development and optimization of a novel t7 polymerase ... · 11.05.2020 · a t7 polymerase can...

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Title 1

Development and optimization of a novel T7 polymerase-independent Marburg virus 2

minigenome system. 3

4

Authors 5

Bert Vanmechelena, Joren Stroobantsb, Kurt Vermeireb, Piet Maesa,# 6

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Affiliations 8

aKU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute, 9

Laboratory of Clinical Virology, Leuven, Belgium. 10

bKU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute, 11

Laboratory of Virology and Chemotherapy, Leuven, Belgium. 12

#Author for correspondence 13

Piet Maes, Rega Institute for Medical Research, Herestraat 49, box 1040, BE3000, Leuven, 14

Belgium, Tel. +32 16 321 309, [email protected] 15

16

.CC-BY 4.0 International licensemade available under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

The copyright holder for this preprintthis version posted May 12, 2020. ; https://doi.org/10.1101/2020.05.11.088211doi: bioRxiv preprint

https://doi.org/10.1101/2020.05.11.088211

http://creativecommons.org/licenses/by/4.0/

2

Abstract 17

Marburg virus (MARV) is the only known pathogenic filovirus not belonging to the genus 18

Ebolavirus. Minigenomes have proven a useful tool to study MARV, but all existing MARV 19

minigenomes are dependent on the addition of an exogenous T7 RNA polymerase to drive 20

minigenome expression. However, exogenous expression of a T7 polymerase is not always 21

feasible and acts as a confounding factor in compound screening assays. We have developed 22

an alternative minigenome that is controlled by the natively expressed RNA polymerase II. We 23

demonstrate here the characteristics of this new system and its applicability in a wide range of 24

cell types. Our system shows a clear concentration-dependent activity and outperforms the 25

existing T7 polymerase-based system at higher concentrations, especially in difficult-to-26

transfect cell lines. In addition, we show that our system can be used for high-throughput 27

compound screening in a 96-well format, thereby providing an attractive alternative to 28

previously developed MARV minigenomes. 29

30



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Introduction 31

Marburg virus (MARV) is a highly pathogenic virus that was first discovered in 1967 in 32

Germany (Siegert, Shu, Slenczka, Peters, & Muller, 1967; Smith, Simpson, Bowen, & Zlotnik, 33

1967). MARV is the only known member of the genus Marburgvirus, one of the five genera in 34

the family Filoviridae (Amarasinghe et al., 2019). The best-known member of this family is 35

Ebola virus (EBOV), which has caused two large outbreaks in the past few years and 36

consequently has been the center of most filovirus-related research in the past decade (Rojas et 37

al., 2020). Similarly to EBOV, MARV outbreaks are typically associated with a very high case-38

mortality rate. While advances have been made for Ebola virus disease in recent years, there 39

are still no licensed therapeutics or vaccines available for Marburg virus disease (MVD)(WHO, 40

2018). Because of this lack of virus countermeasures, WHO has listed MVD as a ‘priority 41

disease’ for research and development (Mehand, Al-Shorbaji, Millett, & Murgue, 2018). 42

However, studies involving live MARV are difficult to perform due to its classification as a 43

biosafety level 4 (BSL-4) pathogen (U.S. Department of Health and Human Services, 2009). 44

An alternative to handling infectious virus is the use of MARV minigenomes to study different 45

aspects of the viral life cycle. In a minigenome system, the leader and trailer region of the 46

genome are placed adjacent to a reporter gene and transfected into target cells alongside the 47

different components of the viral polymerase complex (Wendt, Bostedt, Hoenen, & Groseth, 48

2019). Once the different viral proteins are expressed, they form polymerase complexes and 49

bind to transcripts of the minigenome, leading to the expression of the reporter gene. The use 50

of easily detectable reporters, such as luciferase or green fluorescent protein (GFP), in a BSL-51

2 setting, has enabled the widespread use of minigenome systems for viral research, although 52

their applicability is usually limited to studying a specific phase of the viral life cycle, i.e., virus 53

replication and/or transcription (Hoenen, Groseth, de Kok-Mercado, Kuhn, & Wahl-Jensen, 54

2011). 55



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The first MARV minigenome was developed over 20 years ago, employing a chloramphenicol 56

acetyltransferase as a reporter gene (Muhlberger, Lotfering, Klenk, & Becker, 1998). Since 57

then, several variations of this minigenome have been made, aimed at improving its usability 58

by replacing the reporter gene with different types of luciferases, GFP or even dual reporters, 59

although the general design of the minigenome has been conserved (Schmidt & Muhlberger, 60

2016). In all currently existing MARV minigenome systems, the minigenome is placed 61

immediately downstream of a T7 RNA polymerase promoter that controls the expression of the 62

minigenome and ensures the generation of 5’ ends with minimal overhang. On the 3’ end, a 63

self-cleaving hepatitis delta virus ribozyme (HdVRz) is attached to the minigenome to ensure 64

the generation of defined 3’ ends, which are necessary for replication activity. 65

Although a potent T7 RNA polymerase promoter is now widely used to drive expression in 66

viral minigenome systems, it does have certain drawbacks that limit its usability. As the T7 67

polymerase is derived from bacteriophages, it is not naturally expressed in mammalian cell lines 68

and thus needs to be provided separately (Lieber, Kiessling, & Strauss, 1989). There are several 69

ways to enable expression of a T7 polymerase in mammalian cells, such as co-transfection of 70

an additional plasmid encoding for a T7 polymerase, or infection of cells with T7-expressing 71

recombinant virus (e.g. replication-deficient vaccinia virus (MVA-T7))(Sutter, Ohlmann, & 72

Erfle, 1995). However, plasmid-based expression of a T7 polymerase can be difficult to achieve 73

in certain cell lines, while the use of MVA-T7 can lead to cytotoxicity, limiting the usability of 74

T7 polymerase-driven systems in certain cell lines (Jordan, Horn, Oehmke, Leendertz, & 75

Sandig, 2009; Nelson et al., 2017; Walpita & Flick, 2005). An example of such a cell line is the 76

Rousettus aegyptiacus-derived RoNi/7.1 cell line. This is particularly troublesome when 77

studying MARV, as Rousettus aegyptiacus bats are known to be the natural reservoir of MARV 78

(Towner et al., 2009). A T7 polymerase can also act as a confounding factor in compound 79

screening assays. Most minigenome systems used for antiviral compound screening are limited 80



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to detecting polymerase (co-factor) inhibitors. As such, the classes of compounds that are likely 81

to show activity in these assays (e.g. nucleoside analogs) are also the ones that might inhibit T7 82

polymerase activity, potentially resulting in high rates of false positivity. 83

In this article, we describe the development and optimization of a MARV minigenome system 84

under the control of a mammalian RNA polymerase II promoter. We show that this system is 85

operational in a variety of cell lines and can be used as a high-throughput compound screening 86

assay, providing a valid alternative for the widely used T7 polymerase-based systems. 87

88

Results and Discussion 89

Development of a polymerase II-driven MARV minigenome 90

One of the key applications of minigenomes is the screening for viral polymerase inhibitors, 91

highlighting the need for a system that is independent from exogenous polymerases. The 92

necessary addition of such secondary polymerases, like the T7 polymerase, acts as a difficult-93

to-correct confounding factor. Conversely, in minigenomes using promoters targeting 94

polymerases natively expressed in mammalian cell types, this confounding does not occur, as 95

off-target inhibition of the host’s RNA polymerase will also directly affect cell viability. RNA 96

polymerase II is the enzyme responsible for the generation of mRNA precursors in all 97

eukaryotic cell types and as such provides an interesting alternative to the bacteriophage-98

derived T7 polymerase(Woychik & Hampsey, 2002). Furthermore, it has previously been 99

shown that RNA polymerase II can be used to drive the expression of viral reverse genetics 100

systems (Griffin et al., 2019; Martin, Staeheli, & Schneider, 2006; Nelson et al., 2017; Wang 101

et al., 2015). To generate an RNA polymerase II-driven MARV minigenome, the trailer and 102

leader regions of MARV, flanking an eGFP reporter gene, were cloned into the pCAGGS-103

3E5E-EBOV-eGFP vector, resulting in an antisense MARV minigenome under the control of 104



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the potent CAG promoter (Figure 1A). In addition, the trailer and leader regions of MARV 105

were flanked by a self-cleaving hammerhead ribozyme and a HDV ribozyme, respectively, to 106

ensure defined 5’ and 3’ ends. Transcription of this minigenome by the cellular RNA 107

polymerase II and subsequent cleavage of the ribozyme sequences allows the minigenome to 108

be bound by the viral polymerase (L) and its co-factors (NP, VP30 and VP35), which are 109

provided in trans. The viral polymerase will then transcribe the reporter gene encoded by the 110

minigenome, producing a measurable eGFP signal. Initial transfection tests showed that this 111

CAG-controlled minigenome had only limited activity (~25%) compared to the T7-driven 112

MARV minigenome (Figure 1B). We hypothesized that the lack of minigenome activity (as 113

evidenced by a weak GFP signal) was due to an incompatibility of the used hammerhead 114

ribozyme with the MARV trailer region. As is the case for EBOV, the MARV trailer contains 115

multiple conserved and self-complementary motifs, allowing the formation of secondary RNA 116

structures. One of these structures is an RNA hairpin that is presumably recognized by the viral 117

polymerase and acts as the replication promoter (Crary, Towner, Honig, Shoemaker, & Nichol, 118

2003; Volchkov et al., 1999). Because the formation of this hairpin is crucial for viral 119

replication to occur, the incorporation of a flanking ribozyme sequence that generates defined 120

5’ ends containing the necessary motifs for secondary structure formation is indispensable to 121

ensure minigenome activity and needs to be incorporated into the ribozyme design. As the 122

genome ends of EBOV and MARV are highly conserved, we initially assumed we could reuse 123

the hammerhead design used for EBOV in the MARV minigenome. This design is a type I 124

hammerhead ribozyme, in which stem I is formed by pairing of the first eleven nucleotides of 125

the virus trailer with a complementary motif at the 5’ end of the ribozyme sequence (Figure 126

2A). However, even though the first ~20 nucleotides of the EBOV and MARV genomes are 127

highly conserved, the upstream motifs differ, resulting in the formation of slightly different 128

hairpin structures (Figure 2B). We hypothesized that these minor but distinct differences in 129



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structure and especially in the position of the EBOV and MARV trailer hairpins result in altered 130

minigenome activity. Predictive modelling of RNA secondary structures in the EBOV trailer 131

shows that a hairpin is formed at the very end (nt 1-54) of the sequence and that the addition of 132

a hammerhead ribozyme, incorporating the first eleven nucleotides of the virus trailer, abolishes 133

the formation of this hairpin. Conversely, the MARV hairpin is located slightly downstream (nt 134

12-62) and its formation is predicted to be unaffected by the addition of the ribozyme, thereby 135

resulting in the attempted formation of two large secondary structures separated by only one 136

nucleotide. Presumably, the presence of another large structure in the proximity of the 137

hammerhead ribozyme results in sterical hindrance, preventing the ribozyme from being formed 138

or cleaving itself, thereby yielding an inactive minigenome transcript. 139

To overcome the lack of minigenome activity, we sought to reduce this sterical hindrance by 140

altering the relative size and position of the ribozyme and hairpin structures. However, simply 141

adding a spacer sequence between the ribozyme and the MARV trailer hairpin to increase the 142

distance between these two structures, or otherwise altering the sequence of the 3’ end of the 143

ribozyme was not feasible, as the beginning of the MARV trailer forms part of stem I of the 144

ribozyme. As such, modifications could only be made by mutating the 5’ end of the ribozyme 145

sequence. Initially, we attempted to mimic the EBOV situation, in which the ribozyme and 146

trailer hairpin share part of their sequence, resulting in the preferential formation of the 147

hammerhead ribozyme. However, sequentially increasing the size of the first stem of the 148

hammerhead to incorporate part of the hairpin sequence did not significantly increase 149

minigenome activity (data not shown), presumably because the majority of the hairpin structure 150

could still be formed. Because lengthening the stem of the ribozyme proved unsuccessful, we 151

explored the opposite by shortening the stem as much as possible. The first eleven nucleotides 152

at the 5’ end of the MARV trailer sequence, which represent one of the complementary strains 153

that form stem I of the hammerhead ribozyme, do not belong to the trailer hairpin. Mutating the 154



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nucleotides at the 5’ end of the ribozyme that are complementary to these eleven nucleotides 155

will therefore reduce base pairing in the ribozyme stem and consequently shorten the length of 156

the stem without altering the MARV trailer sequence. As shown in Figure 3, each additional 157

mutation that shortened the ribozyme stem and, consequently, increased the length of the spacer 158

between the ribozyme and the trailer hairpin, resulted in an increase of minigenome activity 159

(Fig 3A), with a maximum at a shortening of four nucleotides, corresponding to a remaining 160

stem length of six base pairs. Further shortening the stem leads to a rapid loss of activity, in 161

accordance with predictive RNA secondary structure modelling calculating the probability of 162

the hammerhead structure being formed. Based on these findings, the six-basepair-stem 163

hammerhead design was selected for further validation. 164

Comparison of the T7 and RNA polymerase II MARV minigenomes 165

To qualitatively compare the optimized polymerase II-driven minigenome with the T7-166

polymerase control, HEK293T cells were transfected with MARV support plasmids and 167

varying amounts of MARV minigenome plasmid. pCAGGS-T7 was added to all wells 168

transfected with the T7 minigenome. Cells were incubated for 72 hours in an IncuCyte 169

incubator, measuring cell density and fluorescence at distinct time points. As shown in Figure 170

4A, eGFP expression for both systems was almost undetectable after 24 hours but increased 171

steadily over time. Both systems’ activity was dependent on the amount of minigenome 172

transfected, with the T7 polymerase system reaching its optimum at lower concentrations (400 173

ng), while the polymerase II minigenome was more efficient when using higher amounts of 174

plasmid (750/1000 ng). Using more than 1000 ng minigenome plasmid did not result in 175

additional gain of activity (data not shown). Taken together, these data show that, at higher 176

plasmid concentrations, the polymerase II-driven MARV minigenome outperforms the T7 177

polymerase system while displaying a similar time-dependent activity. 178



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To explore the cell dependency of the MARV minigenome system, we next compared the 179

activity of the T7 polymerase and polymerase II-driven minigenomes in HEK293T cells and 180

five additional cell lines (Figure 5). One of the drawbacks of T7 polymerase-dependent systems 181

is that the T7 polymerase is not natively expressed in most mammalian cell lines and needs to 182

be supplied either by co-transfection or through transduction with the MVA-T7 vaccinia virus. 183

However, supplementing the transfection mix with an additional (T7) plasmid reduces 184

transfection efficiency, while not all cell lines are equally suited for transduction. This is 185

particularly true for R. aegyptiacus cell lines, which are known to die rapidly when transduced 186

with MVA-T7 (Jordan et al., 2009). As R. aegyptiacus bats are the natural host for MARV, the 187

availability of a minigenome that can be employed in these cell lines is a highly desired tool for 188

MARV research. Therefore, in addition to cell types commonly used in varying types of 189

filovirus research (Albarino et al., 2013; Enterlein et al., 2006; Tsuda et al., 2015), such as 190

African green monkey Vero E6 cells, hamster-derived BHK21 and BSR-T7/5 cells and the 191

human HEK293T and Huh-7 cell lines, we tested our system in the R. aegyptiacus RoNi/7.1 192

cell line, all. For this experiment, all cell lines were transfected with 1000 ng of either the T7 193

polymerase- or polymerase II-driven MARV minigenome, encoding a Renilla luciferase as 194

reporter gene. After 72 hours, cells were lysed and luciferase activity measured. As summarized 195

in Figure 5, the activity of the polymerase II system was comparable to or higher than the 196

activity of the T7 system. Especially in the difficult-to-transfect Vero E6 and RoNi/7.1 cells, 197

the polymerase II system clearly outperformed the T7 system. The only exception was the BSR-198

T7/5 cell line, where the T7 system yielded higher luciferase levels than the polymerase II 199

system. This cell line is a modified clone of the BHK21 cell line that stably expresses T7 200

polymerase (Buchholz, Finke, & Conzelmann, 1999). Presumably, this native expression of T7 201

polymerase contributes to the increased activity of the T7 polymerase-driven minigenome in 202

this cell line. Together, these results show that the polymerase II system can be successfully 203



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used in a wide variety of cell lines suitable for filovirus research, including cells from R. 204

aegyptiacus bats, the natural hosts for MARV. 205

The polymerase II system as a tool for antiviral research 206

One of the primary applications of minigenome systems is their use in antiviral compound 207

screening. Even though they can only detect polymerase (cofactor) inhibitors, their ease of use 208

makes them an often sought-out alternative to working with live virus. To show that the 209

polymerase II system presented here can be used for antiviral screening, HEK293T cells were 210

transfected with the polymerase II minigenome and all pCAGGS MARV support plasmids. Six 211

hours post transfection, cells were transferred to a 96-well plate prefilled with a dilution series 212

of ribavirin. Ribavirin is a broad-spectrum antiviral compound that is used for the treatment of 213

viral hemorrhagic fevers, respiratory syncytial virus infections and hepatitis C virus infections, 214

although its use is not recommended for the treatment of flavivirus and filovirus infections due 215

to a lack of proven clinical benefit (Huggins, 1989). Nonetheless, ribavirin possesses detectable 216

in vitro anti-MARV activity in the polymerase II MARV minigenome system with an IC50 217

value of 23.7 µM (CC50: 90.9 µM), in accordance with previous results obtained using a T7-218

driven MARV minigenome (Figure 6)(Uebelhoer et al., 2014). 219

To efficiently use a minigenome system for compound screening, scaling up is necessary to 220

reduce the monetary and time cost per compound. By performing transfections in T-75 flasks, 221

using the inexpensive PEI as a transfection reagent and subsequently transferring the transfected 222

cells to 96-well plates filled with compound, well-to-well variation is minimized, increasing 223

the reliability of the obtained results. Using this method, the polymerase II MARV minigenome 224

system was shown to have a Z’ score of 0.8, indicating a highly robust assay (Zhang, Chung, 225

& Oldenburg, 1999). 226



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In conclusion, we present here a novel MARV minigenome system. By specifically tailoring 227

the hammerhead ribozyme sequence to that of the MARV trailer, the minigenome could 228

effectively be placed under the control of an RNA polymerase II promoter. The system shown 229

here represents the first MARV minigenome that can work independently from the addition of 230

an exogenous polymerase, allowing the system to be used in a high-throughput manner and in 231

a wide range of cell lines while concurrently avoiding the lower specificity typically observed 232

with T7 polymerase-based systems. 233

234

Materials and methods 235

Cell lines 236

Cell lines used for this study were human embryonic kidney cells (HEK-293T), African green 237

monkey kidney cells (Vero E6; American Type Culture Collection, C1008), hamster kidney 238

fibroblasts (BHK21, BSR-T7/5), human hepatocellular carcinoma cells (Huh-7) and kidney 239

fibroblasts from the fruit bat Rousettus aegyptiacus (RoNi/7.1), kindly provided by Prof. M.A. 240

Müller, Charité, Berlin, Germany. All cell lines were maintained in Dulbecco’s Modified Eagle 241

Medium (DMEM; Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine 242

serum (FBS; Biowest, France), with the exception of the BHK21 cells, which were maintained 243

in Minimal Essential Medium (MEM) Rega-3 (Thermo Fisher Scientific ) + 10% FBS. 200 mM 244

L-glutamine (Thermo Fisher Scientific) was added to the BHK21, Huh-7, BSR-T7/5 and 245

RoNi/7.1 cell medium. Additional supplements were 1% sodium bicarbonate (Thermo Fisher 246

Scientific) for Huh-7 and BHK21 cells, 1% sodium pyruvate (Thermo Fisher Scientific) for 247

RoNi/7.1 cells and 1% Non-Essential Amino Acids (NEAA; Thermo Fisher Scientific) for Huh-248

7 and RoNi/7.1 cells. Used antibiotics were 1% Penicillin-Streptomycin (Thermo Fisher 249

Scientific) for Vero E6, BSR-T7/5, Huh-7 and RoNi/7.1 cells, 0.5% Geneticin (InvivoGen, 250



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France) for BSR-T7/5 cells and 1% Gentamicin (Thermo Fisher Scientific) and 0.2% Fungizone 251

(Thermo Fisher Scientific) for Vero E6 cells. 252

Minigenome design and construction 253

The artificial T7-3M-Luc-5M minigenome and plasmids encoding the MARV L, NP, VP30 and 254

VP35 proteins were kindly provided by Prof. Stephan Becker. The T7-3M-eGFP-5M 255

minigenome was made by replacing the Renilla luciferase gene in the T7-3M-Luc-5M 256

minigenome with an eGFP gene through assembly of PCR fragments using the NEBuilder HiFi 257

DNA assembly cloning kit (New England Biolabs, MA, USA). A modified version of this 258

minigenome, containing a multiple cloning site and a hammerhead ribozyme (HHRz) sequence 259

between the T7 promoter and MARV trailer sequence (T7-HHRz-3M-eGFP-5M), was made 260

by mutagenesis PCR using the site-directed mutagenesis kit (New England Biolabs). Additional 261

mutants of this intermediate vector were generated through site-directed mutagenesis of the 262

HHRz sequence. Each of the resulting MARV minigenomes was then cloned into the 263

pCAGGS_3E5E_eGFP vector, which was a gift from Elke Mühlberger (Addgene plasmid # 264

103054). SacI-HF (New England Biolabs) was used for restriction digestion, followed by 265

vector-insert ligation using the Quick Ligation kit (New England Biolabs) to generate the 266

original and mutated pCAGGS-HHRz-3M-eGFP-5M vectors. Sanger sequencing was used to 267

confirm the sequence of all vectors. pCAGGS-3M-Luc-5M minigenomes were made using the 268

same approach. 269

Transfection and eGFP imaging 270

TransIT transfection reagent (Mirus Bio, WI, USA) and Lipofectamine LTX were used 271

according to the manufacturer’s instructions. TransIT-293 was used for the transfection of 272

HEK293T cells, TransIT-LT1 for the transfection of BHK-21J, Huh-7 and Vero E6 cells and 273

Lipofectamine LTX for the transfection of BSR-T7/5 and RoNi/7.1 cells. One day prior to 274



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transfection, cells were seeded in 6-well plates at sufficient density to reach 80-90% confluence 275

at the time of transfection. The next day, a mixture consisting of 3:1 (DNA:reagent) transfection 276

reagent and 1000 ng pCAGGS-L, 500 ng pCAGGS-NP, 100 ng pCAGGS-VP35, 100 ng 277

pCAGGS-VP30 and 1000 ng pCAGGS-HHRz-3M-eGFP-5M/pCAGGS-HHRz-3M-rLuc-5M 278

or 1000 ng T7-3M-eGFP-5M/T7-3M-rLuc-5M and 1000 ng pCAGGS-T7 was added to each 279

well. Six hours post transfection, cells were trypsinised and transferred to a 96-well plate (5 x 280

104 cells/well), prefilled with 100 µl DMEM +2% FBS/well. When using rLuc as a reporter, 281

plates were incubated at 37°C for 72 hours prior to assessing luciferase activity using the 282

Renilla-Glo Luciferase Assay System (Promega, Madison, WI, USA). Readout of eGFP was 283

done by incubating and monitoring plates at 37°C for 72h in an IncuCyte® (Essen BioScience 284

Inc., Ann Arbor, MI, USA) for real-time imaging, or by incubating at 37°C for 48h and 285

subsequently adding 6 µM Hoechst 33342 nucleic acid stain (Thermo Fisher Scientific) as a 286

background stain for high content imaging analysis, performed on an Arrayscan XTI (Thermo 287

Fisher Scientific). Fluorescence signal was generated by exciting the cells with the following 288

wavelengths: 386-23 nm (Hoechst) and 485-20 nm (eGFP), and images were taken with a 10X 289

objective. Exposure times and objective offsets were kept constant for each imaging plate. A 290

custom analysis protocol was developed in-house using the Cellomics SpotDetector 291

BioApplication. First, a background reduction was performed on both channels to ensure 292

positive signals. Second, nuclei were detected using a dynamic tresholding method based on 293

differences in intensity peaks. Next, eGFP signals were detected using a fixed-intensity 294

threshold based on images acquired from the eGFP positive control. Additional parameters 295

were determined to remove non-nuclei and non-eGFP objects (false positive signals). Lastly, 296

the percentage and number of eGFP positive cells was calculated using the Cellomics software. 297

For compound screening, transfections were done in T-75 flasks using polyethylenimine 298

(Polysciences Inc, PA, USA). Transfections were done according to the manufacturer’s 299



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instructions, using 8 µg pCAGGS-L, 4 µg pCAGGS-NP, 800 ng pCAGGS-VP35, 800 ng 300

pCAGGS-VP30 and 8 µg pCAGGS-HHRz-3M-eGFP-5M for each flask. 301

RNA secondary structure modelling and statistical analyses 302

Secondary structure modelling of the hammerhead ribozyme and MARV/EBOV trailer hairpins 303

was done using the RNAstructure Fold and MaxExpect web servers 304

(https://rna.urmc.rochester.edu/RNAstructureWeb/). Comparisons of minigenome activity and 305

associated statistical analyses were done using GraphPad Prism 8.2.0. Z-factor calculations for 306

compound screening were done using the equation: Z = 1- [(3σC+ + 3σC-)/(µC+ - µC-)](Zhang et 307

al., 1999). Positive control wells were transfected with all elements of the minigenome assay, 308

while the pCAGGS-L plasmid was omitted from the negative controls. 309

310

Acknowledgments 311

The authors wish to thank prof. E. Mühlberger, Boston University, MA, USA for providing the 312

pCAGGS_3E5E_eGFP plasmid, prof. S. Becker, Philipps-Universität, Marburg, Germany for 313

providing the T7-3M-Luc-5M minigenome and MARV support plasmids, and Prof. M.A. 314

Müller, Charité, Berlin, Germany for providing the RoNi/7.1 cell line. The authors also wish to 315

thank Winston Chiu and Leentje Persoons for excellent technical assistance. BV is supported 316

by a FWO SB grant for strategic basic research of the “Fonds Wetenschappelijk 317

Onderzoek”/Research foundation Flanders [1S28617N]. This work is also supported by 318

‘Interne Fondsen KU Leuven / Internal Funds KU Leuven’ project 3M170314 awarded to KV 319

and PM. The funders had no role in study design, data collection and analysis, decision to 320

publish or preparation of the manuscript. 321

322



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Declarations of interest 323

None 324

325

Author contributions 326

BV, JS and PM conceived the experiments. BV and JS performed the experimental work. BV, 327

KV and PM wrote the main manuscript. KV and PM supplied laboratory materials and funding. 328

All authors revised and approved the manuscript. 329

330



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Figures 425

426

Figure 1 427

Exchange of the T7 promoter by a mammalian CAG promotor leads to a significant loss of 428

MARV minigenome activity. (A) Schematic representation of the pCAGGS-HHRz-3M-eGFP-429

5M minigenome, made by replacing the T7 promoter of the T7-3M-eGFP-5M plasmid by a 430

CAG promoter and a 5’ hammerhead ribozyme (HHRz). HdVRz = hepatitis delta virus 431

ribozyme. (B) Comparison of the T7 polymerase and RNA polymerase II eGFP minigenome 432

systems. HEK293T cells were transfected with 1000 ng of the indicated minigenome, as well 433

as all necessary support plasmids. Images were taken 48 hours post transfection. Cells are 434

stained with Hoechst 33342 as background staining (shown in blue), eGFP-expressing cells are 435

shown in green. 436



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437

Figure 2 438

Secondary structure modelling of RNA structures. (A) Type I hammerhead ribozyme used in 439

the initial pCAGGS-HHRz-3M-eGFP-5M minigenome. The arrow indicates the position at 440

which the ribozyme cleaves. This corresponds to the start of the EBOV/MARV trailer, which 441

is integrated into the 3’ end of the ribozyme sequence (marked in black) to ensure the generation 442

of an exact 5’ end. (B) Comparison of the hairpin structures formed at the 5’ end of the EBOV 443

and MARV trailers shows that the EBOV hairpin is positioned at the very end of the sequence 444

while the MARV hairpin is shifted 11 nucleotides inwards. The start of each sequence is 445

indicated with an arrow, while the residues marked in black correspond to the nucleotides 446

incorporated into stem I of the hammerhead ribozyme. 447

448

449



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450

Figure 3 451

Shortening the ribozyme stem increases minigenome activity. (A) Transfection of HEK293T 452

cells with different mutants of the pCAGGS-HHRz-3M-eGFP-5M minigenome shows that 453

minigenome activity can be increased by reducing complementarity between the ribozyme and 454

the MARV trailer. Shortening by 3-5 nucleotides significantly increases activity, with an 455

optimum at a residual stem length of six base pairs. Data from three independent experiments, 456

done in triplicate. Error bars indicate the standard error of mean. Significance testing was done 457

using one-way ANOVA with Dunnett’s test for multiple comparisons: * p<0.05, ** p<0.01, 458

**** p<0.0001. RFU = Relative fluorescence units, obtained by normalizing the amount of 459

cells/well. (B) Schematic comparison of the unmodified (left) and the maximally active (right) 460

ribozyme-trailer junctions. Nucleotides that are mutated to shorten the ribozyme stem are 461

marked in red, their complementary partners that will act as additional spacers between the 462

ribozyme and the MARV trailer in cyan and the rest of the MARV trailer in blue. See also 463

supplementary table S1. 464



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465

Figure 4 466

Comparison of the T7 polymerase- and RNA polymerase II-driven minigenome systems. (A) 467

HEK293T cells were transfected with 1000 ng of T7 polymerase- or polymerase II-driven 468

MARV minigenome and observed for 72 hours in an IncuCyte. Both systems displayed a 469

similar time-dependent gain in eGFP expression, with the RNA polymerase II system reaching 470

significantly higher expression levels. (B) While the T7 polymerase-driven MARV 471

minigenome reaches its maximum activity at lower amounts of minigenome plasmid used, the 472

RNA polymerase II-driven minigenome displays a clear concentration-dependent effect and 473

outperforms the T7 system at higher plasmid concentrations. Data from three independent 474

experiments, performed in triplicate in 6-well plates. Error bars indicate the standard error of 475

the mean. eGFP expression is measured as the total green signal area, normalized for the amount 476

of cells/well. Significance testing was done using multiple t-tests, correcting for multiple 477

comparisons using the Holm-Sidak method: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. 478

See also supplementary table S1. 479



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Figure 5 481

Minigenome activity comparison in different cell lines. Different cell lines were transfected with 482

all support plasmids and 1000 ng of the T7 polymerase-driven or the modified RNA polymerase 483

II-driven MARV minigenome containing a Renilla luciferase. Data is summarized from three 484

independent experiments, performed in triplicate. RLU = Relative luciferase units, background-485

corrected. Significance testing was done using multiple t-tests, correcting for multiple 486

comparisons using the Holm-Sidak method: * p<0.05. See also supplementary table S1. 487

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491

Figure 6 492

Ribavirin displays selective anti-MARV activity in vitro. HEK293T cells were transfected with 493

the modified RNA polymerase II-driven MARV minigenome and, after 6 hours, transferred to 494

96-well plates prefilled with dilutions of ribavirin. After 48 hours, a clear dose-dependent 495

reduction in minigenome activity was observed. Data from three independent experiments, 496

performed in triplicate. Significance testing compared to the untreated control (0 µM) was done 497

by two-way ANOVA with Dunnett’s multiple comparisons test: * p<0.05, **** p<0.0001. See 498

also supplementary table S1. 499

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Supplementary data 507

Supplementary table S1 508

Detailed overview of the statistical test results obtained in figures 3, 4, 5 and 6. 509

510

Source data 511

Figure 3-source data 512

Data used to create the graph in figure 3. 513

Figure 4-source data A 514

Data used to create the graph in figure 4A. 515

Figure 4-source data B 516

Data used to create the graph in figure 4B. 517





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