 15 cm dishes of infected cells was used to infect 15 x 15 cm dishes of confluent BSC40 cells. These were then incubated for two days.

Finally, the infected cells were harvested by scraping in PBS, followed by centrifugation at 300 x g for 5 min. The pellet was stored at -80°C until virus purification.

Virus purification

Sedimentation through 36% sucrose cushion.

The pellet was resuspended in 12 ml of 10 mM Tris pH 9.0 and left on ice for 5 min. Cells were then disrupted on ice in a tight fitting douncer (a homogenizer, consisting of a glass tube with a tight-fitting pestle) using 25 strokes. The resulting cytosolic extract was transferred to a 15 ml Falcon tube and centrifuged for 10 min at 2,000 x g. The supernatant was transferred to a new Falcon tube and the centrifugation and transfer steps were repeated to remove cell debris.

Next, a SW32 tube (Beckman Coulter) was filled with 16 ml of 36% sucrose in 20 mM Tris pH 9.0, and the cytosolic extract (approx. 10 ml) was carefully loaded on top of the sucrose. 10 mM Tris pH 9.0 was added to a final 20 ml volume, with a 2 ml mineral oil layer on top to prevent virus aerosolization. This was centrifuged in a SW 32 Ti Rotor (Beckman Coulter) for 80 min at 4°C and 17,600 rpm. Following centrifugation, the supernatant was aspirated, and the pellet was resuspended in 200 µL of 1 mM Tris pH 9.0, if followed by band-purification. If sedimented virus was to be used, pellet was instead resuspended in 1 ml of 1 mM Tris pH 9.0.

Band-purification with 25-40% sucrose gradient.

A SW41 tube (Beckman Coulter) was filled with approx. 6 ml of 25% sucrose in 10 mM Tris, and this was underlaid with an equal amount of 40% sucrose in 10 mM Tris using a 12 cm needle. A sucrose gradient was then prepared using Gradient Maker (Biocomp), using the following settings: 3:00 min, 81.5°, 18 rpm.

The sedimented virus was sonicated and vortexed and loaded on top of the 25-40% sucrose gradient, which was then centrifuged for 60 min at 9,700 rpm, using a SW40 TI Rotor (Beckman Coulter).

Side of the tube was covered with Scotch tape and a 21 G needle was used to pierce that part of the tube and aspirate an approx. 2.5 ml layer of intracellular mature virions (IMVs). The layer containing IMVs could be identified as a clouded band around the middle of the tube. The IMV layer was placed in a new SW41 tube and 1 mM Tris pH 9.0 was added for a final volume of 12 ml, followed by a 40 min centrifugation at 17,400 using the SW40 TI Rotor. The pellet was resuspended in 300 µL of 1 mM Tris pH 9.0. Plaque assay was then used to determine the plaque forming units /ml.

2.3.3 Plaque assay

BSC40 cells were seeded onto a 6-well plate. Assay was performed when cells reached confluency. 2 µL of assayed virus or sample were diluted in 1 ml DMEM. Then, serial 1 in 10 dilutions, from 10-3 to 10-9, were prepared. 500 µL of appropriate dilution was aliquoted onto each well. The plate was placed at 37˚C for 60 min, with gentle agitation every 10-15 min. Wells were then aspirated, and fresh DMEM was added. Staining with crystal violet (0.5% crystal violet, 2% PFA, in water) was done two days after infection. The number of viral plaques (a viral plaque is the area where the cells moved away from the centre of infection, thus leaving an unstained area) was manually counted, and the final concentration of plaque forming units (pfu/ml) was calculated based on dilution factor, using a well that showed between 20-100 plaques.

2.4 Primary screen

HeLa cells were seeded onto 96-well plates (CellCarrier-96, Perkin Elmer) at a concentration of 20,000 cells per well approximately 16h before the start of the experiment.

Separate 96-well plates (Falcon) with the Prestwick Chemical Library drugs at 20 µM in DMEM were provided by a member of the High Throughput Facility.

Medium was aspirated from the cell plates and 50 μL of drug was aliquoted from the prepared 96-well plates into each cell well, to allow for drug pre-incubation. A control plate used to optimize the screen was prepared using three columns each of No Drug, AraC at 20 µM, BFA at 30 µM, and CHX at 100 µM (final concentrations). Plates were left at room temperature (RT) for 30 min. Next, 50 μL of VACV E-EGFP L-mCherry screening mutant in DMEM was added at an experimentally defined dilution ensuring required plaque number (MOI 0.05). This resulted in a final drug concentration of 10 µM. Wells were aspirated after 60 min at RT, and 50 μL of DMEM was aliquoted to each well, followed by 50 μL of drug to maintain the 10 µM final drug concentration. Plates were placed in the 37°C, 5% CO2 incubator. Ten hours post infection (hpi), 20 μL of 60 μM AraC was added to each well to a final concentration of 10 μM, to prevent late gene expression in secondary infected cells. At 24 hpi, 20 μL of 36% PFA was added to each well for a final concentration of 5% and incubated for 20 min to fix the cells.

Cells were washed once for 5 min in PBS and once for 5 min in 0.5% BSA (Sigma) in PBS. To boost the signal of early EGFP, 50 μL of primary staining/permeabilizing solution (0.5% BSA, 0.5% Triton-X 1000 (Sigma), 1:1000 anti-GFP antibody (rabbit) in PBS) was aliquoted to each well and plates were kept at RT, in the dark, for 60 min followed by 3x 5 min wash with 0.5% BSA in PBS. 50 μL of secondary staining solution (0.5% BSA in PBS, 1:1000 anti-rabbit antibody (Alexa 488, Thermo Fisher Scientific), 1:10000 Hoechst) was aliquoted to each well and kept in the dark, at RT, for 60 min followed by 3x 5 min wash with PBS. Finally, 100 μL of PBS was aliquoted to each well, and plates were stored at 4°C until imaging.

2.4.1 Imaging

OPERA (Perkin Elmer) high-throughput microscope was used to detect EGFP, mCherry, and Hoechst fluorescence with a 20x air objective. 18 plates were imaged, with 9 neighbouring images per well, for a total of 15,552 images. Images of each well were stitched into a single image for further analysis.

2.4.2 Image analysis

Image analysis was done by Janos Kriston-Vizi, LMCB Head Bioinformatician. As a first step in the image analysis, ImageJ was used to find the local maxima on each image in the blue channel (nuclei), then a watershed method was used to detect the entire nucleus. The same maxima locations were then used to measure the intensity in the green channel (early gene expression) and red channel (late gene expression). The number of all cells (based on the nuclei number) and the number of cells showing fluorescence intensity over a threshold

value in the green and red channels were counted. A ratio of green/total and red/total was calculated for each well. Raw data was then placed in an Excel file.

From this point all data analysis was done by me.

A “hit” was determined by a low ratio of green or red over blue count. All hits were sorted in an ascending numerical order, with the assumption that the lower the ratio, the stronger the hit. The best 50 hits were selected, and their images were inspected visually. In a few cases it was apparent that there were few cells present, suggesting cell death and indicating a false positive. Such hits were excluded, and the next best hit was added to the list. The final best 50 hits were selected for secondary screening.

2.5 Secondary screen

HeLa cells were seeded onto 96-well plates (Falcon) at a concentration of 20,000 cells per well, 16 hrs before the start of the experiment.

First, a drug dilution was prepared in 96-well plates in DMEM, which when mixed 1:1 with virus-containing medium gave final dilutions of 50 µM, 10 µM, 5 µM, 1 µM, 0.1 µM, 0.01µM. Alternatively, a 2-fold serial dilution starting at 100 µM was prepared.

In HEK experiments, Cardenolides were used at final concentrations of 25 µM, 5 µM, 1 µM, 0.2 µM, 0.04 µM, and 0.008 µM.

Medium was aspirated, and cells were pre-incubated with 50 μL of drug for 30min, followed by addition of VACV E-EGFP (all hits) or VACV L-EGFP (non-Early hits, following the Early screen results) or Intermediate-EGFP (Late hits only) in DMEM. Alternatively, cells were infected with VACV E-EGFP L-mCherry (all hits). Virus MOI used was experimentally determined to result in 40-60% of cells being infected in the primary infection. After 1h of incubation at 37°C wells were aspirated to remove unbound virus and synchronize the infection, 50 μL of DMEM and 50 μL of 2x drug was added and plates were placed at 37°C,

5% CO2. At hpi = 24 cells were prepared for flow cytometry analysis.

2.5.1 Flow cytometry

For the flow cytometry analysis wells were aspirated, and cells washed once with PBS. 80 μL of trypsin was added for 20 min at 37°C, followed by addition of 80 μL of blocking buffer (5% FBS in PBS). Finally, 80 μL of 8% PFA was added for 15 min to fix cells. At no point were the cells removed from the wells.

Fluorescence in 488 nm and 594 nm channels was measured on a Guava easyCyte HT (Merck). 5,000 events (cells) were recorded, and a gate of “live” cells was selected based on a control well of uninfected cells. The same control was used to create markers of “non- fluorescence”. Anything above the levels detected in uninfected cells was counted as a fluorescence hit, i.e. an infected cell expressing EGFP or mCherry.

GraphPad Prism 7 (GraphPad Software, Inc) was used to analyse the flow cytometry data. A non-linear fit equation (log(inhibitor) vs. response -- Variable slope) was applied to the flow cytometry results to obtain the EC50 (half maximal effective concentration) values. EC90 (90% maximal effective concentration) values were calculated using the Hill slope and EC50 values in GraphPad online calculator. The value of EC90 was then adjusted for a more straightforward dilution-making in the future. In most cases, it was rounded up to the closest 5 or 10 µM, and this value was used as EC90 in all future experiments.

2.6 Cytotoxicity

Cytotoxity of confirmed compounds was measured using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) following the manufacturer’s instructions. Briefly, compounds at their EC90 were added to approx. 80% confluent HeLa cells for 24h. A serial 10-fold dilution of staurosporine (10 mM – 0.0001 mM) was used as a control. After 24h, 50 µL of medium taken off cells was mixed with 50 µL of Pierce LDH Reaction Mix in a new plate, and incubated at room temperature for 30 min, followed by the addition of 50 µL Stop Solution. The absorbance at 480 nm and 680 nm was measured on VersaMax Microplate Reader (Molecular Devices). All values were Min-Max normalized to the lowest and highest values on the plate.

2.7 Early assays

2.7.1 Pre-incubation

VACV L-EGFP at MOI 1,000 was preincubated with Early compounds at their EC90 for 60 min at RT in DMEM. The virus was then diluted 1:100 (MOI 10) in DMEM 0% and used to inoculate HeLa cells in a 96-well plate. After 60 min of binding at RT the inoculate was replaced with DMEM. 20 hpi cells were trypsinised, fixed, and analysed with flow cytometry for EGFP fluorescence.

2.7.2 Binding

50 µL of early compounds at 2x their EC90 were added to HeLa cells in a 96 well plate, on ice. After 15 min 50 µL of VACV A5-EGFP at MOI 50 was added for 60 min, on ice. Cells were then washed with ice cold PBS, fixed with 4% PFA, and scraped within the well with a pipette tip. The plate was analysed with flow cytometry for EGFP fluorescence.

2.7.3 Acid bypass

50 µL of early compounds at 2x their EC90 were added to HeLa cells in a 96-well plate for 15 min. 50 µL of VACV E-EGFP or L-EGFP at MOI 10 was added to each well for binding at RT for 30 min. The inoculate was then replaced with 100 µL DMEM, 20 mM MES, pH 5.0 or pH 7.0 containing Early compounds at their EC90 for 10 min at 37˚C, followed by aspiration and addition of Early hits at their EC90 in DMEM. 6 hpi (E-EGFP) or 8 hpi (L-EGFP) cells were analysed by flow cytometry for EGFP fluorescence.

2.7.4 AraC release assay

Hela cells in 96-well plate were infected with VACV E-EGFP, L-EGFP, or E-EGFP L-mCherry at MOI 10, in presence of 10 µM AraC. After 3h at 37˚C cells were washed 5 times with DMEM, and fresh medium containing Early compounds at their EC90 was added. Plates were incubated for 8h at 37˚C, followed by analysis by flow cytometry.

2.8 Microscopy assays

2.8.1 Entry assay

HeLa cells were seeded onto CELLview Slides (Greiner Bio-One) at 20,000 cells per well 16h before the experiment, to reach confluency. Early hits (50 µL per well) in DMEM were added at 2x their EC90 for 15 min pre-incubation. 1% DMSO, 100 µM CHX, and 60 µM EIPA were used as controls. VACV A5 EGFP or VACV F17-EGFP A5-mCherry was then added at MOI 30 in 50 µL of DMEM. After 60 min incubation at room temperature wells were aspirated to remove non-bound virus, and compounds were re-added at their EC90. 4 hpi cells were fixed with 4% EM-grade PFA for 20 min, followed by a PBS wash.

Staining and labelling was preceded by blocking (without permeabilization) in blocking buffer (5% BSA, 1% FBS, in PBS) for 60 min at RT. Next, L1 mouse (7D11) antibody (1: 1000) in blocking buffer was added for 60 min at RT, followed by a PBS wash. Anti-mouse antibody

(Alexa 647, Invitrogen. 1:1000) and Hoechst (1:10000) in blocking buffer were added for 60 min at RT, followed by a PBS wash.

Images were taken using a 100x oil immersion objective (NA 1.45) on a VT-iSIM microscope (Visitech; Nikon Eclipse TI), using 405 nm, 488 nm, 561 nm, 647 nm laser frequencies for excitation.

Quantification of colocalization of virions was done using ZedMate plug-in on ImageJ. Briefly, ZedMate utilizes the TrackMate plug-in from ImageJ for spot detection, linking them in a Z- stack. This allows for accurate localization of the viral cores. ZedMate then provides the fluorescence intensity for the location of a given particle in other channels. In this case, the colocalization of core and L1 was of interest. Using an Excel and KNIME based workflow, the number of cores colocalizing with L1 was determined. ZedMate and KNIME workflow were created by Artur Yakimovich.

2.8.2 Pre-replication and replication site formation

HeLa cells were seeded onto CELLview Slide (Greiner Bio-One) at 20,000 cells per well 16h before the experiment. VACV A5-EGFP was added at MOI 20, to increase the chances of synchronous infection. After 60 min incubation at RT wells were aspirated and Late hits were added at their EC90. 1% DMSO, 100 µM CHX, and 10 µM AraC were used as controls. After 8h incubation cells were fixed with 4% EM-grade PFA in PBS for 20 min, then washed with PBS.

Staining and antibody labelling was preceded by a blocking and permeabilization step (5% BSA, 1% FBS, 0.5% Triton-X 1000, in PBS) for 60 min at RT. Primary antibody against I3, a viral single strand DNA binding protein, was added in blocking buffer (I3 (rabbit) 1:1000, 5% BSA, 1% FBS, in PBS) for 60 min at RT, followed by a PBS wash. Anti-rabbit antibody (Alexa 647 1:1000), Phalloidin 594 (1:1000), and Hoechst (1:10,000) in blocking buffer were added for 60 min at RT, in the dark, followed by a PBS wash.

Images were taken using iSIM, as previously described for the entry assay.

2.8.3 Edu incorporation

Click-iT™ EdU Alexa Fluor™ 488 Imaging Kit (ThermoFisher Scientific) was used. Cells were seeded onto CellCarrier-96 Ultra Microplates (PerkinElmer), at 20,000 cells per well 16h before the experiment. VACV WT at MOI 20 was added to the cells for 60 min at RT in DMEM. After unbound virus was aspirated, Late hits in a serial 2-fold serial dilution starting at 25 µM in DMEM with 1 µM Edu were added. Controls were also used in a 2-fold serial dilution

starting from: 10% DMSO; 200 µM CHX; 200 µM AraC. After 8h incubation cells were fixed with 4% EM-grade PFA in PBS for 20 min, then washed with PBS.

The Edu labelling was completed following manufactures instructions. First, cells were permeabilized with a blocking and permeabilization buffer (5% BSA, 1% FBS, 0.5% Triton-X 1000, in PBS) for 60 min at RT. The Edu Reaction Cocktail (5 ml) was prepared by mixing 4.3 ml 1x Click-iT™ reaction buffer, 200 µL CuSO4, 12.5uL Alexa 488 Fluor azide, and 500 µL reaction buffer additive. 50 µL of Reaction Cocktail per well was added for 60 min at RT, protected from light. Cells were washed twice with PBS and stained with Hoechst (1:10,000) in PBS for 30 min at RT, then washed twice with PBS. Plates were imaged on OPERA Phoenix using 20x air objective. Alexa 488 and Hoechst fluorescence were measured.

Images were analysed by Artur Yakimovich. Briefly, nuclei were localised, and their fluorescence was excluded from analysis. The total intensity of Alexa 488 was then quantified per cell, allowing for an approximation of the total Edu incorporated by the virus into the pre-replication and replication sites.

2.8.4 Spread hits assay

HeLa cells were seeded onto CELLview Slide (Greiner Bio-One) at 10,000 cells per well 16h before the experiment. VACV A5-mCherry F13-EGFP was added at MOI 20, to increase the chances of synchronous infection. After 60 min incubation wells were aspirated and Spread hits were added at their EC90. 1% DMSO and 35 µM BFA were used as controls. 12 hpi incubation cells were fixed with 4% EM-grade PFA in PBS for 20 min, then washed twice with PBS.

Staining and labelling was preceded by blocking (without permeabilization) in blocking buffer (5% BSA, 1% FBS, in PBS) for 60 min at RT. Next, B5 antibody (mouse, 1:1000) in blocking buffer was added for 60 min at RT, followed by a PBS wash. Anti-mouse antibody (Alexa 647), Hoechst (1:10000) in blocking buffer were added for 60 min at RT, followed by a PBS wash.

Images were taken using iSIM, as previously described.

2.9 24h EEV and IMV yields

HeLa cells at approx. 80% confluency in 6-well plates were infected with WT VACV at MOI 1 for 60 min at RT, with gentle agitation every 10-15 min. Wells were then aspirated and spread hits at their EC90 were added. Plates were incubated at standard conditions for 24h. 24hpi medium was collected for EEVs yields, and the remaining cells were scraped in PBS and collected for MVs yields.

2.9.1 Extracellular enveloped virions

The IMV neutralizing antibody L1 (7D11, 1:250) (Wolffe, Vijaya, and Moss 1995) was added to 20 µL of medium from each sample for a final concentration of 20 µg/mL, and incubated at 37°C for 60 min. A control of DMSO sample was used where no L1 was added for comparison. Following the incubation, a 10-2, 10-3, and 10-4 dilutions were prepared by adding 980 µL medium to the 20 µL of virus, followed by two 10-fold dilutions. A plaque assay was used to quantify the number of plaque forming units.

2.9.2 Intracellular mature virions

Cells harvested from the wells were centrifuged (5 min at 300 x g) and the pellet was resuspended in 100 µL of 1 mM Tris pH 9, followed by lysis by freeze-thawing. A plaque assay was used to quantify the number of plaque forming units.

2.10 Transmission electron microscopy

50,000 Hela cells were seeded on 13 mm slide coverslips placed in wells of a 24-well plate. The next day, cells were infected with MOI 10 of VACV WT for 60 min at 37˚C in DMEM. The medium and unbound virus were aspirated, and spread hits were added at their EC90. After 12h medium was aspirated, and the coverslips were fixed in EM-grade 2% paraformaldehyde/2% glutaraldehyde (TAAB Laboratories Equipment, Ltd.) in 0.1 M sodium cacodylate for 20-30 minutes.

EM was kindly done by Ian White, LMCB deputy electron microscopy leader. Coverslips were secondarily fixed for 1h in 1% osmium tetraoxide/1.5% potassium ferricyanide at 4˚C and then treated with 1% tannic acid in 0.1 M sodium cacodylate for 45min at room temperature. Samples were then dehydrated in sequentially increasing concentration of ethanol solutions and embedded in Epon resin. Coverslips were inverted onto prepolymerised Epon stubs and polymerised by baking at 60˚C overnight. The 70 nm thin sections were cut with a Diatome 45˚ diamond knife using an ultramicrotome (UC7; Leica). Sections were collected on 1×2 mm formvar-coated slot grids and stained with Reynolds lead citrate. All samples were imaged using a transmission electron microscope (Tecnai T12; FEI) equipped with a charge-coupled device camera (SIS Morada; Olympus).

2.11 African swine fever virus

African swine fever virus (ASFV) experiments at the Pirbright Institute involving handling of live virus were performed by Chris Netherton. Experiments not involving live virus were done by me.

Top 50 compounds from the high-throughput microscopy-based screen were used. The ASFV experiment was done before the secondary screens. Confluent Vero cells were seeded at 30,000 cells per well in 96-well plate a day before the experiment. 50 µL of each compound at 20µM per well was added for 30 min at RT. Cells were then infected with Vero adapted ASFV Badajoz 1971 strain (abbreviated to Ba71v) (Enjuanes et al. 1976), at MOI 4 in 50 µL of media. Virus was left to bind for 60 min, on ice. Medium and unbound virus were then aspirated, and medium containing drugs at 10µM was added. 1% DMSO, 100 nM Bafilomycin A (BafA), 10 µM AraC, and 30 µM BFA were used as controls. Plates were placed at 37˚C, 5%

CO2. 24 hpi cells were fixed with 4% PFA. After fixing samples were permeabilized with Triton X-1000 0.1% in PBS for 15 min. They were then blocked with 0.5% BSA in PBS for 30min, followed by labelling with antibodies against an ASFV early protein p30 (mouse, 1:10; in house preparation (Heath, Windsor, and Wileman 2001)) and a late protein p34 (rabbit, 1:1000; in house preparation (Heath, Windsor, and Wileman 2001)) for 60 min at RT. After a wash (3x5 min PBS), secondary antibodies (Alexa 488 anti-mouse and Alexa 568 anti-rabbit, 1:1000) in 0.5% BSA in PBS was added for 60 min at room temp. After a second wash DAPI (1:10,000 in PBS) was added for 15 min at room temp, followed by a final wash. Imaging was done on Leica microscope with a 40x air objective.

2.12 STAT3 experiments

2.12.1 Flow cytometry

A serial 2-fold dilution of stattic, S31-201 (Sigma), and cryptotanshinone (crypto) (Sigma) in DMEM was prepared. First, 50 µL of the compounds were added to HeLa cells in 96-well plates for 15 minutes at 2x concentration, followed by addition of 50 µL of VACV E-EGFP at MOI 10. After 60 min at RT wells were aspirated, and compounds replaced at 1x. 6 hpi cells were prepared for flow cytometry as described before, and the percentage of cells expressing EGFP was quantified.

2.12.2 Pre-incubation

VAVC E-EGFP at MOI 1,000 was preincubated with Stattic at their EC90 for 60 min at room temp. It was then diluted 1:100 (MOI 10) and added to HeLa cells in 96-well for 60 min of binding at room temp. Afterwards the inoculate was replaced with DMEM. 20 hpi cells were trypsinised, fixed, and analysed with flow cytometry.

2.12.3 Addition timepoints

Stattic at EC90 was added at t= -30, t= 0, t= 30, t= 60, and t= 120 [min] relative to infection (t=0) with VACV E-EGFP MOI 10 in HeLa cells. At t= 6h cells were prepared for flow cytometry as previously described, and number of cells expressing EGFP was counted.

2.12.4 STAT3 siRNA knockdown

First, human STAT3 siRNA (assay ID s743) (ThermoFisher Scientific) were validated. Briefly, HeLa cells were transfected with the siRNA (protocol below) for 48 or 72h days at several siRNA concentrations. They were then lysed and analysed by western blot (following the protocol in 2.12.5).

Hela cells were seeded at 5,000 per well in 96-well plates. 20h later, human STAT3 siRNA assay ID s743 (ThermoFisher Scientific) were added. The siRNA mix was made as follows, using Lipofectamine RNAiMAX Reagent (Invitrogen): 1.5 µL RNAiMAX was diluted in 25 µL DMEM, and 0.5 µL siRNA (20µM) was also diluted in 25 µL DMEM. The two were then mixed 1:1 and incubated at RT for 45-60 min. 10 µL of siRNA-lipid mix was added per well. Two days after transfection cell were infected with VACV E-EGFP in presence of serially diluted (2-fold) stattic, starting at 50µM. 8 hpi cells were prepared for flow cytometry analysis.

2.12.5 Western blots

Confluent HeLa cells in 10 cm dishes were infected with VACV WT at MOI 100. Cells were harvested at 0, 30min, 1h, and 2h post infection by scraping. They were centrifuged for 5 min at 300 x g, and the pellet was resuspended in 100 µL of lysis buffer (Cell Signalling) on ice for 30 min. Samples were then centrifuged for 10 min at 20,000 x g to remove cellular debris, and the supernatant was transferred to a fresh tube. It was then mixed with 4x loading dye (Life Sciences) and 30x DTT (Life Sciences) before being boiled for 10 min and sonicated for 5 min. 50 µL of sample per well was loaded into a 4-12% Bolt Bis-Tris Plus Gel (Invitrogen) and run for 4h at 50V in Bolt Mini Gel Tank (Invitrogen). Transfer in was done at 15 V for 45 min in Bolt Mini Gel Tank. Membrane was blocked with 5% BSA in TBS-T for 60 min. Primary

antibody for STAT3 (mouse, Cell Signalling) in blocking buffer was added for overnight incubation at 4˚C. Alpha-tubulin (mouse) was used as the loading control. Membrane was then washed 3x with TBS-T before secondary fluorescent antibodies (IRDye 680RD mouse, IRDye800 rabbit; Licor) in blocking buffer were added for 60 min at RT. Odyssey (Licor) laser imager and Image Studio Lite (Licor) were used to analyse the membrane.

3 Results

3.1 Microscopy-based high-throughput drug library screen

Given the limited number of alternatives to smallpox vaccination and the potential risks associated with it, there is a need for new cellular targets, new drugs, and new tools to study poxviruses. To this end a commercial drug library consisting of 1,280 FDA approved compounds (Prestwick Chemical Library) was screened. In order to screen for inhibitors of the complete virus replication cycle a recombinant virus expressing EGFP under the control of a VACV early promoter, and mCherry under the control of a VACV late promoter (E-EGFP L-mCherry; Mercer, unpublished) was used. This approach took advantage of VACV temporal gene expression (Fig. 3.1). The virus was designed following the general methods used to create recombinant viruses described in the introduction, and is similar to published viruses (Dower et al. 2011). The progression of early and late gene expression for the VACV E-EGFP L-mCherry is shown in Fig.3.2. Briefly, VACV in a primary infected cell goes through early gene expression, EGFP is expressed, and the cell turns green. As late VACV genes are expressed

Figure 3.1. VACV replication cycle and its inhibitors From entry to EEV release or cell lysis, the VACV replication cycle takes place entirely in the cytoplasm. CHX blocks protein synthesis. AraC interferes with DNA synthesis. BFA inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus, disrupting the Golgi.

(and the early gene expression is downregulated), the cell turns red due to mCherry being expressed. The virus spreads from the primary infected to surrounding cells which then follow the same sequence. This creates a small plaque with a red centre cell surrounded by green cells. This temporal change can be measured by both microscopy and cytometry (Fig. 3.2 B). To be able to identify defects in spread the plaques need to be isolated from each other, so the multiplicity of infection (MOI; the number of plaque forming units [pfu] per cell) was determined experimentally for each virus batch. Based on the temporal characteristics of the virus, it was decided that addition of AraC 10 hpi would block late gene expression in

Figure 3.2. E-EGFP L-mCherry virus plaque formation and gene expression over time. (A) As the infection progresses, the initially infected cell goes through early (green) and late (red) gene expression. Then, as the surrounding cells are infected, they follow the same progression of infection. Images taken at 8 hpi, 12 hpi, 16 hpi. (B) VACV E-EGFP L- mCherry temporal characteristic of gene expression during infection, over a 10h period. In both experiments, cells were infected at MOI 5. (B) Fluorescence was analysed via flow cytometry, counting the cells expressing EGFP or mCherry; or microscopy, measuring the mean fluorescence intensity of the well.

Means of 6 technical repeats from a single experiment ± s.d. are shown.

secondary infected cells. This stops the progress of infection in the secondary infected cells, preventing them from infecting further cells and resulted in them remaining green.

To show this would allow for the identification of compounds that inhibit VACV at different stages of the virus replication cycle, the screen was tested using a set of control compounds

Figure 3.3. Primary screen controls. (A) 1% DMSO causes No inhibition. 100 µM CHX blocks Early gene expression. 20 µM AraC blocks Late gene expression. 35 µM BFA blocks viral spread. (B) All cells were counted via nuclear staining. Cells expressing EGFP and mCherry over a threshold intensity were counted, and a ratio of green:all and red:all was calculated and plotted for each control. Each dot represents a well in a 96-well plate.

(described in section 1.3.2). This experimental set up allowed for an identification of three phenotypic inhibition readouts within a single screen: early inhibition with CHX (no fluorescence detected), late inhibition with AraC (EGFP only in primary infected cell) and spread inhibition with BFA (mCherry with residual EGFP in primary infected cells and no secondary infected cells) (Fig. 3.3). CHX blocks VACV early gene expression by inhibiting protein synthesis. AraC blocks VACV late gene expression by inhibiting viral DNA replication and replication site formation. BFA inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus resulting in its collapse, thereby preventing IMVs from being wrapped to form IEVs. As a result, viral spread by CEVs and EEVs is blocked. The controls were analysed the same way as the actual screen data, described below. With controls established the screen of the Prestwick Chemical Library was performed. The drug library compounds were added to cells at 20 µM in medium ~30 min before infection in case the effect of the drug required preincubation. This was followed by addition of virus in medium at a MOI of ~0.05. AraC was added 10 hpi and cells were fixed 24 hpi. Samples were then labelled with anti- EGFP antibody, to boost the early protein signal, and stained with Hoechst. Plates were imaged using a high-throughput microscope, and automated image analysis was performed. Analysis entailed localizing all cell nuclei (Hoechst signal maxima), and then recording green and red fluorescence at the same location. A hit could be determined by calculating the ratio of cells expressing EGFP or mCherry in a well (Fig.3.4). The best 50 hits were selected, and their images were inspected visually. In a few cases it was apparent that the number of cells was drastically lower than expected, suggesting cell death and indicating a false positive. Such hits were excluded, and the next best hit was added to the list. The final best 50 hits were selected for secondary screening (supplementary Fig. 6.1).

3.2 Flow-cytometry based confirmation screen

A flow cytometry assay was developed to, firstly, confirm the hits and identify any false positives, and secondly, to establish effective concentrations (EC50 and EC90) values for further experiments. The three controls were used once again: CHX, AraC, and BFA, in conjunction with VACV E-EGFP L-mCherry (Fig. 3.4A). Briefly, HeLa cells were infected for 16h with MOI 5, which resulted in 40-60% primary infection, in presence of serially diluted drugs. CHX completely inhibited early and late gene expression. AraC inhibited late gene expression but allowed for early gene expression in primary infected cells (40-60%). BFA blocked viral spread while allowing for both early and late gene expression in primary infected cells. The experiment was repeated with VACV E-EGFP and L-EGFP recombinant viruses following the

Figure 3.4. Prestwick Chemical Library screen results. The workflow of the screen is shown in A. Briefly, HeLa cells were infected with recombinant VACV E-EGFP L-mCherry at MOI 0.05, to keep the number of primary infected cells low, in the presence of compounds at 10 µM. 10 hpi AraC was added to stop late gene expression in secondary infected cells, resulting in the mini-plaque, where the primary infected red cell in the centre is surrounded by secondary infected green cells, if the infection was not inhibited. (B) All cells were counted via nuclear staining. Cells over expressing EGFP and mCherry over a threshold intensity were counted, and a ratios of green:all and red:all was calculated and plotted for each control. Each dot represents a single well in a 96-well plate. same protocol to confirm the results. A non-linear fit equation (log(inhibitor) vs. response -- Variable slope) was applied to the results to obtain the EC values.

This secondary screen was then applied to the VACV inhibitors identified in the primary screen. Hela cells were pre-treated with hit compounds and infected with either E-EGFP, L- EGFP, or E-EGFP L-mCherry, in the presence of hit compounds. 16 hpi cells were analysed by

flow cytometry for fluorescence. In the primary screen more Early hits were identified, therefore all 50 hits were first examined using the E-EGFP recombinant. Hits that did not inhibit infection at the expected level were then tested using the L-EGFP virus, to differentiate Late and Spread hits and false positives. Additionally, E-EGFP L-mCherry virus was also used on all confirmed hits to visualise the inhibition of both early and late gene expression simultaneously.

27 initial hits were confirmed: 14 Early inhibitors, 3 Late inhibitors, and 7 Spread inhibitors. All 27 hits displayed a dose-dependent reduction of infection, and EC50 and EC90 values were calculated from the VACV E-EGFP L-mCherry data (Table 3.1; individual graphs in Supplementary). Showing the efficacy of the primary screen, CHX, AraC, amiloride, gefitinib, and mitoxantrone were among the confirmed hits. These are known VACV inhibitors previously used in VACV research (Mercer and Helenius 2008, Mercer et al. 2010a). Mitoxantrone was excluded (Deng et al. 2007) from further analyses, while the other compounds remained as internal controls.

EC50 Applied Early Code Clinical use Cytotoxicity [µM] EC90 [µM]

Aminacrine E1 topical 5.99 25 25%

Astemizole E2 10 µM 7.63 25 30%

Avermectin B1a E3 18 µM 19.3 25 31%

Benzethonium Topical 0.1- E4 6.84 25 68% chloride 0.2%

Chlorhexidine E5 Topical 0.2% 16.8 25 31%

Daunorubicin E6 0.31 µM 1.15 5 38%

Digitoxigenin C5 n/a 0.64 10 9.8%

Digoxigenin C7 n/a 3.00 10 9.9%

Digoxin C8 2.5 µM 0.46 10 11%

Fludarabine E7 10 µM 4.48 25 8.1%

Gefitinib E8 0.36 µM 14.2 25 9.5%

Lanatoside C C10 2.0 µM 0.63 10 10%

Proscillaridin A C13 n/a 0.01 10 10%

Raloxifene E9 0.0011 µM 20.9 25 9.1%

EC50 Applied Late Code Clinical use Cytotoxicity [µM] EC90 [µM]

Ciclopirox L1 1.0 µM 2.84 5 7.2%

Gemcitabine L2 89 µM 0.06 5 8.4%

Trifluridine L3 1% eye drops 0.84 5 6.4%

EC50 Applied Spread Code Clinical use Cytotoxicity [µM] EC90 [µM]

Amethopterin S1 1.3 µM 1.20 25 9.3%

Colchicine S2 1.0 µM 0.05 25 11%

Pinaverium S3 16 µM 20.0 50 12% bromide

Zardaverine S4 n/a 0.01 5 10%

5 µM – 250 Progesterone S5 3.24 25 11% µM*

Norgestimate S6 n/a** 7.04 25 11%

15 µM*** Testosterone S7 3.45 25 9.0% 0.75 µM****

Table 3.1. Confirmed hits. Early, Late, and Spread hits identified by the primary screen and confirmed by the secondary screen. Applied EC90 was established by calculating the EC90 value and choosing a convenient drug dilution higher than the EC90. Cytotoxicity was measured at applied EC90.

* natural concentration in females, depending on menstrual cycle/pregnancy

** rapidly metabolised (Kuhl 2005); *** natural concentration in males; **** concentration in females

Sources for clinical use:

E2 - (Woodward 1990); E3 - (Kobylinski, Foy, and Richardson 2012); E6 - (Liston and Davis 2017); C8 - (2017); E7 – (Avramis et al.1998) ; E8 - (Liston and Davis 2017); C10 (Karjalainen and Ojala 1975); E9 - (Liston and Davis 2017)

L1 - (Minden et al. 2014); L2 - (Liston and Davis 2017); L3 - (Wilhelmus 2015)

S1 - (Liston and Davis 2017); S2 - (Ben-Chetrit, Bergmann, and Sood 2006); S3 - (Ren et al. 2011)

3.3 Cytotoxicity

Figure 3.5. Cytotoxicity of confirmed hits. Cell death was measured using a lactate dehydrogenase assay (LDH), and staurosporine was used as cell death control. Cells were incubated in medium with compounds at their EC90 for 24h. A portion of medium was then used to analyse the amount of LDH released due to by cell membrane breakdown. Raw signal intensity was min-max normalized to the highest (10 µM) staurosporine (1.0) reading and DMSO control (0).

Means of 3 independent experiments ± s.d. are shown.

Even though the screened compounds are FDA approved drugs with known toxicological data, I needed to make sure that at the level of cell culture the concentrations that effectively block VACV are not toxic. A cell death assay using lactate dehydrogenase (LDH) release was used to establish cytotoxicity. LDH is an enzyme present in the cytosol and is only released into the cell culture medium when the structural integrity of the plasma membrane is breached. This extracellular LDH can then be quantified using series of enzymatic reactions that result in red formazan production directly proportional to the amount of LDH released. The formazan fluorescence can then be quantified as an indicator of toxicity using a plate reader. Staurosporine, an inhibitor of protein kinases which causes cell death via apoptosis, was used as a positive cell death control, and DMSO was used as a negative control.

In this cytotoxic assay, HeLa cells were incubated in DMEM with compounds at their EC90 for 24h. As seen in Fig. 3.5, most of the hits were not toxic at their EC90. There were some

exceptions within the Early compounds, specifically aminacrine (E1), avermectin (E3), benzethonium chloride (E4), chlorhexidine (5), and daunorubicin (E6).

Each class of hits encompasses multiple stages of the replication cycle. In the following sections I am going to discuss results of experiments that employed targeted assays with the aim of elucidating and narrowing down the particular stage at which each compound inhibits the VACV replication cycle.

3.4 Early hits

Figure 3.6. Early gene expression block by CHX.

VACV early genes are transcribed within viral cores and translated on host ribosomes, as described in the introduction. Thus, the Early hits must block infection prior to or at the level of early protein translation (Fig.3.6). This means the drug acts at one of the following steps:

• direct effect on virus particle • binding to cell surface • internalization into the cell • fusion with the endosome and entry into the cytoplasm • core activation • early gene transcription and translation

CHX, which was used as an Early control and is described in 1.3.2, is part of the Prestwick Library and was one of the Early hits. The expression of early genes is followed by genome uncoating marked by the appearance of pre-replication sites that consist of the unpackaged viral genomes and associated protein – if those are detectable, a compound is no longer an Early hit.

A brief description of the Early hits follows:

Aminacrine (9-Aminoacridine; 9AA) is an DNA and RNA intercalator (Sakore et al. 1977). The antiviral potential of acridones has been investigated and the range of viruses inhibited by this compound is very broad e.g. adenovirus, CMV, HSV, HCV, DENV (Sepulveda et al. 2013),

as well as encephalomyocarditis virus and poliovirus (Gasparian et al. 2010) and bovine viral diarrhea virus (Loddo et al. 2018). It was also identified as a hit in a screen for EBOV protein VP35 inhibitors (Luthra et al. 2017).

Astemizole is an H1-histamine receptor antagonist (Vanwauwe et al. 1981, Richards et al. 1984), that was identified as a potential antimalarial (Chong et al. 2006) and antiprion agent (Karapetyan et al. 2013). It has also been suggested to have an effect on cellular calcium levels (Sauve et al. 1991, Ariasmontano and Young 1993). Astemizole was identified as a hit in a screen against MERS-CoV (Dyall et al. 2014).

The discovery of avermectin as an anti-parasitic drug was awarded with a Noble prize in 2015 (Nobelprize.org 2015). In invertebrates it targets the glutamate-gated chloride channels (Yates and Wolstenholme 2004). Avermectin has recently been identified as an inhibitor of Porcine Sapovirus (Ohba et al. 2017), Chikungunya Virus (Varghese et al. 2016), Zika Virus (Barrows et al. 2016), Venezuelan Equine Encephalitis Virus (VEEV) (Lundberg et al. 2013), and several flaviviruses (Mastrangelo et al. 2012).

Benzethonium chloride is a synthetic quaternary ammonium salt and is used as a general topical disinfectant. It has known antimicrobial properties (Kawana et al. 1997), and it has also been identified as a potential anticancer drug (Yip et al. 2006). To date, there is no mention of antiviral activity in the literature.

Chlorhexidine, similarly to benzethonium chloride, is an antibacterial disinfectant, also used in dentistry and in mouth washes (Elkerbout et al. 2018). It has been shown to inhibit the replication and cytolytic activity of herpes virus (Park and Park 1989) and HIV (Montefiori et al. 1990).

Daunorubicin, also known as daunomycin, is a DNA intercalator that blocks transcription, and is often used in combination with AraC for the treatment of acute myeloid leukemia and Kaposi’s sarcoma (Chen, Fathi, and Brunner 2018). It was a hit in a screen developed to find drugs that either inhibit or evade VP35 IFN-antagonist function in Ebola infection (Luthra et al. 2017, Kouznetsova et al. 2014).

Fludarabine is a prodrug to purine analogue, F-ara-A (9-β-D-arabinosyl-2-fluoroadenine), which can affect DNA synthesis and translation: inhibition of ribonucleotide reductase, incorporation into DNA causing a repression of DNA polymerisation, and inhibition of DNA primase and DNA ligase. It is used in treatment of acute leukaemia and other cancers. (Gandhi and Plunkett 2002, Burke, Borland, and Litosh 2016). It was also shown to affect STAT1 (Frank, Mahajan, and Ritz 1999). Fludarabine, in combination with azidothymidine, has been shown to inhibit murine AIDS in mice (Fraternale et al. 2000).

Gefitinib (Iressa) is a epidermal growth factor receptor (EGFR) inhibitor, and is predominantly used in treatment of brain, breast, colorectal, and lung cancers (Penne et al. 2005). It has been shown to inhibit entry and spread of VACV (Mercer et al. 2010a, Langhammer et al. 2011). Gefitinib has also been shown to have an inhibitory effect on the DENV (Duran et al. 2017).

Raloxifene is a selective estrogen receptor modulator (SERM), and treatment with raloxifene has been shown to have beneficial effect on bone strength, cholesterol levels, and breast cancer risk (Seeman 2001, Thiebaud and Secrest 2001). It has also been shown to inhibit infection and replication of HCV (Takeda et al. 2012, Murakami et al. 2013) and EBOV (Kouznetsova et al. 2014).

3.4.1 Cardenolides

Three of the Early hits were cardenolides (Digitoxigenin, Digoxigenin, Digoxin) and two were bufadienolides (Proscillaridin and Lanatoside C). Both groups are a type of cardiac glycosides, but for simplicity they will be collectively referred to as Cardenolides. Cardenolides have been used as a treatment for heart issues even before modern medicine found out their mechanism of action (Schoner and Scheiner-Bobis 2007, Babula et al. 2013), which is the inhibition of sodium-potassium pump (Na+K+ ATPase). This prevents sodium from exiting the cell, leading to an increase in intracellular calcium. Specifically digoxin has been the first choice for heart failure, however this has changed after it was discovered that it had an

Figure 3.7. Cardenolides general structure.

associated mortality risk (Vamos, Erath, and Hohnloser 2015). All cardenolides share the structural backbone of a steroid (Fig. 3.7), with variation on their side chains.

Previous research has demonstrated that these compounds have very broad antiviral properties, and some cardenolides have been shown to inhibit VACV. Table 3.2 lists inhibited viruses together with corresponding cardenolides and relevant references. 9 additional Cardenolides were obtained and analysed using the secondary, flow cytometry assay following the same procedure as initially identified compounds. The new compounds have all shown to inhibit VACV at an early stage in a dose dependent manner. Thus, a new sub- class of the Early class was created, with 14 Cardenolides (Table 3.3), leaving 9 Early hits.

Included in Compound Virus inhibited References this project Convallatoxin HBV, HIV, HCMV (Okuyama-Dobashi et al. 2015), (Laird et al. 2014), (Gardner et al. 2015) Yes Cymarine VACV, HIV (Deng et al. 2007), (Laird et al. 2014) Yes (Su et al. 2008), (Cheung et al. 2014),(Dodson et al. 2007), (Okuyama-Dobashi Digitoxin HSV, DENV, HIV, HCMV Yes et al. 2015), (Laird et al. 2014), (Kapoor et al. 2012), Digitoxigenin HSV (Su et al. 2008) Yes Digoxigenin HSV, HIV (Su et al. 2008), (Laird et al. 2014) Yes HSV, VZV, HCMV, (Su et al. 2008), (Hartley et al. 2006), (Cheung et al. 2014), (Dodson et al. 2007), Adenovirus, DENV, VACV, (Okuyama-Dobashi et al. 2015), (Deng et al. 2007), (Zhyvoloup et al. 2017, Digoxin Yes HIV, HCMV, CHIKV, ZIKV, Laird et al. 2014), (Kapoor et al. 2012), (Ashbrook et al. 2016), (Wong et al. EBOV 2013), (Barrows et al. 2016), (Kouznetsova et al. 2014) Gitoxin HIV (Laird et al. 2014) Yes Gitoxigenin HSV, HIV (Su et al. 2008), (Laird et al. 2014) No Lanatoside C DENV, VACV (Cheung et al. 2014), (Deng et al. 2007) Yes Neriifolin VACV (Deng et al. 2007) Yes (Su et al. 2008), (Dodson et al. 2007), (Deng et al. 2007), (Laird et al. 2014), Ouabain HSV, VACV, HIV, HCMV Yes (Kapoor et al. 2012) Ouabagenin HSV (Dodson et al. 2007) No

Periplocymarin HIV (Laird et al. 2014) No Sarmentogenin HIV (Laird et al. 2014) Yes Strophanthins HSV (Su et al. 2008), (Dodson et al. 2007) No Strophanthidol HSV (Su et al. 2008) No Strophanthidin HIV (Laird et al. 2014) Yes Bufalin DENV, HBV, HIV (Cheung et al. 2014), (Okuyama-Dobashi et al. 2015), (Laird et al. 2014) Yes Proscillaridin A HBV (Okuyama-Dobashi et al. 2015) Yes

Table 3.2. Cardenolides in the literature.

EC50 Cardenolides Code Applied EC90 [µM] Cytotoxicity [µM]

Bufalin C1 0.06 10 13%

Cinobufagin C2 0.12 10 11%

Convallatoxin C3 0.04 10 9.3%

Cymarine C4 0.11 10 9.3%

Digitoxigenin C5 0.64 10 9.8%

Digitoxin C6 0.14 10 10%

Digoxigenin C7 3.00 10 9.9%

Digoxin C8 0.46 10 11%

Gitoxin C9 4.70 10 8.7%

Lanatoside C C10 0.63 10 10%

Neriifolin C11 0.07 10 10%

Ouabain C12 0.10 10 10%

Proscillaridin A C13 0.01 10 10%

Sarmentogenin C14 1.30 10 9.4%

Table 3.3. Cardenolides confirmed by flow cytometry. Cardenolides identified in the literature were examined using the secondary flow- cytometry based screen, following the same procedure as Early hits. Applied EC90 was established by calculating the EC90 value and choosing a convenient drug dilution higher than the EC90. Cytotoxicity was measured at applied EC90 (Fig. 3.6). Most Cardenolides are not in clinical use.

3.4.2 Direct effect on virus particle

Certain compounds, termed virucidals, act by directly inactivating virus particles. Detergents, for example prevent viral infection by disrupting or extracting the lipid from the envelopes of enveloped viruses. To determine if the identified Early inhibitors have virucidal activity VACV E-EGFP in DMEM was preincubated with compounds at their EC90 for 60 min. The virus and inhibitor containing medium was then diluted 100-fold, reducing the concentration of the inhibitors to below their minimal effect concentration. These dilutions were then used to infect HeLa cells. At 6hpi, cells were harvested, and infection was analysed by flow cytometry (Fig. 3.8). For these assays, 70% ethanol, which destroys the VACV lipid membrane (Rabenau, Rapp, and Steinmann 2010) was used as a positive control for virucidal effects. In the absence of inhibitors ~60% of cells in the population were infected. Ethanol, as expected, effectively killed VACV particles and prevented VACV infection. Analysis of the % of cells infected by viruses that had gone through pre-treatment with the various compounds indicated that none of the early hits possessed significant direct virucidal activity.

Figure 3.8. Direct effect of Early hits on virus particle. VACV E-EGFP was incubated with Early inhibitors at their EC90 for 60 min. The virus was then diluted 100-fold, to ensure the compounds were below their effective concentration, and used to infect HeLa cells. After 6h cells were harvested and analysed with flow cytometry. 70% ethanol was used as positive control. Dashed line shows DMSO expression levels. **** p < 0.001

Means of 3 independent experiments ± s.d. are shown.

3.4.3 Binding to cell surface

Since previous experiment showed that the viral particles were not defective after being exposed to hit compounds, the next move was to assess the impact of the Early inhibitors on virus binding. It has been shown that at 4˚C VACV can bind to cells, but entry is virtually blocked (Locker et al. 2000).

Initially, this was tested by incubating various dilutions of VACV A5-EGFP (EGFP-labelled core protein, with strong fluorescent signal) with cells, using the protocol described below. There was a demonstrable difference between cells where no virus was added compared to samples with virus (Fig. 3.9). There was also a direct relationship between the amount of virus added and mean fluorescence intensity detected.

For this assay, cells were first preincubated with drug-containing medium at 4˚C for 15 min. VACV A5-EGFP was added at MOI of 50 and allowed for binding at 4˚C for 60 min. After binding, cells were washed thoroughly to remove any unbound virus, scraped within the wells and analysed by flow cytometry to detect the fluorescent viral particles bound to cells (Fig 3.10). There was a significant difference in the mean fluorescence intensity between the negative “no virus” control and positive “no drug” controls. Analysis of the various Early inhibitors and Cardiac glycosides showed no significant drop in fluorescence intensity compared to the “no drug” control. From this, it was concluded that none of the inhibitors work by preventing virus-binding to host cells.

Figure 3.9. Binding assay test. VACV A5-EGFP 2-fold serial dilution starting at MOI 100 was incubated with HeLa cells at 4˚C for 60min. Cells were then washed and scraped within their wells and analysed via flow cytometry. There was a significant difference between the No Virus control and the samples containing virus, with a dilution-dependent drop-off.

Means of 5 technical repeats from a single experiment ± s.d. are shown.

Figure 3.10. VACV binding to cells. VACV A5-EGFP at MOI 50 was incubated with HeLa cells at 4˚C for 60 min. After binding wells were thoroughly washed with PBS. Cells were detached by scraping and analysed with flow cytometry. Data was normalised, with DMSO = 1 and No Virus = 0. No significant differences were observed for any of the hits.

Means of 4 independent experiments ± s.d. are shown.

3.4.4 Acid bypass of internalization and fusion

The initial and confirmatory screens in combination with the above assays indicated that all of the Early hits and Cardenolides block VACV infection after virus binding but before early gene expression. As VACV uses macropinocytosis as a major route of cell entry (Mercer and Helenius 2008), these results made it likely that the early inhibitors were blocking VACV macropinocytosis or fusion. To assess the impact of these inhibitors on VACV entry an acid bypass assay was used. This assay is designed to bypass virus endocytosis by dropping pH to force virus fusion with the plasma membrane. By depositing viral cores directly into the cytosol, it can be assumed that if an inhibitor can be bypassed it must block infection at the level of endocytosis or fusion, and if it cannot be bypassed then the inhibition must occur after fusion is completed (Fig. 3.11).

This assay was used to further define what stage of the VACV replication cycle was blocked by the identified Early hits and Cardenolides. First VACV E-EGFP was bound to cells in DMEM pH 7.0 at 4˚C for 30 min. The medium was then replaced with DMEM pH 5.0 at 37˚C for 10 min to allow for fusion of virus to plasma membrane. In no-fusion control medium was

Figure 3.11. VACV acid bypass. By briefly dropping the pH of media from pH 7.0 to pH 5.0 viral fusion at the plasma is induced, causing deposition of viral cores directly into the host cytosol. If a compound blocks the internalization or endosomal fusion of the virus particle, this pH drop will bypass those steps, circumventing the inhibition. replaced with DMEM pH 7.0. This was followed by once more changing to DMEM pH 7.0 for 6h at 37˚C. All steps were performed in the presence of the various compounds at their EC90. After the final incubation, samples were harvested and analysed by fluorescent flow cytometry. CHX was used as negative bypass control and EIPA, IPA3, and staurosporine were used as positive controls, since these drugs inhibit fusion (Mercer et al. 2010). As expected, the inhibitory effect of CHX was not bypassed. For compounds that inhibit the endocytic pathway, recovery of infection at pH 5.0 relative to pH 7.0 would be expected, and can be seen in the results for EIPA, IPA3, and staurosporine. The results indicate that acid bypass was only possible in the presence of daunorubicin (E6) and raloxifene (E9), as well as all the 14 cardenolides (C1-C14) (Fig. 3.12). Being sodium-potassium pump inhibitors, it is likely that they might affect the endocytic pathway. The data suggests that all the other hits block early gene expression at a post-fusion step, as the infection was still strongly inhibited despite the pH drop.

However, when VACV L-EGFP, that is a virus expressing EGFP under a late gene promoter, was used as an additional control Cardenolides no longer allowed for infection recovery (data not shown). This pointed to a secondary block, occurring after early gene expression.

Figure 3.12. Acid bypass. VACV E-EGFP or L-EGFP at MOI 10 was bound to HeLa cells in DMEM pH 7.0 at room temperature for 30 min. Medium was then replaced with DMEM pH 5.0 at 37˚C for 10 min to allow for fusion of viral envelope with the plasma, resulting in a direct deposition of viral cores into the cell cytosol. In the no-fusion (pH 7.0) controls medium was not replaced. All steps were performed in the presence of the various compounds at their EC90. EGFP expression was measured using flow cytometry, and the difference between expression of fusion – no-fusion was analysed. * p < 0.05, ** p < 0.01, ***p < 0.005, **** p < 0.001.

Means of 3 independent experiments ± s.d. are shown.

Figure split in two for clarity. Experiments were performed simultaneously for all Early and Cardenolides hits and controls.

3.4.5 AraC release – secondary, Late inhibition?

The acid bypass assay demonstrated that inhibition of early gene expression by Cardenolides could be bypassed, however late gene expression was not. This suggests that Cardenolides also inhibit late gene expression, independently from and at a step after early gene expression. To further confirm this finding an assay where the compounds are added after early gene expression has occurred was required. As described in section 1.3.2, AraC blocks DNA replication after genome uncoating, allowing for early gene expression while blocking late gene expression. When AraC is washed out, the replication cycle is “released” and resumes as normal.

For this assay cells were infected with VACV E-EGFP L-mCherry in the presence of AraC. After 3h at 37˚C, cells were washed thoroughly to ensure all AraC was removed, and medium with compounds at their EC90 was added for 6h at 37˚C. This allowed for early gene expression but blocked the viral lifecycle before genome replication. This way Early hits were added after early gene expression, but before late gene expression. Samples were analysed for fluorescence using flow cytometry (Fig. 3.13). Results demonstrated that with DMSO early

Figure 3.13. AraC release: independent Late inhibition. HeLa cells were infected with VACV E-EGFP L-mCherry at MOI 10 for 3h at 37 ˚C in presence of AraC. Wells were then washed five times with DMEM, to remove AraC, followed by addition of DMEM with compounds at their EC90 for additional 6h at 37 ˚C. Cells were then harvested and analysed with fluorescent flow cytometry to determine the percentage of cells expressing EGFP and mCherry. Due to the variability of gene expression this experiment was used only to determine the trends.

Means of 3 independent experiments ± s.d. are shown.

and late gene were expressed as normal. CHX blocked both early and late gene expression, while AraC blocked only late gene expression, with early genes expression measured at a level identical to DMSO. Cardenolides showed strongly reduced late gene expression, without a strong inhibition of early genes. Aminacrine (E1), benzethonium chloride (E4), daunorubicin (E6), and fludarabine (E7) all inhibited late gene expression. Astemizole (E2), chlorhexidine (E5), and gefitinib (E8) had somewhat intermediate effect, while avermectin (E3) and raloxifene (E9) did not block late gene expression. Due to the variability of the results this experiment was used just to identify the trends of gene expression. A 24h yield experiment was done to find any significant changes in virus titres in presence and absence of AraC.

AraC release – 24h yield.

As an alternative to the use of fluorescent viruses, a standard 24 yield experiment can be used to identify viral inhibition in the presence or absence of compounds. Briefly, cells were infected with VACV WT in presence or absence of AraC for 3h, analogous to section 3.4.5. Following the media exchange infection was allowed to progress for 24h at 37˚C. After 24h cells were scraped in media and a used to infect monolayers of BSC-40 cells, following a titration. Plaques were counted and a 24h yield was calculated. Due to their similarity, only 5 representative Cardenolides were used.

In absence of AraC all early hits significantly reduced the number of plaque forming units after 24h infection (Fig. 3.14A). Compounds E1, E2, E4, E6, E7, and Cardenolides reduced the viral by 3 log or more, some being as efficient as CHX. Others, like E3, E5, E8, and E9 were less efficient, but still reducing the pfu/mL by at least 1 log compared to DMSO and BFA controls.

When compounds were added after the AraC wash out, which allowed for early gene expression in infected cells, the results of the previous experiment were expanded and more accurately quantified (Fig. 3.14B). Specifically, Aminacrine (E1), astemizole (E2), benzethonium chloride (E4), daunorubicin (E6), and fludarabine (E7) significantly reduced VACV 24h yield, while avermectin (E3), chlorhexidine (E5), gefitinib (E8), and raloxifene (E9) did not. Additionally, Cardenolides still strongly inhibited VACV replication, despite being added after early gene expression.

This suggests that all the hits able to block VACV replication following AraC release have two stages of inhibition, one before early gene expression, and one after. This could be due to a single mechanism that has a more “global” effect, or two mechanisms that act on different part of the viral replication. Since 14 Cardenolides have been identified, we decided to further investigate those hits and their mechanism of action. 83

Figure 3.14. AraC release, 24h yield. A) Hela cells were infected with MOI 1 of VACV WT, in presence of Early hits and controls. After 24h supernatant and cells were collected for total virus yield. 24h yield was determined by titre as previously described. B) Cells were infected with VACV WT in presence of AraC, allowing for early gene expression, but preventing late gene expression by blocking viral DNA replication. After 3h AraC was removed by washing, and fresh media containing Early hits was added. Infection was allowed to continue for 24h before being analysed as described in A. * p > 0.5

Means of 3 independent experiments ± s.d. are shown.

3.4.6 Is inhibition by Cardenolides ATPase dependent?

As previously described, Cardenolides are known sodium-potassium ATPase inhibitors, and that mechanism underlines their use a heart medication. To evaluate if the same mechanism 84

is responsible for their action as VACV inhibitors, a way to circumvent the ATPase inhibition was required. Previous research demonstrated that digoxin can inhibit HIV in Jerkat cells, but not when they are expressing mouse Na+/K+ ATPase (mATPase), which is insensitive to Cardenolides (Zhyvoloup et al, 2017). Therefore, a HEK 293T cell line (HEK) stably expressing mATPase (kind gift from Prof Fassati) was used to test the hypothesis that the inhibition of VACV infection by Cardenolides is dependent on their known mechanism of action.

Briefly, HEK cells stably expressing mATPase (HEK mATPase) and their parent HEK cell line stably expressing an empty vector (HEK) were infected with VACV E-EGFP or L-EGFP at MOI 10 for 60 min at RT. The inoculant was then removed, and media containing DMSO control, or serial dilutions of CHX or Cardenolides was added. Infection was allowed to progress for 16h at 37˚C. Samples were then analysed via flow cytometry to count cells expressing EGFP (infected cells).

The results depicted in figure 3.15 indicate that both HEK and HEK mATPase can be infected with VACV E-EGFP and L-EGFP, and that green fluorescence can be used to quantify the inhibitory effect of CHX. VACV Early gene expression was inhibited by Cardenolides in HEK, but not in HEK mATPase cells (with the exception of Gitoxin). The level of inhibition was much less pronounced than in HeLa cells used previously in this thesis. It is possible that HEK cell line is less sensitive to Cardenolides, or that the inhibitory effect of Cardenolides isn’t as pronounced. Unfortunately, due to time constraints it was impossible to perform extensive testing of Effective Concentrations (EC50 and EC90) to determine the optimal drug concentration or toxicity. Instead a serial dilution starting at 25 µM and ending at 0.008 µM was used (in HeLa cells the applied EC90 was 10 µM). In addition, the error bars (S.D.) are quite large, leading to a lack of statistical significance. This was caused by the large variance between infection levels in the biological repeats, despite a clear trend of inhibition (where present). This is likely due to the biological repeats being performed on different days, with fresh dilutions of virus, fresh dilutions of compounds, and freshly seeded cells. Nevertheless, comparing the result for Gitoxin, a Cardenolides that has been shown to have a relatively low inhibitory effect on VACV (EC50 tenfold higher than most Cardenolides tested), where there was no difference between the two cells lines in VACV E-EGFP, and other Cardenolides, where there was a difference, indicates that ATPase inhibition is required for early gene expression.

Moreover, all the Cardenolides had a clear inhibitory effect on VACV L-EGFP in both cell lines. This indicates that ATPase is not necessary for late gene expression inhibition. The variation in the levels of infection between the two cells lines potentially supports the notion that ATPase plays role in Early inhibition. When early expression is inhibited, there are two stages

where the virus replication is inhibited: Early and late. Without early inhibition there is only the Late block, resulting in somewhat higher infection levels. This results also support the hypothesis that Cardenolides have two independent inhibitory steps, as shown by the AraC release experiments.

Figure 3.15. VACV Cardenolide-dependent inhibition in HEK cells expressing mouse Na+/K+ ATPase. HEK cells stably expressing an empty vector or mouse Na+/K+ ATPase (mATPase) were infected with VACV E-EGFP or L-EGFP at MOI 10 for 16h in presence of serially diluted compounds. CHX: 100 µM, 20 µM, 4 µM, 0.8 µM, 0.16 µM, 0.032 µM. Cardenolides: 25

µM, 5 µM, 1 µM, 0.2 µM, 0.04 µM, 0.008 µM. DMSO was used at the highest concentration found in drug dilutions. Flow cytometry results were normalised to DMSO = 1. Gitoxin has previously been shown to have lower inhibitory effect on VACV, and was used as a negative control. Four representative Cardenolides are shown here; all results can be found in the Supplement, figure S.6.2.

Means of 3 independent experiments ± s.d. are shown.

3.4.7 Microscopy of VACV binding, internalization, and fusion.

To confirm these results with a different approach, a microscopy-based virus entry assay was set up. As all Cardenolides have so far given comparable results, an assumption was made that they all work via the same mechanism. Because the microscopy assay is not as high- throughput as the cytometry-based assays, only four Cardenolides were selected for imaging: bufalin (C1), digitoxin (C6), digoxin (C8), and ouabain (C12).

For this assay, cells were infected with recombinant VACV that packages a mCherry-tagged core protein (A5) and an EGFP-tagged lateral body protein (F17) (Schmidt et al. 2013). Infected cells were left unpermeabilized, and immuno-stained for the virion membrane protein L1. Using this assay should have made it possible to distinguish between bound uninternalized virions (3-colour colocalization); virions that were internalized and thus protected from L1-antibody staining, but did not yet fuse with the endosome leaving the LBs still attached (2-colour colocalization); and virions that have internalized and have undergone fusion, depositing the mCherry core into the cytoplasm (red only, no colocalization). Unfortunately, the variability in the experiment caused the results to lack any significance. This could have been caused by the software being unable to distinguish and correctly count the particles. Possibly caused by inadequate antibody binding and low fluorescence intensity. Alternatively, the number of particles attaching to cells in each experiment might been fluctuating too much.

To simplify the experiment, a simpler 2-colour assay using a recombinant virus that packages an EGFP-tagged core protein (A5) was employed. Cells were infected with A5-EGFP virus and the unpermeabilized cells stained for L1 to differentiate between virions that failed to internalize from the ones that did (Fig. 3.16 A). Cores were localized and quantified using an ImageJ plugin (Fig. 3.16 B), and a ratio of all viral cores and cores colocalizing with L1-antibody was calculated (Fig. 3.16C).

Aminacrine (E1), daunorubicin (E6), and fludarabine (E7) can be identified with strong confidence as compounds that block core activation but not viral entry. Remaining hits appear to block viral internalization to a certain degree, however none of them is as potent as the entry control ethyl-isopropyl amiloride (EIPA). This could indicate that the macropinocytosis/endocytic pathway is inhibited, but most of the inhibitory effect takes place after internalization, leaving only some virus particles on the cell surface.

Figure 3.16. Microscopy-based entry assay. VACV A5-EGFP at MOI 30 was added to HeLa cells for 4h in the presence of controls or Early hits at their EC90. After fixation samples were labelled with L1-antibody, Hoechst, and Phalloidin without permeabilization and imaged. (A) shows controls, 100 µM CHX for core stabilization, where most virions enter the cell but are not activated, and 60 µM EIPA for entry block, where most virions remain outside of the cell. (B) Quantification of imaging was done using ZedMate by spot tracking using EGFP signal to localise virions and counting L1- positive colocalizations. “Inside cell” – A5, green; “Outside cell” – A5+L1, yellow. (C) A ratio of A5+L1/A5 tracks was calculated. Results significantly different from EIPA marked ** p < 0.01; * p < 0.05. Average values of two independent experiments (10 images/condition) ± s.d. are shown.

3.4.8 Early hits – conclusions

Based on the results obtained so far, the inhibition stage for the Early hits has been narrowed down for most of the hits. What can be said with high certainty is that none of the hits had a direct virucidal effect on the virus particle, and they did not affect binding of virions to the cells. Interestingly, it appeared that some hits had a secondary, late inhibition effect (Table 3.4).

Aminacrine (E1) blocks the core activation stage, as the microscopy assay gave results very similar to CHX and acid bypass did not rescue infection. Since aminacrine is a DNA intercalator, it is not surprising that it would also block late gene expression.

Infection in the presence of astemizole (E2) could not be bypassed, and the microscopy entry assay indicated an internalization block. These results suggest that astemizole (E2) somehow affects VACV infection at the plasma membrane. How this compound impacts both fusion and uptake needs further investigation.

Avermectin (E3) consistently appeared as an entry/internalization block, with an infection rescue after the acid bypass and high ratio of particles located on cell surface. It can be said that it blocks at the internalization/fusion step.

Benzethonium chloride (E4) and Chlorhexidine (E5), in the experiments that did work, appear as internalization/fusion blockers. However, if further optimization does not help with the cytotoxicity, these hits might need to be removed because of this issue.

Daunorubicin (E6) and fludarabine (E7) were both very clear blockers of core activation, being unaffected by acid bypass and showing a high ratio of internalized virions. Both compounds also blocked late gene expression in the AraC release experiments, which is consistent with daunorubicin being a DNA binding molecule, and fludarabine being a nucleotide analogue.

Gefitinib (E8) presented an unexpected acid bypass result, considering it has been previously shown to inhibit entry (Mercer et al. 2010a); here there was no recovery. However, the fluorescence microscopy seems to confirm gefitinib as an entry blocker. Hopefully with further repeats of the acid bypass this can be resolved.

Raloxifene (E9) has shown some infection recovery in the acid bypass, and the microscopy assay also classifies it as a likely internalization/fusion block.

Cardenolides (C1-C14) that have been identified all appear to work in the same way, except for gitoxin (C9), which seems to have either a much higher EC90 than other Cardenolides or is simply not as efficient in blocking. Other compounds in this group however, have shown a clear internalization block, as the infection recovery after the acid bypass was clearly visible

with an early EGFP expressing VACV, a result that was supported by the microscopy assay. Interestingly, they have all shown a secondary late gene expression block, as seen in the AraC release experiment and the mechanism of this step appears to be different than the early block, as discovered in the HEK mATPase experiment. This group of compounds will certainly be the target of further investigation with the goal of clearly understanding the mechanism of both the early and late blocks.

Blocks Block bypassed Core Late Compound Virucidal? binding? by pH drop? deposited? inhibition?

E1 Aminacrine

E2 Astemizole

E3 Avermectin

E4 Benzethonium Cl n/a

E5 Chlorhexidine n/a

E6 Daunorubicin

E7 Fludarabine

E8 Gefitinib

E9 Raloxifene

Cardenolides

Table 3.4. Early hits summary table. Orange – No. Green – Yes. Light green – inconclusive, but likely Yes. n/a – cytotoxicity issues during the experiment.

3.5 Late hits

Figure 3.17. Late gene expression block by AraC.

The secondary screen has confirmed three inhibitors of VACV late gene expression that did not block early gene expression. Given the strict temporal nature of the VACV lifecycle (Fig. 3.17) the block in late gene expression could be due to

• a defect in genome uncoating • DNA replication • intermediate gene expression • late gene expression

As a reminder, the three late inhibitors were ciclopirox olamine (L1), gemcitabine (L2), and trifluridine (L3) (Fig. 3.18). The EC50 values for the compounds were 2.84 µM, 006. µM, and 0.84 µM, respectively.

Ciclopirox olamine is a topical, synthetic antifungal drug shown to reduce HIV replication in human peripheral blood mononuclear cells (Hoque et al. 2009) and HSV-1 shedding in mice (Bernier and Morrison 2018). HIV inhibition occurs at the level translation. It is not yet known by what mechanism ciclopirox reduces HSV-1 replication.

Gemcitabine and trifluridine are both nucleoside analogues, that are known to interfere with DNA replication when they are incorporated into a newly synthesized strand, thereby blocking further transcription (Plunkett et al. 1995). They are both used as anti-cancer drugs (Gesto et al. 2012, Lenz, Stintzing, and Loupakis 2015) and have been employed as antivirals. Gemcitabine was identified as an inhibitor of at least 13 different viruses, most of which are ssRNA viruses. These include MERS-CoV, SARS-CoV, ZIKA, HCV, Poliovirus, Influenza A virus, HIV, MuLV, CVB3, EV71, HRV, and SINV. Gemcitabine was also reported to be effective against a dsDNA virus, HSV-1 (Shin, Kim, and Cho 2018). Trifluridine was shown to be effective

Figure 3.18. Late gene inhibition. Results of the secondary screen. Hela cells were infected with VACV L-EGFP MOI 10 for 16h in presence of Late compounds at various dilutions; Ciclopirox (L1), Gemcitabine (L2), and Trifluridine (L3). Cells were then analysed for EGFP fluorescence by flow cytometry, and values were normalized to expression at lowest drug concentration (=no inhibition).

Means of 3 independent experiments ± s.d. are shown. in treating HSV-1 keratitis (Roozbahani and Hammersmith 2018) and complications of the smallpox vaccine if spread to the eyes (Pepose et al. 2003).

3.5.1 Intermediate gene expression

VACV intermediate genes encode VACV late gene transcription factors and as such are required for late gene expression (Yang et al. 2013). Since the primary and secondary screen have confirmed late gene inhibition, the next step was to test for intermediate gene expression, to determine if the compounds were specifically targeting late gene expression or intermediate transcription and/or translation.

To test this an experiment following the same scheme as the secondary screen was performed. Briefly, HeLa cells were infected with VACV I-EGFP, a mutant expressing EGFP under the control of VACV intermediate promoter, in presence of Late compounds at 37˚C. 16 hpi cells were fixed and analysed by flow cytometry for EGFP fluorescence. The result was a dose-dependent inhibition of EGFP expression for all three drugs, with EC50 values of 18.5 µM (ciclopirox), 0.220 µM (gemcitabine), and 39.1 µM (trifluridine) (Fig. 3.19). These values are more than 10-fold higher than seen for inhibition of Late gene expression. This is likely explained by the fact that while DNA replication is, to a degree, necessary for intermediate gene expression, it is the early proteins that act as intermediate transcription factors. Since early genes were successfully expressed, it is possible that some intermediate gene

Figure 3.19. Intermediate gene inhibition. Hela cells were infected with VACV I-EGFP (EGFP under a VACV intermediate promoter) at MOI 10 for 16h in presence of Late compounds at various dilutions; ciclopirox (L1), gemcitabine (L2), and trifluridine (L3). Cells were then analysed for EGFP fluorescence by flow cytometry, and values were normalized to expression at lowest drug concentration (=no inhibition).

Means of 3 independent experiments ± s.d. are shown. expression will inevitably occur. None-the-less, that intermediate genes were blocked points to an earlier stage of the virus lifecycle as the target of these compounds.

3.5.2 Pre-replication sites and replication sites formation

As there appeared to be a defect in both intermediate and late gene expression in presence of each inhibitor, the next logical step was to test whether VACV genomes were release and/or replicated in the presence of the hits. For this HeLa cells were infected with VACV A5- EGFP at MOI 10 in the presence of the compounds and fixed at 8 hpi. Samples were then immuno-stained with antibody against viral protein I3, which binds to the ssDNA found in both pre-replication and replication sites (Rochester and Traktman 1998, Mercer et al. 2012), and analysed using fluorescent microscopy. Pre-replication and replication sites can be differentiated by size: pre-replication sites are seen as multiple small cytoplasmic puncta, while replication sites form large cytoplasmic structures nearly the size of the host nucleus. Cells infected in the presence of DMSO formed large replication sites positive for both I3 and A5-EGFP, which is expressed as a late gene during infection. CHX was used as a negative pre- replication site formation control, as it blocks genome release. AraC was used as a positive pre-replication site formation control, as it does not prevent genome release. The microscopy imaging showed that ciclopirox (L1), gemcitabine (L2), and trifluridine (L3) all

Figure 3.20. Initiation site and replication site formation inhibition. HeLa cells were infected with VACV A5-EGFP at MOI 10 in presence of indicated compounds at their EC90 or controls: 1% DMSO, 100 µM CHX, 20 µM AraC. 8 hpi cells were fixed and stained with I3 antibody, a DNA stain, and phalloidin. I3 binds to ssDNA found in pre-replication and replication sites. Fluorescence imaging revealed that with no inhibition (DMSO), large replication sites are formed where late genes (A5-EGFP) are expressed. Block of genome uncoating (CHX) leaves the cores (A5-EGFP puncta) intact. Inhibition of DNA replication (AraC) results in block at the pre-replication site stage. Images of all three Late hits were comparable to AraC, indicating a DNA replication block. allow for pre-replication site formation, while completely preventing DNA replication site formation (Fig. 3.20). As the effectiveness of the compounds was virtually 100% no quantification was implemented. These results indicate that all three Late hits inhibit VACV replication at the level of DNA replication.

3.5.3 Edu incorporation

To confirm and extend these findings a closer look was given to on-going replication in the presence of these inhibitors using EdU click chemistry (Wang et al. 2013). EdU is a modified thymidine analogue that is efficiently incorporated into newly synthesised DNA. The incorporated EdU is then detected by copper(I)-catalyzed azide-alkyne cyclo-addition

Figure 3.21. EdU incorporation. HeLa cells were infected with VACV WT at MOI 10 in presence of serially diluted indicated compounds and 1 µM EdU. 8 hpi cells were fixed, and EdU was labelled with Alexa Fluor 488-azide using Click chemistry. Image analysis included exclusion of the nuclei and quantification of mean fluorescence of incorporated Edu per cell.

Means of 3 independent experiments ± s.d. are shown. reaction with Alexa Fluor 488-azide. This allows for image-based analysis of DNA replication (Yakimovich et al. 2017).

HeLa cells were infected with VACV WT in the presence of serial diluted ciclopirox, gemcitabine, or trifluridine in DMEM with 1 µM Edu. At 8 hpi samples were fixed, incorporated Edu was labelled with Alexa Fluor 488-azide using click chemistry, and DNA was stained with Hoechst. After imaging on a high-throughput microscope the data was analysed. The nuclei were excluded from the analysis, as the goal was to measure fluorescence within the pre-replication and replication sites. Mean fluorescence per cell was quantified. (Fig. 3.21). As expected, while DMSO had little impact on VACV DNA replication, the positive controls CHX and AraC completely blocked DNA replication preventing the incorporation of EdU. Both ciclopirox and trifluridine reduced the mean fluorescence intensity of EdU incorporation in a dose dependent manner, while gemcitabine was the most potent inhibitor, attenuating the synthesis of VACV DNA at nM concentrations. These results are consistent with previous analysis of intermediate and late gene expression in which gemcitabine was the most effective inhibitor.

3.5.4 Late hits – conclusions

The results obtained indicate that all three Late hits (ciclopirox (L1), gemcitabine (L2), and trifluridine (L3)) inhibit VACV infection after genome uncoating, at the stage of pre-initiation site formation, by blocking DNA replication and subsequent replication site formation.

It is likely that both gemcitabine and trifluridine, which are nucleoside analogues, inhibit the infection by incorporating into newly synthesising viral DNA, analogous to the effect exhibited by AraC. This blocks the VACV lifecycle at the very early stage of DNA replication, right after genome uncoating. Since ciclopirox is not a nucleoside analogue, it must be using a different mechanism than the other two Late hits. This is partially supported by the finding that the effect of gemcitabine and trifluridine tapers slowly as their concentration decreases, which is expected of a small molecule being incorporated into the DNA, as opposed to the activity of ciclopirox which drops quite rapidly supporting an alternative mechanism of inhibition.

3.6 Spread hits

Of the 27 confirmed hits in the screen, 7 affected the spread of Extracellular Virions (EVs), which include Cell-associated Extracellular Virions (CEVs) and Enveloped Extracellular Virions (EEVs), within the first 24h of infection. As the screening readout for spread was secondary infection of cells surrounding the primary infected cell, the steps that could be inhibited include (Fig. 3.22):

• the formation of Intracellular Mature Virions (IMVs) • their transport to the wrapping site • their wrapping and formation of IEVs • egress and release of CEVs and EEVs

Amongst confirmed Spread hits was a previously identified VACV inhibitor, mitoxantrone. Consistent with our results this inhibitor has been shown to have no impact on early or late VACV gene expression, but to affect virus morphology (Deng et al. 2007). Having already been published, this compound was not included in targeted experiments.

The remaining Spread hits include:

Amethopterin (S1), also known as methotrexate, is used as a chemotherapy agent, immunosuppressant, and in treatment of rheumatoid arthritis (Lopez-Olivo et al. 2014). It is an antimetabolite that inhibits dihydrofolate reductase (DHFR), which is an enzyme crucial in the nucleotide synthesis (Rajagopalan et al. 2002). As such, amethopterin inhibits production of RNA, DNA, and proteins. This in turn resulted in inhibition of adenovirus (Wirjawan and Wigand 1978), HSV-1 (Wigand and Hassinger 1980, Prichard, Prichard, and Shipman 1993), both human and murine CMV (Shanley and Debs 1988), and flaviviruses (Fischer et al. 2013)

Colchicine (S2) has been used in gout treatment for more than a 1,000 years, and has recently been applied to dermatology, cardiology, immunology, and oncology (Dasgeb et al. 2018). Colchicine acts by binding to tubulin dimers, and blocking microtubule polymerization (Andreu and Timasheff 1982). As VACV makes use of microtubules throughout the replication

Figure 3.22. Spread block by BFA.

cycle, this is clearly significant to VACV infection. Colchicine has been used in VACV and ASFV studies to demonstrate the viral dependence on the microtubules for directed intracellular movement (Dematos and Carvalho 1993, Carter et al. 2003). As such the drug was used as an internal control during the follow-up assays.

Pinaverium bromide (S3) is a calcium channel blocker that acts selectively on the gastrointestinal tract (Christen and Tassignon 1989), and is used as a treatment for the symptoms of irritable bowel syndrome (Guslandi 1994). No antiviral activity has been directly attributed to pinaverium bromide.

Zardaverine (S4) is a dual-selective PDE3/4 phosphodiesterase inhibitor (Schudt et al. 1991). Its intended use is for bronchodilatation, however clinical results were inconsistent, from high effect to no effect (Brunnee et al. 1992, Ukena et al. 1995). Phosphodiesterases hydrolize cAMP and cGMP; cAMP plays an important role as a secondary messenger in cell signalling, and as such have been considered as targets for novel therapeutics (Lugnier 2006). There is evidence that cAMP levels may affect HIV-1 replication (Nokta and Pollard 1992).

Progesterone (S5) is a sex steroid hormone and the major progestogen in the body. It was found to inhibit orthopox virus in late stages of infection in Vero cells and in live rabbits (Pfahler, Reimann, and Munz 1987). Interestingly, VACV SalF7L (A44L) gene product encodes a protein with 31% identity to 3β-HSD, the enzyme that catalyses the synthesis of progesterone from pregnenolone, and its deletion led to attenuated virulence (Moore and Smith 1992, Reading, Moore, and Smith 2003). It was also found to inhibit HIV-1 replication in placental cells and T cell blasts by reducing TNF (Munoz et al. 2007). In addition, a later study has demonstrated that progesterone inhibits HIV-1 infection in human THP-1 monocytes and monocyte-derived macrophages by blocking macropinocytosis (Chapuy- Regaud et al. 2013).

Norgestimate (S6) is a progestin, a synthetic progestogen, and an agonist of the progesterone receptor. It is used for birth control and hormone replacement therapy (Kuhl 2005). It is probable that the block of VACV infection by both compounds is modulated by the same mechanism.

Testosterone (S7) is the male sex hormone and an anabolic steroid. It plays crucial role during the development, as well as in later life. As a drug, it is mainly used in hormone or testosterone replacement therapy, and as doping in sports (Bassil, Alkaade, and Morley 2009). There were no examples in the literature of testosterone inhibiting any viruses.

3.6.1 24h IMV and EEV yield

A single virion that successfully infects a cell can result in thousands of new virions being produced by the host. A simple way to determine if a drug successfully inhibits viral morphogenesis as opposed to protein expression is to infect cells for 24h at a low MOI, and then quantify the number of infectious particles produced. In VACV this can be done using a plaque assay. As VACV produces two infectious forms, two different methods of collecting the viral progeny are employed: the EEVs are collected with the medium, as they are released from the cells; while the IMVs, IEVs, and CEVs are collected by harvesting the cells and lysing them, thus releasing the virus. The virus samples are then serially diluted and used to infect BSC40 monolayers, where viral plaques will be formed – each plaque originates from a single virion, hence the plaque forming unit (pfu/mL) measurement can be used to quantify the virus replication (Fig. 3.23 A). This allows for a straightforward initial analysis that could determine if the defect occurs pre- or post-IMV formation – an abrogation of EEVs with normal counts of IMVs would suggest a post-formation block.

First, to determine if any of the spread hits block the formation of IMVs or EEVs 24h viral yields were performed. Briefly, HeLa cells were infected with VACV WT at MOI 1 in the presence of the various inhibitors. At 24 hpi EEVs were collected by carefully removing the medium and spinning out any cells and neutralizing contaminating IMVs and disrupted EEVs with an antibody against L1 (Schmidt 2011, Wolffe, Vijaya, and Moss 1995). IMVs were harvested by lysing the infected cells. Plaque assays were performed in BSC40 cells in order to quantify the number of IMVs and EEVs produced in the presence of controls or the Spread hits.

All hits resulted in a significant reduction in IMV and EEV production, with some hits nearing detection limit (Fig. 3.26 B). In samples treated with BFA there was a small drop of IMVs, and an almost complete abrogation of EEVs. The block occurred after IMV formation, as expected.

The number of IMVs has dropped by 95% in cells treated with amethopterin (S1). Colchicine (S2), which binds to tubulin and disrupts microtubule polymerization has shown a trend similar to BFA. This means the wrapping is inhibited by blocking the transport of IMVs into the wrapping sites. Pinaverium bromide (S3) showed a similar drop of both IMVs and EEVs as did zardaverine (S4), though the latter was much more effective.

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Figure 3.23. 24h IMV and EEV yield. (A) Example of a plaque experiment. 3 dilutions of viral samples are shown. (B) HeLa cells were infected with VACV WT at MOI 1 for 24h in presence of Spread hits at their EC90 or controls. Extracellular virions were collected by removing the medium and L1 antibody was used to neutralize any IMVs and disrupted EEVs. IMVs were collected by harvesting and lysing cells. Plaque assays in BSC40 cells were performed to quantify the number of infectious particles produced in presence of compounds.

Means of 5 independent experiments ± s.d. are shown.

Progesterone (S5), norgestimate (S6) and testosterone (S7) showed a drop in IMV yield by 80%, 84%, and 75%, respectively. An equivalent drop in EEV yields was also observed, indicating that the production of infectious particles was inhibited.

3.6.2 Fluorescent phenotyping of infected cells

VACV recombinants where specific proteins are tagged with fluorescent proteins offer an excellent opportunity to visually identify the location and distribution of viral proteins and particles, as well as their colocalization with other proteins or cellular compartments. Alternatively, antibodies can be used instead of fluorescent proteins to ask similar questions. Following the finding that virus production was inhibited in some of the hits, the 101

experimental focus turned to the various forms of the virus: IMVs, IEVs, and CEVs, and what was their fate in the presence of Spread hits.

To see if any phenotypic changes in the formation or distribution of IMVs, IEVs, or CEVs could be identified, HeLa cells were infected with a VACV A5-mCherry F13-EGFP, a VACV recombinant expressing A5 core protein tagged with mCherry and a EEV membrane protein F13 tagged with EGFP, in the presence of the Spread inhibitors. At 12 hpi cells were fixed, left unpermeabilized and immuno-labelled for the EEV protein B5 to detect CEVs on the cell surface. This set up allowed for the identification of IMVs containing the red A5 core protein (1 colour); IEVs containing the red core and the F13-EGFP EEV membrane protein (2 colour colocalization); and CEVs, containing the red core, F13-EGFP, and labelled with B5 (3 colour colocalization) (Fig. 3.24).

The DMSO control showed the expected phenotype. Large cytoplasmic replication sites containing vast numbers of red IMVs. The wrapping sites exhibited numerous colocalizing red and green particles, and there were fully formed IEVs in the cytoplasm. CEVs were also detected on the cell surface.

BFA control resulted in, as expected, the collapse of the Golgi into the ER, resulting in abrogation of protein transport within the cell and a phenotype where viral proteins accumulate in random locations throughout the cell.

In the presence of amethopterin (S1) several very dense areas of A5 were detected. These did not have the same appearance as those present in DMSO control, and there were virtually no A5-positive particles around the cells, no particles colocalizing with EGFP or far red channel suggesting that IMV, IEV, and CEV formation was largely abrogated in these cells.

Colchicine (S2) showed the expected lack of colocalization between A5 and F13, as the particles were not being transported into the wrapping sites. The wrapping sites themselves appeared largely disrupted, and the number of A5-positive particles was predictably high.

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Figure 3.24. Immunofluorescence imaging of Spread hits inhibition. HeLa cells were infected with VACV A5-mCherry F13-EGFP at MOI 10 for 8h in the presence of Spread hits. After fixing, samples were labelled with B5 (EEV membrane protein) antibody without permeabilization and imaged. This allowed for visualisation of core particles/IMVs (red-positive puncta – white stars in DMSO magnified image), IEVs (red+green – white arrows), or CEVs (red+green+magenta – white triangles). Representative images for each compound are shown.

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Infection in the presence of pinaverium bromide (S3) presented an interesting redistribution of the A5-positive particles within the replication sites. It appears that replication and late gene expression was still occurring, but replication sites were somewhat disrupted and spread out around the cell, rather than “packed” in one location.

Infection in the presence of zardaverine (S4) seemed to have little effect on the distribution of the replication sites or their structure while significantly lowering the total number of A5- particles, both in replication and wrapping sites.

The three “steroids” (progesterone (S5), norgestimate (S6), and testosterone (S7)) showed very similar results. First, there was significantly less A5 containing particles in the replication sites. And second, while there was some limited red-green colocalization in what appears to be wrapping sites, there were virtually no IEVs in the cells, and therefore no CEVs. This means there were either no IEVs produced, or they were stuck in the wrapping sites.

3.6.3 EM investigation of VACV morphogenesis

VACV was the first virus imaged with a transmission electron microscope (EM), and there is over seven decades of experience in the field (Nagler and Rake 1948). The structures of various morphological stages of VACV are well characterized, allowing for relatively easy identification. As a reminder, Fig. 1.10 from the Introduction has been reproduced here (Fig. 3.25).

There have been some significant abnormalities identified by the fluorescent imaging of the cells infected with VACV in presence of Spread hits. It was decided that an even closer look was necessary to investigate if any defect in virus morphogenesis were present. An analogous experiment to the fluorescence microscopy imaging was performed, where HeLa cells were infected with VACV WT for 8-12h in presence of Spread hits, followed by EM imaging.

In the EM images of our DMSO control (Fig. 3.26) we were able to identify the expected changes in the cell brought upon by VACV replication. First, viral factories devoid of cellular organelles could be found surrounded by a membrane of ER-origin and mitochondria. All the early stages of VACV particle assembly could be found there, crescents IV, IVNs, and IMVs. Second, wrapping sites which had accumulated IMVs and IEVs were located near the cell nucleus and contained a high of concentration of membranes and vesicles as expected. The cells themselves appeared to be in good condition, the nuclear membrane was intact, and little to no aberrant vacuolation could be detected. Other organelles, e.g. mitochondria

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Figure 3.25. EM imaging of VACV particle formation. Red – crescents. Blue – Immature Virions. Yellow – Immature Virions with Nucleoid. Green – Intracellular Mature Virions. endosomes and lysosomes also appeared normal. Therefore, cells infected with VACV WT in presence of DMSO serve as a good control to which the inhibitors of VACV spread can be compared.

In the EM images of amethopterin (S1; Fig. 3.27), particles which appear to progress to the stage of IVs can be seen in the viral factories, however the number of IVNs is very low, and IMVs are virtually absent. In this case, wrapping sites are missing. This points to a block in VACV morphogenesis at the IV to IVN transition. When compared to DMSO-treated cells, no significant signs of cytotoxicity were seen.

EM imaging of colchicine (S2; Fig. 3.28) confirmed that while IMVs were present, virtually no IEVs could be seen. This would agree with the earlier results and the mechanism of action of colchicine. Additionally, the number of visible IMVs was low than WT as previously reported (Ploubidou et al. 2000), indicating earlier stages of the VACV lifecycle also suffer from reduced microtubule transport. Finally, wrapping sites were dispersed throughout the cytoplasm as opposed to being located at the MTOC as seen in infected cells. Treated cells showed some cytotoxicity, as expected from a compound that blocks the assembly and polymerization of microtubules. Specifically, some abnormalities in the shape of mitochondria appear to be present. Additionally, the clearing of organelles from the viral factories seems to be less efficient compared to DMSO.

In Pinaverium bromide (S3; Fig. 3.29) treated cells normal looking IVs with a lower number of IVNs were present. The wrapping sites contained a small number of IMVs relative to other

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samples, but they seemed disordered. A high number of vacuoles were present, perhaps adding to the effect that the compound had on aberrant wrapping. Overall, the EMs showed visible cytotoxicity in the form of large vacuoles, suggestive of Pinaverium bromide-mediated cellular damage.

Zardaverine (S4; Fig. 3.30) appears to have caused a total block of virus morphogenesis prior to crescent formation. There was a complete lack of IVs, IVNs, and IMVs in this sample. Large replication sites, with cellular organelles were displaced, were still present, consistent with the late block. More electron-dense areas, possibly viral DNA, or the late protein seen in the fluorescent imaging (Fig 3.24), were observed. Cells showed minor signs of cytotoxicity, specifically small vacuoles and some changes in mitochondria morphology, however these did not appear to be serious relative to control samples.

EM imaging of progesterone (S5; Fig. 3.31), norgestimate (S6; Fig. 3.32), and testosterone (S7; Fig 3.33) all showed that IVs, IVNs, and even IMVs were present in the viral factories albeit to a significantly lower than observed in control samples. Organelle displacement from replication sites appeared less efficient, though it was hard to quantify the effect. Small vesicles, or possibly vacuoles, were present in the wrapping sites and any IMVs found in the vicinity failed to be wrapped. This observation is in agreement with both the IF and 24h yield results (Fig. 3.23, Fig. 3.24).

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Amethopterin

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Colchicine

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Pinaverium bromide Pinaverium

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3.6.4 Spread hits – conclusions

The identified Spread hits block the VACV lifecycle at various steps. Amethopterin (S1) appears to block at the IV stage, with replication factories full of particles, but very few IVNs visible and no IMVs. This confirms the 24h yield results, and explains the immunofluorescence (IF) data, where large clusters of A5 were seen, with no IEVs or CEVs. Colchicine (S2) unsurprisingly blocks wrapping of IMVs, as expected based on its effect on microtubules, and had noticeable cytotoxic effect. The lower 24h yields might be explained by general disruptions to the intracellular transport. Pinaverium bromide (S3) reduced the number of IMVs significantly, and EEVs proportionally to the IMVs. However, wrapping appeared to be affected the most – even if IEVs were in the wrapping sites, they could not egress. However, pinaverium bromide has had a significant cytotoxic effect. Zardaverine (S4) abolished morphogenesis almost completely – 24h yield dropped by ~90% – with large replication sites with little late protein and no IVs seen in the EM. This would indicate that the particles seen in the IF imaging are not functional IMVs but just A5 protein aggregates. The last three hits: progesterone (S5), norgestimate (S6), and testosterone (S7) all seem to primarily reduce the number of infectious particles at each stage, without blocking the assembly at a particular stage. They also reduced the number of IEVs, as seen on both IF and EM. It is possible that the compounds act at multiple stages, with varying degrees of efficiency, but taken together the resulting block is very efficient. Since these hits belong to the class of steroid hormones, it is likely that they all block infection at the same stage.

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3.7 African swine fever virus screen

As stated in the introduction, African swine fever is a disease with an economically significant burden. Development of antivirals targeting ASFV could directly help animals, as well as support further vaccine development and basic biology research. Both ASFV and VACV belong to the order of nucleocytoplasmic large DNA viruses (NCLDVs), and as such they share many aspects of their replication cycles. Since VACV has lower biosafety requirements than ASFV, the hope of this effort was that by screening a large library of compounds for VACV inhibitors the efficacious hits could be tested in ASFV. By having already narrowed down the number of potential hits the experiments are made more manageable. With this in mind, the hits from the primary screen were tested for inhibitory effects on ASFV.

Briefly, Vero cells were infected with ASFV Ba71v (Enjuanes et al. 1976) at MOI 4 for 24h in presence of the top 50 hits at 10 µM (Supplementary table 6.1), as the experiments at Pirbright were done before the secondary confirmation screen. After fixation, cells were permeabilized and labelled with P30 and P34 antibodies. P30 is a protein expressed early in the ASFV lifecycle, while P34 is expressed late. By using these two antibodies it was possible to detect early and late gene expression (Fig. 3.34). Nuclei were stained with DAPI, to localize cells. Images were analysed manually, and as such only very large changes were considered as having an inhibitory effect. Due to the stringent safety procedures at the Pirbright Institute, I was not permitted to perform any of the experiments involving live virus, and they were done by Chris Netherton. I was allowed to handle the samples once they were fixed, and I performed the fluorescent labelling and staining, as well as imaging. After optimization the screen was done once (Fig. 3.35).

One compound, astemizole, caused a reduction in ASFV early gene expression. Astemizole also inhibited early gene expression in VACV. It was somewhat surprising that there were no other Early inhibitors, considering ASFV is also believed to enter via a pH-dependent endocytic pathway.

Four compounds that inhibited late gene expression were identified: amethopterin (S1) aminacrine (E1), ciclopirox (L1), and gemcitabine (L2). It is interesting that one Spread and one Early hits in VACV resulted in a Late block in ASFV. It is likely that ciclopirox and gemcitabine inhibit ASFV via the same mechanism as they inhibit VACV.

No inhibitors of ASFV spread were identified.

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Figure 3.34. ASFV screen controls. VERO cells were infected with ASFV at MOI 4 in the presence of 1% DMSO, 100 nM Bafilomycin A (BafA), 10 µM AraC, 30 µM BFA (controls). 24 hpi cells were fixed and labelled for early protein P30 (green) and late protein P34 (magenta), and nuclei were stained with DAPI (white). BafA blocked virus entry, and thus early and late gene expression. AraC blocked viral DNA replication and late gene expression. BFA had partial effect on blocking viral spread.

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Figure 3.35. ASFV screen hits. Preliminary hits from the Prestwick Chemical Library were screened for their inhibitory effect on ASFV. 1 hit blocked early protein expression, and 4 hits blocked Late protein expression.

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3.8 Signal transducer and activator of transcription 3

Stattic, a STAT3 phosphorylation inhibitor, has been identified as a VACV inhibitor in a high- throughput screen of the LOPAC library performed in the Mercer lab by Rosalie Helig and Sam Kilcher (unpublished). Here their work was continued by confirming the results and testing the effect of two additional compounds that prevent the phosphorylation of STAT3, S31-201 and Cryptotanshinone (Crypto), on VACV infection.

Upon phosphorylation at Y705 STAT3 forms dimers and translocates into the nucleus. Therefore, blocking the phosphorylation of STAT3, its activity should be abolished. If all three compounds block VACV replication, it is likely that the inhibition is a result of blocking STAT3 from getting into the nucleus.

Considering the role STAT3 plays in the JAK1/STAT signalling pathway, one would expect VACV to favour STAT3 inhibition, since it uses VH1 to block STAT1/1 and STAT1/2 phosphorylation and activity. It is therefore counterintuitive that inhibition of STAT3 phosphorylation would result in a block of VACV replication.

3.8.1 Vaccinia virus inhibition

Briefly, HeLa cells were infected with VACV E-EGFP in the presence of the three compounds in a serial dilution. 8 hpi cells were fixed and analysed for fluorescence by flow cytometry. All three compounds inhibited VACV infection in a dose dependent manner, indicating that VACV requires the phosphorylation of STAT3 for successful infection (Fig. 3.36). Stattic was the most potent of the three, with EC50 = 7.9 µM, followed by Crypto (EC50 = 46 µM) and S31-201 (EC50 = 230 µM). The compounds were simultaneously tested for toxicity (following the same protocol as described in section 3.3). While static and S31-201 became cytotoxic around the concentration where they reached their peak inhibition, crypto proved to be toxic at an earlier stage. However, the toxicity results for crypto could have been skewed, because the toxicity assay employs fluorescence at a wavelength similar to crypto auto-fluorescence.

3.8.2 Pre-incubation

To find out if the compounds had a direct virucidal effect on the virus particle a preincubation experiment was done. Briefly, VACV E-EGFP was preincubated in DMEM with compounds at their EC90 for 60 min at room temp, diluted 1:100 in DMEM, and used to infect HeLa cells 6 hpi cells were analysed for fluorescence by flow cytometry. Neither of the compounds has shown any virucidal effect, with no significant change in infectivity (Fig. 3.37 A).

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Figure 3.36. VACV inhibition by STAT3 phosphorylation inhibitors and their toxicity. HeLa cells were infected with VACV E-EGFP at MOI 10 in the presence of stattic, crypto, or S31-201 in serial dilution. 8 hpi cells were fixed and analysed for EGFP fluorescence using flow cytometry. Toxicity was tested using the LDH assay following 24 h incubation of HeLa cells in presence of compounds, as previously described. Means of at least 3 independent experiments ± s.d. are shown.

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Figure 3.37. Stattic preincubation and addition timepoints. (A) VACV E-EGFP at MOI 1,000 was preincubated with compounds at their EC90 for 60 min at RT. After 1:100 dilution the preincubated VACV was used to infect HeLa cells at MOI 10. 6 hpi cells were analysed for EGFP fluorescence using flow cytometry. (B) HeLa cells were infected with VACV E-EGFP at MOI 10 while Stattic was added at the indicated time, relative to virus addition. 6 hpi cells were analysed for EGFP fluorescence using flow cytometry.

Means of at least 2 individual experiments in triplicates ± s.d. are shown.

3.8.3 Addition timepoints

The LOPAC screen results indicated that stattic was an early inhibitor with no virucidal effects. The next step was to confirm this with a time-point of addition experiment. Stattic was added to cells 30 min before infection with VACV L-EGFP, at the same time as infection, and 30/60/120 min after infection (Fig. 3.37 B). While it was still quite effective when added at the same time as the virus (<20% cells infected), addition 30 min before infection was most efficient (<5% cells infected). Adding 60 min or later after infection resulted in decreasing inhibitory effect, with addition 120 min after infection showing almost no inhibition. This confirmed that stattic blocks VACV infection early in the lifecycle and likely has no effect on post-replicative stages.

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3.8.4 Phosphomimetic cell line

Having confirmed that three different STAT3 phosphorylation inhibitors are VACV infection inhibitors, we obtained a STAT3 (Y705F/Y705F) DLD-1 cell line where the tyrosine was mutated into phenylalanine, therefore making the phosphorylation at this site impossible. The parental cell line was used as a control. As with other experiments, cells were infected with VACV E-EGFP for 6h in presence of serially diluted stattic, then analysed by flow cytometry. Even with a “phospho dead” STAT3 cell line stattic still inhibited VACV infection (Fig. 3.38). This was a surprising finding, considering there were 3 drugs with supposedly analogous mechanism of action, yet this experiment seems to show that all 3 might act via a different mechanism. There was a noticeable difference between the Y705F cell line and the parental cell line, with stattic being somewhat more effective in the latter, showing its effect at a lower concentration. This effect vanished at the highest levels of inhibition. Y705 cells were more similar to HeLa controls. While we did not have time to further investigate this variance, it could possibly be explained by the general cellular effect of STAT3 not being phosphorylated.

Figure 3.38. VACV inhibition in Y705F/Y705F cell line. DLD Y705F/Y705F phosphomimetic cell line, it’s parental cell line, and HeLa cells were infected with VACV E-EGFP at MOI 10 in the presence of serially diluted stattic. 8 hpi cells were fixed and analysed for EGFP fluorescence using flow cytometry.

Means of at least 3 independent experiments ± s.d. are shown.

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3.8.5 STAT3 siRNA knockdown

Since it appeared that phosphorylation at Y705 was not necessary for stattic-dependent VACV inhibition despite earlier evidence, a different approach was chosen. In case a different STAT3 phosphorylation site was important, or inhibition of STAT3 via an alternative mechanism was taking place, an siRNA knockdown of STAT3 was used. First, the knockout was verified via Western blot (Fig. 3.39 A). Seeing a very efficient knockdown at 48 h and 20 nM, an inhibition test followed. Briefly, HeLa cells in 96-well plates were transfected with the siRNA for two days, followed by infection with VACV E-EGFP in presence of stattic and analysed by flow cytometry 8 hpi. Remarkably, stattic was still able to block VACV infection despite lacking STAT3 (Fig. 3.39 B). The results suggest that the inhibition of VACV by stattic is not STAT3 dependent, and the compound works by a different mechanism.

A B

Figure 3.39. siRNA knockdown of STAT3. (A) siRNA targeting STAT3 were tested at indicated concentrations for 48h. si Control 20 nM (B) STAT3 siRNA-transfected HeLa cells (20nM), and siRNA control transfected HeLa cells (20 nM) were infected with VACV E-EGFP at MOI 10 in presence of serially diluted stattic. 8 hpi cells were fixed and analysed for EGFP fluorescence using flow cytometry.

Means of 3 repeats in a single experiment ± s.d. are shown.

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4 Discussion

High-throughput microscopy-based screening of the 1,280 compounds making up the Prestwick Chemical Library, presented in this thesis, has successfully identified multiple novel VACV inhibitors. By using a VACV E-EGFP L-mCherry recombinant, the screening strategy allowed for a preliminary determination of the viral replication stage that was inhibited – i.e. Early, Late, or Spread. Targeted assays have been used to further elucidated the inhibitory step for each efficacious compound, e.g. viral entry, DNA replication, virion morphogenesis (Fig. 4.1). Moreover, demonstrating the potential application of the strategy to other nucleocytoplasmic large DNA viruses, five potential inhibitors of African Swine Fever virus were identified by screening of the initial VACV hits at the Pirbright Institute.

Continuation of the work of Rosalie Heilig and Sam Kilcher on the compound stattic identified two additional STAT3 inhibitors that block VACV infection. While the exact mechanism requires elucidation, it appears that the compounds do not work by inhibiting STAT3, as they still blocked VACV infection when STAT3 was knocked-down using siRNAs.

In the ensuing sections the results presented in this thesis will be placed in context of VACV drug discovery, followed by a discussion on the individual hits identified.

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demonstrates demonstrates the

Chemical Library drug screen. drug Library Chemical

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Figure 9 Early, 3 Late, and 7 Spread hits were identified, alongside 14 Cardenolides which also blocked Early. This diagram block. compounds the that lifecycle viral ofthe step specific the down narrow to designed ofassays results

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4.1 Vaccinia virus high-throughput screens

The recent approval of TPOXX as a smallpox antiviral by the FDA (FDA 2018, Grosenbach et al. 2018) demonstrates both the practicality of high-throughput VACV screens as an early stage drug discovery tool, as well as the continued need for orthopox antivirals. The TPOXX screen used the cytopathic effect, that poxvirus infection induces in cells, to detect inhibition. This assay simply reports whether the virus has spread or not (Yang et al. 2005). A later VACV high-throughput microscopy-based drug screen, using EGFP expression as a reporter, identified mitoxantrone as a VACV inhibitor alongside 12 other hits (Deng et al. 2007). This screen also used virus spread as a sole end-point readout. To date the only VACV screen using a recombinant virus expressing temporally distinct fluorescent proteins did not expand on the hits identified. In addition, this screen used a plate reader to detect fluorescence as opposed to using a microscopy-based assay (Dower et al. 2011). The screen of LOPAC (Heilig and Kilcher, unpublished) used the E-EGFP L-mCherry virus as analysed using flow cytometry. As such, the screen presented in this thesis, to my knowledge, is the only one to use a high- throughput microscopy-based approach capable of differentiating inhibition at distinct stages of replication. Neither the Yang et al. nor the Dower et al. screens name the source or composition of their libraries, while Deng et al used 2,000 compounds from Microsource and 880 compounds from Prestwick Chemical. They did not specify if all those compounds were FDA-approved, however, since 7 of their 13 hits were identified in my screen (5 Cardenolides, mitoxantrone, and cytarabine), it is likely that all 880 were FDA-approved. This makes the screen reported here one of the largest to identify inhibitors of VACV replication and/or spread using FDA-approved compounds.

4.2 Prestwick Chemical Library screen

A compound can inhibit viral infection in two ways. It can have an effect on a cellular factor, thereby stopping the virus from using a cellular mechanism which it requires to complete its replication cycle; or it can have an effect on a viral protein or function, directly prevented viral infection. BFA and colchicine are examples of the former. TPOXX and mitoxantrone are examples of the latter, both targeting viral proteins, F13 and the DNA ligase, respectively (Yang et al. 2005, Deng et al. 2007). The distinction between the two possibilities would be one of the main goals of any follow up on hits identified in the Prestwick screen. In some cases, the answer might be clear, e.g. nucleotide analogues are likely to block viral DNA replication. The mechanism of action of other compounds may be more complex. The most direct way to answer this question would be to attempt to create resistant viruses, followed by marker rescue or whole-genome sequencing to identify the mutated target, following the

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example of the Yang et al and Deng et al. experiments trying to pinpoint cellular mechanisms can be run in parallel, e.g. siRNA knockdowns of suspected proteins and other inhibitors of cell pathways.

Apart from generating resistant viruses, other experiments could be applied to all the identified hits. All compounds could be tested for synergistic effects. Would using an Early and a Late, or a Late and a Spread compound, which block infection at different stages, give more potent results? Could the compounds be used at a lower – potentially less toxic or with fewer side effects – concentration when used in combination?

Once the mode of action is determined, the next step would involve determining if the drugs work on other (pox)viruses. The small-scale screen performed on ASFV, was an attempt to answer such a question. Both TPOXX and mitoxantrone have demonstrated broad activity against poxviruses (Yang et al. 2005, Deng et al. 2007). However, poxviruses are closely related, and their genomes are highly conserved (Iyer et al. 2006), so one would expect a VACV inhibitor to have at least some effect on other poxviruses. Other LNCDVs might be similar enough for compounds with a cellular target to retain their inhibitory effect. Alternatively, after identifying the mechanisms of action of individual hits, other viruses could be selected based on the use of common cellular pathways. For example, compounds inhibiting VACV by blocking macropinocytosis could also be tested against other viruses using this endocytic entry mechanism.

Following testing inhibition in other (pox)viruses, animal studies would be the next step forward, as they would answer the crucial question: can the compounds protect against VACV, or other (pox)virus infection in vivo? While TPOXX proved to be a great success story, with activity in mice and non-human primates, mice treated with mitoxantrone had survival rates no different than those treated with placebo. This serves as an illustration that even compounds with well understood mechanisms of action may not work in a “real life” situation.

4.2.1 Limitations

There are three main limitations identified in the design of the primary screen in this project: lack of repeats, use of AraC, and use of the VACV E-EGFP L-mCherry recombinant.

Due to time and financial constraints the screen was not repeated. A biological triplicate with technical duplicates would allow for a far more robust analysis. Apart from running statistical analysis, simply taking only hits identified in all three replicates into the secondary screens would have eliminated some false positives. It is also possible that some false negatives, that

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is hits that were missed, would emerge. The screen sensitivity would be increased, giving more robust results. It would also help with the high variability of the controls.

The idea behind using AraC to stop late gene expression in secondary infected cells was to identify potential spread inhibitors that reduce the size of plaque, rather than completely block its formation. For example, an inhibitor could reduce the plaque size by 70%. Unfortunately, the variability of the controls made the distinction impossible.

Why is the presence of AraC disadvantageous? Imagine that only 1 in 1,000 cells is infected by the addition of virus. Then the single infected cell spreads virus to its 50 neighbours. In this screen, AraC was added at this stage to block further infection. Without AraC, the infection would continue, with more cells becoming infected. As 24h is enough for 3 full replication cycles in the absence of AraC, each of the 50 infected cells would go through the viral lifecycle and potentially infect 25 more cells (less than the primary infected cells, since many of its neighbours are already infected). After 24h a plaque would potentially consist of 50+50*25+50*25*25=32,550 cells. In practice this would mean that virtually all cells in a well would be infected in the untreated control. In comparison, an Early inhibitor would block early gene expression in the primary infected cells and no fluorescence would be detected. A Late inhibitor would result in only 1 cell in 1,000 becoming green; and a Spread inhibitor would produce only 1 red cell in 1,000. The dynamic range of the screen would have been improved drastically, eliminating many false positives.

While the strategy of using a VACV recombinant with a fluorescent protein under different promoters is viable and provides more information (Early/Late/Spread inhibition in a single screen), there are certain drawbacks. While Late genes are expressed in high amounts and the mCherry signal is very strong, early genes are expressed in very small amounts, and the E-EGFP signal needs boosting with an antibody. This requires blocking, labelling with primary antibodies, washing, labelling with secondary antibodies, and further washing. Each step adds potential compounding errors, which may explain the high variability of the cntrols. In the case of an VACV E/L-EGFP (expressing EGFP under both early and late gene promoters), all that would be required is fixation and nuclear staining. The signal would be strong and easily detected, and no further steps would be required. Therefore, if the primary screen is run only once, an E/L EGFP (or even only a Late-EGFP) recombinant might be more practical. The secondary screens would still need to be performed to further identify Early, Late, and Spread inhibitors, as they were in this project.

With more time other experiments could be improved. Cytotoxicity, for example, was done only at EC90 instead of a full compound titration. Similarly, the secondary screen could be

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done with a higher number of intermediate dilutions and on a wider range of concentrations (e.g. 1 M – 1 nM) for a more precise result.

Figure 4.2. Experimental workflow from hit identification to in vivo testing. A generalised workflow of drug discovery, from initial hit identification, through confirmation and characterization, to testing against other viruses and in an animal model.

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4.2.2 Early hits

Inhibition of early gene expression can be caused by inhibiting any of the preceding lifecycle steps. The virion could be rendered defective by a virucidal drug, or its binding to the cell surface could be blocked. Internalization or plasma membrane fusion could be inhibited. Transcription and translation of early genes could be blocked. Following the identification of Early inhibitors, targeted assays have been used to pinpoint which of these steps are blocked by each of the compounds.

Based on the results, no virucidal compounds have been found, nor have any of the compounds tested affected binding to cells. Internalization and fusion were the most common targets (Fig. 4.1), with transcription/translation of early genes second. With both classes there is still the need for further elucidation of the exact step of the virus lifecycle that is inhibited, as well as the mechanism of action.

While the acid bypass assay is a great tool to identify entry (internalization and fusion) inhibitors, it does not differentiate between the two. This is where the microscopy-based entry assay can provide a more detailed understanding, by distinguishing between fused and non-fused virus particles. When optimized, this experiment should clarify the results, further classifying the hits into internalization (macropinocytosis) inhibitors or fusion inhibitors.

While astemizole was shown to inhibit both MERS-CoV and SARS-CoV, the authors did not specify the stage or mechanism of inhibition (Dyall et al. 2014). In addition to being a histamin-1 antagonist astemizole is also reported to affect Eag1 and Erg K+ channels (Garcia- Quiroz and Camacho 2011). The requirement for potassium, sodium, and calcium balance will be revisited when discussing other entry hits.

Avermectin, the Noble-prize winning anti-parasite drug, has been shown to inhibit a wide range of viruses, including flaviviruses (Mastrangelo et al. 2012). In these RNA viruses, it was shown to target the NS3 helicase activity. Considering that avermectin was an Early hit in VACV, and VACV brings its own helicase, the mechanism of inhibition may be different than in VACV. Interestingly, ivermectin was shown to inhibit nuclear import of HIV integrase by inhibiting importin α/β (Wagstaff et al. 2011). This mechanism was also shown to be responsible for the inhibition of VEEV (Lundberg et al. 2013). Neither of the two mechanisms explains the inhibitory effect seen in VACV.

Benzethonium chloride, being a disinfectant, was expected to have a virucidal effect. This was not the case. Unfortunately, due to toxicity issues the specific step at which benzethonium blocks infection was not identified. However, in a small-molecule anti-cancer

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screen treatment of cells with benzethonium resulted in cytosolic Ca2+ increase (Yip et al. 2006).

Chlorhexidine has been reported to have moderate virucidal effects on HSV (Park and Park 1989) and HIV (Montefiori et al. 1990). For VACV, although no virucidal activity was found, the inhibitory effect was much stronger than seen for HSV or HIV, suggesting an alternative mechanism of inhibition. Being an ionic salt, it could be hypothesised that a disruption of plasma potential could have an effect on macropinocytosis.

The inhibitory effect of gefitinib on VACV has been published and is well understood (Mercer et al. 2010a, Langhammer et al. 2011).

Raloxifene, a selective estrogen receptor modulator (SERM) was identified as an EBOV entry inhibitor, alongside other SERMs: tamoxifen, clomiphene, and toremifene (Johansen et al. 2013). It would be interesting to see if these compounds also inhibit VACV, confirming the SERM-based mechanism of action.

Additional experiments would be required to clarify whether the block of cores deposited into the cytoplasm occurs at the level of transcription or translation. Cycloheximide blocks infection after the RNA is exported from the core (Moss and Filler 1970). At what stage do aminacrine, daunorubicin, and fludarabine block early gene expression? RT qPCR for early mRNAs should provide the answer. Interestingly, a recent study demonstrated that Hoechst, which was routinely used in this project to stain the nuclei of fixed cells, blocks VACV infection (Yakimovich et al. 2017). Hoechst binds the minor groove of double-stranded DNA, and blocked late gene expression at low concentrations, but also early gene expression at high concentrations. Aminacrine, daunorubicin, and fludarabine also inhibited late gene expression in the AraC release experiment, which is consistent with their suggested mechanism of action.

Of special interest amongst the Early hits would be Cardenolides. These sodium-potassium ATPase inhibitors cause an increased concentration of Ca2+ in the cells. As mentioned earlier, this disrupted cellular calcium balance may have a domino effect on other pathways, and most likely affects the endocytic entry pathway, macropinocytosis. Other compounds affecting calcium levels were shown to inhibit viral infections. For example, verapamil, a calcium-channel blocker, inhibits VACV and measles replication (Leao-Ferreira et al. 2002), influenza A replication (Nugent and Shanley 1984), and along with other channel blockers – amiodarone and dronedarone – blocks flavivirus entry (Gehring et al. 2014).

AraC experiments, combined with experiments in HEK cells expressing mouse Na+/K+ ATPase, demonstrated that Cardenolides are likely to have two mechanisms of action. The inhibition

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of early gene expression seems to be dependent on the ATPase inhibition, while the block of late gene expression does not. This warrants further experiments to elucidate the specific mechanism of the late block, and the significance of other Early compounds also having secondary inhibition effects.

4.2.3 Late hits

The steps between early gene expression and late gene expression include genome uncoating and pre-replication site formation, viral DNA replication, and intermediate gene expression. All three Late hits allowed for uncoating and pre-replication site formation but blocked viral DNA replication. While it was not surprising to find nucleoside analogues in this hit class (gemcitabine and trifluridine), ciclopirox was a novel finding. It was shown to affect DNA repair, replication and cellular transport in yeast (Leem et al. 2003), which might be a clue to its mechanism of action. In bacteria, it was shown to affects galactose metabolism and lipopolysaccharide biosynthesis (Carlson-Banning et al. 2013). Finally, it was shown to inhibit mTOR by activation of AMPK, a kinase required for VACV infection (Sen et al. 2013, Zhou et al. 2016). AMPK activation has been linked to inhibition of flaviviruses (Moser, Schieffer, and Cherry 2012). Taken together, these results suggest a list of targets for further experiments that could elucidate the mechanism of action by which ciclopirox blocks VACV infection.

4.2.4 Spread hits

Spread hits all lowered the number of EEVs exiting the cell and can be divided into IMV morphogenesis blockers (amethopterin, zardaverine), wrapping blockers (colchicine, pinaverium bromide), and the three steroids, which appear to block VACV replication cycle at multiple stages. Further work on all Spread hits would most likely focus on discerning whether a cellular function is affected, like in the case of colchicine, or if a viral protein is inhibited, like in the case of mitoxantrone. Elucidating the inhibitory mechanisms of the steroid hits could be particularly interesting.

Amethopterin seemingly blocked IV to IVN transition, which is the point where the viral DNA is encapsidated. Since amethopterin is supposedly inhibiting the production of nucleotides and protein via DHFP inhibition, one would expect the block to occur at the level of late gene expression. However, judging by the IF imaging, late protein expression was not affected. In flaviviruses, infection in the presence of amethopterin was rescued by addition of folinic acid, a thymidine precursor (Fischer et al. 2013). This suggests a pathway block rather than a direct

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effect on a viral protein. A replication of this experiment with VACV could provide valuable information.

Zardaverine is a dual-selective phosphodiesterases 3 and 4 inhibitor, and increases intracellular cAMP (Sun et al. 2014). cAMP increase has an inhibitory effect on VACV infection, likely via activation of protein kinase A (PKA) and unknown downstream steps (Leao-Ferreira et al. 2002). The study also detected a decrease in DNA synthesis and late protein synthesis, but not a complete block. This small difference could be explained by experimental differences. A follow up would involve a confirmation of increasing cAMP levels and use of PKA inhibitors to restore infection.

That pinaverium bromide acting as a Spread inhibitor was unexpected, considering it is a calcium channel blocker, and as discussed earlier, calcium channel blockers (verapamil, amiodarone, dronedarone) appear to have an effect on entry. No mention of pinaverium bromide in literature outside of the context of irritable bowel syndrome or smooth muscle relaxation was found. Pinaverium acts on the surface membranes of smooth muscle cells (Christen 1990). This leads to two unanswered questions: why doesn’t pinaverium block entry? And how does it block wrapping? It is possible that the mechanism could be unrelated to pinaverium’s action on calcium channels.

Steroid hormones are transcription regulators, usually taking a long time to effect changes in the cell, and with long lasting effect. Following entry to the cell, they bind nuclear receptors, and are then transported into the nucleus where they bind to DNA and induce target gene transcription (Brinkmann 2011). However, steroid hormones can also bind to G-protein coupled receptors (Wang, Liu, and Cao 2014), which is much faster. Considering that VACV was blocked post-replication, at least 4-6 hpi, either pathway may be responsible.

The only example found in the literature directly linking steroid hormones and virus inhibition was the inhibition of HCV by estradiol (Hayashida et al. 2010, Magri et al. 2017). Estradiol is the major female sex hormone, and it was shown to impair HCV lifecycle at multiple stages. However, progesterone and testosterone did not show any inhibitory effect (Magri et al. 2017).

Looking at sex differences in poxvirus infections, pregnant rabbits have been found to be less likely to have local or generalized lesions following a rabbit pox inoculation (Rosahn, Hu, and Pearce 1936). Pseudopregnant rabbits and rabbits receiving estrogenic hormone were more resistant to VACV infection (Sprunt and McDearman 1940). A more recent study, on the other hand, has shown that male mice were nearly twice as resistant as female mice (Geurs et al. 2012). Studies of smallpox vaccines in humans seem to have opposing results, depending on the vaccine. Dryvax elicited a stronger response in women (Kennedy et al. 2009), while 133

IMVAMUNE (Troy et al. 2015) has shown that men had higher peak titres than women. These results, while interesting, are indicative of differences in antibody production and/or efficacy, while the results reported in this thesis indicate that the response was directly based on concentration of steroid hormones. Considering the wide role steroid hormones play, elucidating the mechanism of action responsible for inhibiting VACV in cells would be a daunting undertaking.

4.3 African swine fever virus

Due to animal loss and trade restrictions, ASFV is a real and significant economic threat to countries with high pork production. As there is currently no vaccine or treatment for ASFV, it is important to open new avenues of research. One such avenue is drug repurposing through testing of compounds in similar, but less dangerous viruses. By combining those two approaches, this high-throughput screen of 1,280 FDA-approved compounds on VACV allowed for the narrowing down of potential ASFV inhibitors to 50 compounds. After screening these 50 compounds against ASFV, 5 hits were identified. The identification of 5 compounds can be considered a significant success, since the candidate hits were initially identified in a different cell line using a different virus.

It would be helpful to repeat the screen using the confirmed compounds, and to optimize the screening procedure with more attention to data analysis made possible with more replicates. A higher number of images and repeats would certainly improve the quality of the screen. For follow-up analysis, flow cytometry approaches, similar to those used for VACV, could be used to confirm the ASFV inhibitors and to establish EC values, toxicity, and the stage(s) of infection inhibited.

Identified and confirmed inhibitors could be used for basic ASFV research on the ASFV lifecycle.

4.4 Signal transducer and activator of transcription 3

The role of STAT3 in the JAK1/STAT signalling pathway is similar to that of STAT1 and STAT2; that is, it is required for the transcription of interferon stimulated genes, products of which are antiviral proteins. VACV encodes a protein that specifically inhibits the phosphorylation STAT1 and STAT2 - VH1. By analogy, one would expect a similar mechanism when it comes to STAT3, that VACV would inhibit phosphorylation of STAT3. The block of VACV replication in the presence of a compound blocking the phosphorylation of STAT3 is therefore counterintuitive. While it has been shown that STAT3 can be transported to the nucleus

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without Y705 phosphorylation (Liu, McBride, and Reich 2005), this still does not hint as to why stattic would block VACV replication.

Stattic has been identified as a selective STAT3 inhibitor (Schust et al. 2006), hence when it was identified as a VACV inhibitor it was assumed that STAT3 was required for productive infection. This assumption was only reinforced when two additional STAT3 inhibitors, S31- 201 (Siddiquee et al. 2007) and cryptotanshinone (Shin et al. 2009) were identified as VACV inhibitors. This only served to magnify the surprise when a cell line containing a phospho- dead version of STAT3 and a siRNA knockdown of STAT3 had no impact on stattic’s inhibitory activity. This result could have two explanations. One, all three drugs have the same off- target effect, and their VACV inhibitory activity is not directed against STAT3. Two, a viral protein is similar enough to STAT3 that all three compounds block infection via inhibition of this hypothetical viral protein. At the same time, previous literature has shown that STAT3 does have an effect on VACV infection (Mahony et al. 2017), however the results were the opposite – STAT3 was required for the inhibition of VACV replication. It is of note that that different viruses and cell lines were used in these studies, which could potentially explain the differences. Results presented here would indicate the inhibitory effect of stattic is likely not related to the JAK1/STAT pathway.

Further experiments would need to first confirm the initial results presented here, particularly the siRNA knockdown. Preliminary results suggest phosphorylation at Y705 increases early in infection. Thus, it would be central to investigate the phosphorylation of STAT3 in the presence of VACV and stattic. It is conceivable that the two – inhibition by stattic and Y705 phosphorylation – are unrelated phenomena.

4.5 Summary

By screening an FDA-approved drug library using a high-throughput microscopy-based approach I have identified compounds inhibiting VACV at different stages of the viral life cycle. Identified hits were verified with flow-cytometry-based secondary screens, and I have further elucidated the inhibitory step for each hit using targeted assays. By screening the initial hits against ASFV, I have identified 5 potential inhibitors and demonstrated that VACV can serve as a pre-screening tool for this currently untreatable pathogen. I have also expanded the examination of the mechanisms of action of VACV inhibition by stattic.

The results presented in this thesis are the first step in identifying potential FDA-approved VACV inhibitors, using the principles of drug repositioning. They may also lead to additional insights into poxviruses host-pathogen interaction through determining the mechanism of action of the identified inhibitors. 135

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6 Supplementary

Hit Confirmed Compound Confirmed class? class hit? Astemizole Aminacrine Ciclopirox olamine Colchicine

Cycloheximide Digitoxigenin Early Digoxigenin Digoxin Gemcitabine Lanatoside C Proscillaridin A

Cytarabine Fludarabine Late Trifluridine (S)-(-)-Cycloserine Adrenosterone Amethopterin (R,S) Amiloride hydrochloride dihydrate Amlexanox Amorolfine hydrochloride Avermectin B1a Balsalazide Sodium Betamethasone Chlorhexidine Daunorubicin hydrochloride Debrisoquin sulfate

Diacerein Esmolol hydrochloride Etanidazole Spread Furazolidone Furosemide Gefitinib Indatraline hydrochloride Isoflupredone acetate Isopyrin hydrochloride Liranaftate Loracarbef Mecamylamine hydrochloride Methyl benzethonium chloride Mitoxantrone dihydrochloride Niacin Nicotinamide

160

Norgestimate Oxybenzone Pinaverium bromide Pirenperone Progesterone Promethazine hydrochloride Raloxifene hydrochloride Rebamipide Sulfaguanidine Sulfanilamide Sulmazole Testosterone propionate Troxipide Zardaverine Table 6.1. Hits identified in the primary screen. Early, Late, and Spread hit as they were identified in the primary screen. Green – Yes. Orange – No. False positives were not confirmed in the secondary screen. Certain hits were reassigned to a different hit class following secondary screens.

Early hits:

161

Cardenolides:

Spread hits:

162

Figure 6.1 Early, Cardenolides, and Spread hits titres.

163

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Figure 6.2 VACV Cardenolide-dependent inhibition in HEK cells

164