1

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Differential recognition of Old World and New World arenavirus envelope glycoproteins 2

by subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) 3

4

Dominique J. Burri1, Joel Ramos da Palma1, Nabil G. Seidah2, Giuseppe Zanotti3, Laura 5

Cendron4, Antonella Pasquato1*, and Stefan Kunz1* 6

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1 Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, 8

Switzerland 9

2 Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 10

Montreal, Canada (Affiliated to the University of Montreal) 11

3 Department of Biomedical Sciences, University of Padua, Padua, Italy 12

4 Department of Biology, University of Padua, Padua, Italy 13

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* Corresponding authors. Mailing address: Institute of Microbiology, University Hospital Center 16

and University of Lausanne, Lausanne CH-1011, Switzerland. Phone: +41-21 314 7743, Fax: 17

+41-21 314 4060, E-mail: Antonella.Pasquato@chuv.ch; Stefan.Kunz@chuv.ch, 18

Running title: arenavirus glycoprotein processing 19

Word count: Abstract: 224 words 20

Text: 4323 words 21

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00072-13 JVI Accepts, published online ahead of print on 27 March 2013

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ABSTRACT 23

The arenaviruses are an important family of emerging viruses including several causative agents 24

of severe hemorrhagic fevers in humans that represent serious public health problems. A crucial 25

step of the arenavirus life cycle is maturation of the envelope glycoprotein precursor (GPC) by 26

the cellular subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Comparison of the 27

currently known sequences of arenavirus GPCs revealed the presence of a highly conserved 28

aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in Old World and Clade 29

C New World arenaviruses, but not in New World viruses of Clades A and B, or cellular 30

substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function 31

analysis, we found that residueY285 of SKI-1/S1P, distal of the catalytic triad, is implicated in 32

the molecular recognition of the aromatic “signature residue” at P7 in the GPC of the Old World 33

Lassa virus (LASV). Using a quantitative biochemical approach, we show that Y285 of SKI-34

1/S1P is crucial for efficient processing of peptides derived from Old World and Clade C New 35

World arenaviruses, but not Clade A and B New World arenavirus GPCs. The data suggest that 36

during co-evolution with their mammalian hosts, GPCs of Old World and Clade C New World 37

viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four amino acid 38

recognition sequences and currently occupy an extended binding pocket. 39

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INTRODUCTION 42

The arenaviruses are a diverse family of emerging viruses that includes several important human 43

pathogens. Based on serological and genetic evidence, the arenaviruses are classically 44

subdivided into the Old World (OW) and New World (NW) group (1). The OW arenaviruses 45

include the prototypic lymphocytic choriomeningitis virus (LCMV) and the highly pathogenic 46

Lassa virus (LASV). The New World (NW) arenaviruses are found in the Americas and 47

comprise Clade A, B, and C. All human pathogens in the NW family belong to Clade B and 48

include Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia (2), and the recently 49

discovered Chapare virus (3). Among the arenaviruses, LASV is the most prevalent pathogen, 50

causing several hundred thousand infections per year with a high mortality rate among 51

hospitalized patients (4). A highly predictive factor for disease outcome in human arenavirus 52

infection is the viral load (5), indicating a close competition between viral spread, replication and 53

the patient’s immune system. Anti-viral drugs that limit viral replication and spread may provide 54

the patient’s immune system a window of opportunity to develop anti-viral immune responses, to 55

control, and ultimately clear the virus. 56

Arenaviruses are enveloped negative strand RNA viruses with a non-lytic life cycle (6). 57

Their genome consists of two single-stranded RNA species, a large segment encoding the virus 58

polymerase (L) and a small zinc finger motif protein (Z), and a small segment encoding the virus 59

nucleoprotein (NP) and glycoprotein precursor (GPC) (7). The GPC precursor is synthesized as a 60

single polypeptide chain. Upon processing by cellular signal peptidases, the unusually stable 61

signal peptide (SSP) remains associated with the GP and plays a role in subsequent maturation 62

and transport (8, 9). GPC is post-translationally processed into GP1 and GP2 by the cellular 63

subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) (10-12), a member of the proprotein 64

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convertase family involved in the regulation of cholesterol homeostasis, ER stress response, and 65

lysosome biogenesis (13, 14). The resulting tripartite SSP/GP1/GP2 complex represents the 66

functional unit of host cell attachment and fusion. The GP1 part is located at the tip of the mature 67

virion spike and undergoes interactions with cellular receptors. The transmembrane GP2 contains 68

the machinery for membrane fusion and resembles fusion-active GPs of other enveloped viruses 69

(15). Maturation of GPC by SKI-1/S1P is strictly required for the production of infectious 70

particles and viral spread (10-12, 16). Proof-of-concept studies with protein- and peptide-based 71

SKI-1/S1P inhibitors revealed that targeting of GPC maturation represents a novel and promising 72

anti-viral strategy (17, 18). More recently, the small molecule SKI-1/S1P inhibitor PF-429242 73

(19, 20) was shown to efficiently block arenavirus cell-to-cell propagation (21, 22) and was able 74

to clear virus from persistently infected cells (21). However, considering the important role of 75

SKI-1/S1P for normal physiology, general inhibitors of SKI-1/S1P’s catalytic activity are likely 76

to cause unwanted side effects (21). A major thrust of our current research is therefore the 77

development of inhibitors that specifically interfere with SKI-1/S1P processing of viral GPCs, 78

but not with the activation of cellular substrates. 79

Processing of arenavirus GPCs by SKI-1/S1P relies on the RX(hydrophobic)X motif 80

which is conserved among all members of arenaviruses and represents the minimal and sufficient 81

requirement for recognition (13, 23). Although the four P1 to P4 residues at the cleavage site are 82

generally believed to determine the protease specificity, efficiency of GPC processing can be 83

modulated by flanking residues (24). Indeed, in vitro investigations of synthetic peptides 84

mimicking LASV GPC processing site showed that aromatic residues at position P7 strongly 85

enhance cleavage by SKI-1/S1P (23). Similarly, F259A replacement at P7 of LCMV GPC 86

impaired maturation (10). Based on this indirect evidence, it has been hypothesized that the 87

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catalytic pocket of SKI-1/S1P may interact with additional substrate residues distal from the 88

actual cleavage site (23). In the present study, we further investigated this aspect. Comparison of 89

the currently known sequences of arenavirus GPCs revealed the presence of a conserved 90

aromatic residue at position P7 relative to the SKI-1/S1P cleavage side in OW and Clade C NW 91

arenaviruses, but not in NW viruses of Clades A and B, or cellular substrates of SKI-1/S1P. 92

Combining molecular modeling and biochemical analysis, we found that the residueY285 of 93

SKI-1/S1P, located distal of the catalytic triad, is implicated in the molecular recognition of the 94

aromatic “signature residue” at P7 and is crucial for efficient processing of OW and Clade C NW 95

arenavirus GPCs. 96

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MATERIALS AND METHODS 99

Cell lines and Viruses 100

Chinese hamster ovary (CHO)-K1 cells were cultivated in DMEM/Ham’s F12 1:1 (Biochrom 101

AG, Berlin, Germany) supplemented with 10% FBS and penicillin/streptomycin. SKI-1/S1P 102

deficient CHOK1 cells (SRD12B) (25) were supplemented with 5μg/ml Cholesterol (Sigma), 103

20μM sodium oleate (Sigma) and 1mM Sodium Mevalonate (Sigma). All cell lines were grown 104

at 37°C, 5% CO2. 105

Antibodies 106

Monoclonal antibodies (mAb) anti-V5 was purchased from Invitrogen. MAb anti-5xHis was 107

used to detect SKI-1/S1P-BTMD (Qiagen, Chatsworth CA, USA). MAb M2 anti-FLAG was 108

from Sigma Aldrich. Polyclonal rabbit anti-mouse secondary antibodies conjugated to HRP 109

(Dako, Glostrup, Denmark) was also used. 110

In silico modeling of SKI-1/S1P 111

The full length human SKI-1/S1P amino acid (aa) sequence was submitted to the I-Tasser server 112

(http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (26, 27). The crystal structure of the PC furin 113

(pdb 1P8J) was used to assign template constrains. Prodomain of preproSKI-1/S1P was manually 114

removed using PyMol. The resulting structure was submitted to the energy minimization server 115

ModRefiner (http://zhanglab.ccmb.med.umich.edu/ModRefiner/) (28). Peptide docking was 116

achieved by substitution of aa composing the prodomain segment covering the catalytic site in 117

the original model (non-refined) by the IYISRRLL sequence, followed by energy minimization. 118

All pictures were produced using PyMol (http://www.pymol.org/). 119

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Plasmids and transfections 120

Plasmids coding for wild type (WT) and mutants SKI-1/S1P (pIR SKI-1/S1P FL WT, pIR SKI-121

1/S1P H249A, pIR SKI-1/S1P-BTMD) were previously reported (13, 29). All the constructs 122

were cloned into a pIRES-EGFP vector (Invitrogen), a V5 tag was added in C-terminus except 123

for SKI-1/S1P-BTMD (6xHis tag). SKI-1/S1P Y285A FL and SKI-1/S1P-BTMD Y285A 124

mutants were generated by site-directed mutagenesis (Stratagene) according to the 125

manufacturer‘s protocol. Primers forward 5‘-126

CCAATAATCAGGTATCTGCCACATCTTGGTTTTTGG-3’ and reverse 5‘-127

CCAAAAACCAAGATGTGGCAGATACCTGATTATTGG-3‘were used. The cDNA coding 128

for LCMV GP and LASV GP are described elsewhere (18). All transfections were performed 129

with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Briefly, 2 x105 130

cells were seeded in a 24-well plate 24h prior transfection. Palsimd DNA (0.8 μg) was dissolved 131

in OPTI-MEM medium (Gibco BRL, NY) and mixed with OPTI-MEM-Lipofectamin 2000 132

(2μl/transfection), incubated 15 min at RT, and added to the cells. After 4 hrs, transfection 133

solutions were replaced by fresh medium. 134

Western blotting 135

Cells were washed twice with cold PBS and lysed in SDS-PAGE sample buffer (62.5mM Tris-136

HCl pH6.8, 20% glycerol, 2% SDS, 10% DTT) supplemented with mini complete protease 137

inhibitor cockteil (Roche). Samples were boiled for 10 min at 95°C and centrifuged for 5 min at 138

13‘000 rpm prior loading. Proteins were separated on Tris-HCl ready precast 10% 139

polyacrylamide gel (BioRad) and blotted on nitrocellulose membrane. Unspecific binding site 140

were blocked 1h in blocking solution (PBS, 0.2% Tween 20 (wt/vol) with 3% non-fat milk 141

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(wt/vol)) at room temperature. Membranes were incubated with primary antibody (mAb anti-142

FLAG 1:1000) at 4°C under constant agitation. After overnight incubation, membranes were 143

washed 3 times in washing solution (PBS, 0.2% Tween 20 (wt/vol)) and incubated 1h at RT in 144

secondary HRP-conjugated antibody (1:3000). After 3 washes, membranes were developed with 145

the LiteABlot kit (EuroClone, Pero, Italy). 146

Real-time quantitative PCR (RT-qPCR) 147

Total RNA was isolated using RNeasy Mini Kit by Qiagen. The lysate was homogenized with a 148

Qiashredder kit (Qiagen). The RT reaction was performed using Quantitect Reverse 149

Transcription Kit by Qiagen with 1 μg of template RNA. PCR was done with SYBR® green 150

Reagents. Specific primers were used to determine the level of expression of genes downstream 151

SREBP and ATF6. TaqMan probes specific for heat shock 70 kDa protein A5 (HSPA5; 152

Hs99999174_m1), 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS1; 153

Hs00940429_m), low-density lipoprotein receptor (LDLR; Hs00181192_m1), and 154

glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs99999905_m1) were obtained from 155

Applied Biosystems (Foster City, CA). Real-time PCR analyses were performed in triplicate 156

(real-time PCR ABI PRISM 7000 sequence detection system Applied Biosystems) using the 157

following protocol: 10 min 95°C (one cycle), 15 s 95°C, followed by 30 s 58°C (40 cycles). 158

Pharmacological treatment 159

Induction of genes regulated by SREBP was triggered by cholesterol deprivation culturing 160

CHOK1 in Ham’s F-12 DMEM 1:1, 5% LPDS, 50 μM sodium mevalonate, 50 μM mevastatin 161

(Enzolifescience) for 18 hours, as reported previously (30). The induction of genes downstream 162

ATF6 was triggered by the addition of 5 µg/ml tunicamycin (Sigma-Aldrich) for 4 h before lysis 163

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as described previously (31). Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used as vehicle 164

control. 165

Determination of in vitro activity of soluble SKI-1/S1P variants 166

To assess the in vitro activities of SKI-1/S1P-BTMD WT and SKI-1/S1P-BTMD Y285A, 167

constructs were expressed in HEK293 cells and enzymatic activity detected in concentrated 168

conditioned medium using a reliable enzymatic assay as described (32). Briefly, enzyme and the 169

SKI-1/S1P substrate Ac-RRLL-MCA (custom synthesis, GenScript, USA) (32) or a library of 170

custom synthesized peptides were mixed. The reactions were carried out at room temperature in 171

buffer solution 25 mM Tris-HCl pH 7.5, 25mM MES and 1 mM CaCl2 and fluorescence detected 172

in a TriStar LB 941 multimode microplate spectrofluorometer (Berthold Technologies, Bad 173

Wildbad, Germany). The peptides Ac-YISRRLL-MCA, Ac-FISRRLL-MCA, Ac-AISRRLL-174

MCA were purchased from GenScript Co. at >92% HPLC purity. Soluble SKI-1/S1P WT and 175

Y285A mutant were produced as described above. Peptides at 25μM were incubated with 10μl of 176

conditioned HEK293 medium containing either SKI-1/S1P WT or YA soluble enzymes in final 177

100μl volume containing CaCl2 2mM, 25mM MES, 25mM Tris at pH 7.5. Cleavage of 178

substrates was monitored by measuring fluorescence as described above. 179

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RESULTS 181

Aromatic residues at P7 position are conserved in GPCs of OW and NW arenaviruses of 182

Clade C 183

Although the four amino acids at the P1 to P4 positions of the SKI-1/S1P processing site of 184

arenavirus GPCs generally determine the enzyme specificity, earlier studies provided the first 185

evidence that flanking residues can modulate substrate cleavability (23) affecting the energy 186

levels of the transient enzyme-substrate complex. In order to identify further amino acids 187

potentially implicated in the recognition of arenavirus GPCs by SKI-1/S1P, we aligned the 188

sequences of various GPCs, including the processing site (P1-P4) and the 5 amino acids 189

upstream (P9-P5) (Fig. 1). Sequences were grouped based on their classification within the 190

OW/NW sub-groups and the relative Clades. Our sequence comparison revealed that R at P4 is 191

absolutely conservd in all sequences and that R at the P3 position is the only amino acid that is 192

accepted in OW and NW Clade C GPCs. In contrast, NW Clade A and B GPCs can 193

accommodate different residues in that position, although the requirement for a hydrophilic side 194

chain seems to be conserved (Fig. 1). This corroborates earlier work, which revealed that OW 195

arenaviruses GPCs mimic the SKI-1/S1P autoprocessing site C “RRLL” while NW 196

arenaviruses GPCs resemble the SKI-1/S1P autoprocessing B “RSLK” (32). At position P5, S 197

or T are frequent among all arenavirus GPCs (Fig. 1). The hydroxyl side chain at P5 may confer 198

some advantage, since it is also found in all cellular substrates, with the exception of ATF6 (33). 199

Our alignment further revealed that all currently known GPCs of OW and NW Clade C 200

arenaviruses share a highly conserved aromatic residue at P7 position, which is absent from 201

Clade A and B NW arenavirus GPCs, as well as cellular substrates (Fig. 1). The remarkable 202

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conservation of the aromatic “signature residue” at P7 in all known OW and Clade C NW viruses 203

warranted further investigation. 204

205

Modeling of the SKI-1/S1P catalytic pocket identified Y285 as potential residue interacting 206

with LASV GPC Y253 207

To gain insight into the nature of the residues within and surrounding the catalytic pocket of 208

SKI-1/S1P, we used an in silico modeling approach as the structure of SKI-1/S1P has not yet 209

been resolved. The amino acid sequence of human SKI-1/S1P (Uniprot entry Q14703) was 210

submitted to I-Tasser server (27) which generated a series of predicted models by multiple 211

alignments with already known 3D structures. Furin, a closely related PC whose catalytic 212

domain shares more than 30% identity with SKI-1/S1P, was used as reference template. Each 213

model was then manually investigated to assess its accuracy and reliability. Based on the relative 214

position of the catalytic triad (D218, H249, and S414), only one model among the three 215

generated showed a conformation compatible with a catalytic function. This model was then 216

edited to remove the prodomain to highlight the residues within and surrounding the catalytic 217

pocket and refined using ModRefiner (http://zhanglab.ccmb.med.umich.edu/ModRefiner/) and 218

FG-MD (34). Since the available data suggested that aromatic residues at P7 are preferred over 219

hydrophobic ones (23), we hypothesized that the side chain of an aromatic amino acid exposed 220

on the SKI-1/S1P catalytic groove (S7 position) may be engaged in an interaction with the 221

corresponding residue at P7, as, among non-covalent bonds, aromatic-aromatic interactions 222

involving the π-electron systems are energetically favored. We thus scanned the surface of the 223

SKI-1/S1P catalytic groove and identified Y at position 285 as a possible candidate contact 224

residue, being exposed, free from further interactions, and located in a region accessible to the 225

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substrate’s P7 residue (Fig 2A). To confirm the accessibility of the substrate at P7 to Y285, 226

peptide-docking analysis was performed. The LASV GPC-derived peptide IYISRRLL was 227

virtually docked into the catalytic groove of the SKI-1/S1P model. The results suggested that the 228

aromatic residue in P7 of GPC may indeed interact with Y285 of SKI-1/S1P (Fig. 2A). 229

Next, we generated the mutant SKI-1/S1P Y285A using site-directed mutagenesis (Fig. 230

2B). Protein expression and maturation were assessed by transient transfection in SKI-1/S1P-231

deficient SRD12B cells. Wild-type SKI-1/S1P and the catalytically dead mutant H249A were 232

used as positive and negative controls, respectively. In cell lysates the expression levels of SKI-233

1/S1P Y285A mutant were comparable to the WT, as assessed by Western blot. Interestingly, 234

autoprocessing at site C of SKI-1/S1P Y285A was less efficient with a consequent accumulation 235

of the B’B intermediate form (Fig. 2C). In line with published data, the control mutant H249A 236

showed no autoprocessing (Fig. 2C). 237

In order to characterize the catalytic activity of the SKI-1/S1P Y285A mutant, we tested 238

its ability to process the transcription factor SREBP2, a cellular substrate of SKI-1/S1P. Upon 239

consecutive cleavage by SKI-1/S1P in the Golgi lumen and the protease site-2 protease (S2P) at 240

the cytoplasmic face, the cytosolic part of SREBP translocates into the nucleus where it activates 241

the trancription of target genes, whose expression can be quantified by RT-qPCR. Following 242

transfection of WT SKI-1/S1P (positive control) or SKI-1/S1P Y285A into SKI-1/S1P deficient 243

SRD12B cells (25), processing of the sterol regulatory element binding protein (SREBP2) was 244

induced by sterol depletion and RT-qPCR performed to monitor the induction of the downstream 245

gene HMGCS1. No significant differences between SKI-1/S1P WT and the Y285A mutant were 246

observed (Fig. 2D). In addition, we tested the ability of SKI-1/S1P Y285A to process another 247

cellular substrate, the activation transcription factor (ATF)-6 involved in the cellular ER stress 248

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response. Upon induction of ER stress, e.g. by tunicamycin, an inhibitor of protein N-249

glycosylation, ATF6 is cleaved by SKI-1/S1P and S2P. The released nATF6 translocates to the 250

nucleus where it activate ER-stress response genes, including HSPA5. Similar levels of HSPA5 251

induction were detected in SKI-1/S1P Y285A and WT transfected cells after exposure to 252

tunicamycin (Fig. 2D). Taken together, these results show that replacement of Y285 of SKI-253

1/S1P with A, although reducing the efficacy of zymogen processing, it still generates enough of 254

a fully functional enzyme against key cellular substrates. 255

256

SKI-1/S1P Y285 is crucial for recognition of the aromatic “signature residue” at P7 in 257

arenavirus GPCs 258

The modeling data at hand suggested that residue Y285 of SKI-1/S1P may contribute to the 259

molecular interaction of aromatic residues at P7 position of some arenavirus GPCs with the 260

catalytic pocket of the enzyme, thereby promoting substrate processing. To validate our 261

prediction experimentally, we generated FLAG-tagged variants of LASV GPC that contained Y, 262

F, or A in position P7. The LASV GPC variants were co-transfected with SKI-1/S1P Y285A or 263

WT into SKI-1/S1P null SRD12B cells. As a control, we used the GPC of the Clade B NW virus 264

JUNV that lacks an aromatic residue in P7 (Fig. 1). To mimic the likely molar excess of GPC 265

over SKI-1/S1P in infected cells, expression plasmids were used at a ratio of 3:1 in favor of the 266

GPC. At 48 hrs post-transfection, cells were lysed and GPC processing examined by Western 267

blotting using an anti-FLAG antibody. In line with previous results, the Y253A mutation in 268

LASV GPC resulted in reduced cleavage by WT SKI-1/S1P (Fig. 3). In contrast, substitution of 269

LASV GPC Y253 by F did not affect SKI-1/S1P processing, emphasizing the importance of the 270

aromatic nature of the residue at P7, but not Y per se. Consistent with our modeling-based 271

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predictions, processing of WT LASV GPC by SKI-1/S1P Y285A was impaired (Fig. 3). As 272

expected, processing of LASV GPC Y253A by SKI-1/S1P Y285A was also markedly reduced. 273

In contrast, the processing of JUNV GPC that contains an L in P7 position was not affected by 274

the Y285A mutation in SKI-1/S1P (Fig. 3). These data provided the first evidence for a possible 275

interaction between Y285 of SKI-1/S1P and aromatic residues in P7 of a GPC substrate 276

In order to quantify the enzymatic parameters of SKI-1/S1P Y285A towards different 277

viral and cellular substrates, we employed a robust and reliable biochemical assay using a 278

soluble recombinant form of SKI-1/S1P. To this end, we introduced the Y285A mutation in a 279

soluble SKI-1/S1P form truncated before the transmembrane domain (SKI-1/S1P BTMD) by 280

site-directed mutagenesis (Fig. 4A). The Y285A mutant and WT SKI-1/S1P BTMD were 281

transiently transfected into SRD12B cells, followed by expression of recombinant soluble 282

proteins in serum-free medium. Detection of the soluble SKI-1/S1P variants in the cell 283

supernatant by Western blot revealed significantly reduced secretion of SKI-1/S1P BTMD 284

Y285A when compared to the WT (Fig. 4B). This was expected considering the previously 285

observed impaired processing of the B/B’ form of the full length SKI-1/S1P Y285A into the 286

mature C form (Fig. 2C). The specific activity of SKI-1/S1P BTMD present in conditioned 287

supernatants was measured using the well-characterized peptide substrate Ac-RRLL-MCA, 288

which contains the critical four amino acid SKI-1/S1P recognition sequence RRLL present in the 289

C autoprocessing site and LASV GPC (32), as described in the Materials and Methods. Despite 290

the apparently lower concentration of SKI-1/S1P BTMD Y285A in conditioned supernatants 291

(Fig. 4B), the enzymatic activity detected in our peptide-based assay seemed overall higher, 292

indicating a robust specific activity of SKI-1/S1P BTMD Y285A against the basic substrate 293

sequence RRLL (Fig. 4C). 294

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Next, we compared the activity of SKI-1/S1P BTMD Y285A and WT against the LASV 295

GPC-derived peptide Ac-IYISRLL-MCA, the peptide Ac-FYISRLL-MCA derived from the 296

GPC of the OW virus Mopeia (MOPV), and the peptide Ac-QKSIAVGRTLK-MCA from the 297

GPC of the Clade B NW virus Tacaribe (TACV). The TACV-derived peptide was chosen over 298

the JUNV peptide due to its superior properties as a substrate in our in vitro enzymatic assay 299

(32). Briefly, SKI-1/S1P BTMD Y285A and WT were incubated with increasing concentrations 300

of the substrates and enzymatic activities assessed by monitoring the released AMC fluorescence 301

over time. Data were plotted as initial rate (RFU/sec) vs substrate concentration. The Michaelis-302

Menten curves for the LASV and MOPV GPC-derived peptide showed a dramatic reduction of 303

the reaction velocity when the substrate was digested with SKI-1/S1P BTMD Y285A (Vmax-304

YA) in comparison to the WT enzyme (Vmax-WT) (Fig. 4D, Table 1). In contrast, Vmax of 305

TACV GPC-derived peptide reached similar values with either SKI-1/S1P BTMD WT or the 306

Y285A mutant (Fig. 4D, Table 1). To highlight the specific contribution of the aromatic residue 307

in P7, we compared LASV GPC-derived peptides of different length. Processing by SKI-1/S1P 308

BTMD Y285 revealed reduced processing of longer peptides including the P7 position when 309

compared to the four amino acid substrate peptide RRLL (Fig. 4E, Table 1). As expected, the 310

length of the TACV GPC-derived peptides did not affect efficiency of processing. 311

To extend our findings, we examined the processing of peptides derived from the GPCs 312

of the Clade A virus Pichinde (PICV) and the Clade C virus Latino (LATV) by SKI-1/S1P 313

BTMD Y285A and WT. As shown in Fig. 5, the Y285A mutant showed markedly reduced 314

reaction velocity for substrates derived from the Clade C virus LATV containing an aromatic 315

residue at P7 position, while peptides derived from the Clade A virus PICV, lacking aromatic 316

residues at P7, were only mildly affected by the mutation (Fig. 5, Table 1). 317

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Lastly, we sought to confirm the biochemical interaction between the aromatic “signature 319

residue” at P7 of the GPC with Y285 if SKI-1/S1P in a complementary manner using 7mer 320

peptides mimicking the cleavage site of LASV GPC (P1-P6) and carrying either WT Y or F/A at 321

P7, coupled to the fluorogenic MCA group at the C-terminus (Ac-YISRRLL-MCA, Ac-322

FISRRLL-MCA, Ac-AISRRLL-MCA). Peptides at 25 μM concentration were digested in vitro 323

by soluble SKI-1/S1P WT and Y285A in buffer solution at pH 7.5 and processing evaluated by 324

monitoring the release of AMC over time. For each peptide, fluorescence values were 325

extrapolated after 3 hrs of digestion. In line with previous reports (23), Ala at P7 affected the 326

interaction with the wild type enzyme, resulting in decreased peptide processing when compared 327

to Ac-YISRRLL-MCA and Ac-FISRRLL-MCA (Fig. 6, Table 1) . The opposite was observed 328

when the three peptides were digested by SKI-1/S1P Y285A: efficiency of cleavage of both Ac-329

YISRRLL-MCA and Ac-FISRRLL-MCA was greatly affected resulting in a significant decrease 330

of fluorescence detection. In contrast, the digestion of Ac-AISRRLL-MCA was unaffected Fig. 331

6, Table 1), supporting the hypothesis that Y285 of SKI-1/S1P does not represent a key contact 332

residue for the recognition of substrates lacking an aromatic residues at P7 position. 333

Corroborating our molecular modeling, the experimental data indicate that residueY285 of SKI-334

1/S1P, distal from the catalytic triad, is likely implicated in the molecular recognition of the 335

aromatic “signature residue” at P7 and is crucial for efficient processing of OW and Clade C NW 336

arenavirus GPCs. 337

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DISCUSSION 339

Using a combination of molecular modeling, mutagenesis and biochemical assays, we 340

investigated the molecular recognition of arenavirus GPCs by SKI-1/S1P and reached the 341

following conclusions: 1) The GPCs of OW and Clade C NW arenaviruses contain a conserved 342

aromatic residue at position P7 of the SKI-1/S1P cleavage site, which is absent from other 343

cellular and viral substrates. 2) In docking experiments, residueY285 of SKI-1/S1P, located 344

distal from the catalytic triad, likely contributes to the molecular recognition of the aromatic 345

residue at P7 of LASV GPC. 3) Site-directed mutagenesis of SKI-1/S1P and biochemical assays 346

provided evidence that Y285 is crucial for efficient processing of OW and Clade C NW 347

arenavirus GPC sequences containing an aromatic “signature” residue at P7. 348

Substrate recognition by cellular PCs is generally thought to be dependent on the amino 349

acid residues comprising the consensus sequence (14, 35). In substrates for basic amino-acid 350

specific PCs, residues flanking the basic amino acids at positions P1 and P4 of the recognition 351

sequences can influence the specificity for the protease (36). Classical examples in viral PC 352

substrates are mutations nearby the furin processing site of the hemagglutinin of Hong Kong and 353

H5N1 strains of avian influenza that can accelerate the rate of cleavage and increase the 354

infectivity of virus particles (37, 38). Several examples of critical residues beyond the P1-P4 355

positions of cellular PC-substrates have also been reported. In the substrate proGdf11, the 356

presence of an N at position P1’ after the scissile bond markedly reduces cleavage by PACE4, 357

furin and PC7 while being selectively permissive to PC5/6 (39). Processing of nodal, a regulator 358

of the fate of pluripotent cells, is dramatically enhanced by L and G at P2’and P3’ positions 359

respectively, providing further support for the concept that residues distal from the cleavage site 360

can modulate the processing efficiency (40). 361

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Sequence alignments of all currently known arenavirus GPCs revealed that GPCs of OW 362

and Clade C NW viruses contain conserved aromatic residues Y or F at position P7, which are 363

absent from other NW arenavirus GPCs or cellular substrates. The extent of conservation 364

indicated a strong selective pressure at work and suggested that the RRLL motif alone may be 365

sub-optimal in terms of binding to the catalytic pocket of SKI-1/S1P and may need an aromatic 366

residue at P7 for stabilization and optimal cleavage. 367

Starting from the hypothesis that OW and Clade C NW arenavirus GPC may have 368

evolved to fit into an extended pocket of SKI-1/S1P, we performed molecular modeling of the 369

catalytic domain and performed docking experiments with LASV GPC-derived peptides. These 370

molecular modeling studies pinpointed Y285 of SKI-1/S1P as a putative contact residue involved 371

in the recognition of the aromatic “signature residue” at P7 of the vial GPC. Sequence 372

alignments of SKI-1/S1P revealed a high degree of conservation of aromatic residues in 373

positions corresponding to 285 of the human form, including in rodent species that are natural 374

hosts for arenaviruses. Substituting Y285 of SKI-1/S1P for A selectively reduced processing of 375

LASV GPC, but not JUNV GPC that contains an L in P7. Using a robust and reliable 376

biochemical assay, we found that SKI-1/S1P Y285A was specifically and markedly impaired in 377

processing of peptides derived from OW and Clade C NW arenavirus GPCs, but not peptides 378

from Clade A and B NW viruses or the SKI-1/S1P B autoprocessing site (32). In sum, our study 379

provides evidence that during co-evolution with their mammalian hosts, OW and Clade C NW 380

arenaviruses expanded the molecular contacts between their GPCs with SKI-1/S1P beyond the 381

classical four amino acid recognition sequence and currently occupy an extended binding pocket, 382

including residue Y285. The specificity of the interaction between Y285 of SKI-1/S1P and 383

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aromatic P7 residues for LASV GPC processing, but not cleavage of cellular substrates, makes 384

this interaction further a promising target for the development of specific anti-viral drugs. 385

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ACKNOWLEDGEMENTS 387

The authors thank Michael S. Brown and Joseph L. Goldstein (University of Texas Southwestern 388

Medical Center, Dallas, TX) for the S1P-deficient SRD12B cells. This research was supported 389

by the Swiss National Science Foundation grant FN 31003A-135536 (S.K.), funds from the 390

University of Lausanne (S.K.), as well as a Canada chair research grant # 216684 (NGS). 391

392

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510

511

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LEGENDS TO FIGURES 513

514

FIG. 1. Alignment of SKI-1/S1P recognition motifs within arenaviruses GPCs. Amino acid 515

sequences of NW and OW arenavirus GPCs were aligned using ClustalW. Aligned sequences 516

were then submitted to the WebLogo server (41). Size of the amino acid represents the frequency 517

of occurrence of this amino acid at this position in the alignment. Aromatic amino acids in P7 518

position of OW and Clade C of NW arenavirus GPC are highlighted in light gray. The vertical 519

arrow indicates the location of the cleavage. Positions upstream the cleavage site (P1-P9) are 520

also indicated. 521

FIG. 2. Molecular modeling and characterization of the mutant SKI-1/S1P Y285. (A) Left 522

panel: Cartoon representation of the structural model of SKI-1/S1P. Positions of the catalytic 523

residues D212, H249 and S414 are highlighted in red. The position of Y285 in the extended SKI-524

1/S1P catalytic groove is indicated. Right panel: Surface representation of the SKI-1/S1P model. 525

Positions of the catalytic residues H249 and S414 are in red. Modeled position of Y285 is in 526

orange. The in silico docking result shows the conformation of the LASV GPC derived peptide 527

IYISRRLL shown in yellow. (B) Schematic representation of WT and Y285A SKI-1/S1P. The 528

prodomain is shown in gray and the catalytic domain in light gray, with critical residues of the 529

catalytic triad highlighted. The transmembrane domain is represented by a gray box with an 530

arrow, followed by the cytoplasmic tail. The processing site for signal peptidase (site A) and 531

autoprocessing sites B/B’, as well as C are highlighted. The C-terminal V5 tag is indicated and 532

the residues at position 285 are highlighted in red. (C) Expression and autoprocessing of SKI-533

1/S1P variants. SKI-1/S1P WT and the mutants Y285A and H249A were transfected in SKI-534

1/S1P deficient SRD12B cells. Expression and autoprocessing was assessed by Western blotting. 535

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SKI-1/S1P H249A, an inactive mutant, was used as negative control. Positions of SKI-1/S1P 536

precursor (pSKI-1/S1P) as well as the BB’ and C forms of the enzyme are indicated. (D) Normal 537

processing of the cellular substrates SREBP2 and ATF6 by the SKI-1/S1P Y285A mutant. Cells 538

were subjected to cholesterol depletion to monitor the SREBP2-mediated upregulation of the 539

genes for 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS1) and ER stress to assess 540

the ATF6-mediated induction of heat shock 70-kDa protein 5 (HSPA5). SRD12B cells were 541

transfected with SKI-1/S1P WT, the Y285A mutant, or vector only. After 48 hrs, genes 542

downstream of SREBP2 were induced by the treatment of cells with 50 μM mevastatin. To 543

induce genes downstream of ATF6, cells were treated with 5 μg/ml tunicamycin for 4 hrs. 544

CHOK1 cells were used as positive control. After treatment, cells were washed twice with PBS, 545

and total RNA was isolated to perform RT-qPCR analyses as described in Materials and 546

Methods. Data were normalized by using the calibrator gene hydroxymethylbilane synthase 547

(HMBS). Data are presented as fold induction above levels for mock (DMSO)-treated cells 548

(means + SD; n = 3). Data were analyzed using two-way ANOVA followed by Bonferroni post 549

test with GraphPad Prism. The lack of statistical significance between SKI-1/S1P WT and 550

Y285A (NS) is indicated. 551

552

FIG. 3. The Y285A mutation in SKI-1/S1P affects processing of LASV GPC but not JUNV 553

GPC. SRD12B cells were co-transfected with the indicated FLAG-tagged LASV GPC and V5-554

tagged SKI-1/S1P variants using a 3-fold excess of plasmids encoding the GPCs. As a negative 555

control, Flag-tagged JUNV GPC was used. After 48 hrs, cells were lysed and subjected to SDS-556

PAGE followed by Western blotting. GPC and GP2 were detected using anti-FLAG antibodies. 557

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Processing efficiencies are defined as the ratio of GP2/GPC and normalized to the efficiencies of 558

processing of the WT LASV and JUNV GPC by the WT SKI-1/S1P set at 100. 559

560

FIG. 4. The Y285A mutation in SKI-1/S1P specifically affects processing of peptides 561

derived from OW and Clade C NW viruses. (A) Schematic representation of the soluble form 562

of SKI-1/S1P Y285A. SKI-1/S1P Y285A-BTMD was generated by the removing the 563

transmembrane domain and cytosolic tail of full length SKI-1/S1P Y285A. (B) Expression of 564

SKI-1/S1P-BTMD WT and Y285A. HEK293T were transfected with either the SKI-1/S1P-565

BTMD or the SKI-1/S1P Y285A-BTMD mutant. Medium was then replaced by serum-free 566

medium. Expression of the SKI-1/S1P soluble variants was detected in the medium by Western 567

blot. The position of the active enzyme is indicated by an arrow. (C) Enzymatic activity of SKI-568

1/S1P-BTMD WT and Y285A mutants. Cell culture medium used in panel B was used for in 569

vitro determination of SKI-1/S1P enzymatic activity. Samples were incubated with 10 μM Ac-570

RRLL-MCA in pH 7.5 buffer solution, and the released fluorescent 7-amino-4-methyl-571

coumaride (AMC) (relative fluorescence units [RFU]) was monitored over 6 hrs. Data are 572

triplicates + SD. (D) Processing of arenavirus GPC-derived peptides by SKI-1/S1P Y285A and 573

WT. The LASV GPC-derived peptide Ac-IYISRRLL-MCA, the MOPV-derived peptide Ac-574

FYISRRLL-MCA, or the TACV GPC-derived peptide Ac-QKSIAVGRTLK-MCA were 575

incubated with normalized amounts of either SKI-1/S1P-BTMD WT or Y285A. Fluorescence 576

was monitored and analyzed as described in Material and Methods. Michaelis-Menten curves 577

show initial rates (Vi) in function of substrate concentration (in μM). One out of three 578

representative experiments is shown. (E) Impaired processing of extended LASV GPC-derived 579

peptides by SKI-1/S1P Y285A. The indicated peptides were digested with SKI-1/S1P Y285A 580

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and WT as in (D). The percentage of Vmax SKI-1/S1P Y285A/ Vmax SKI-1/S1P WT for the different 581

substrates were then calculated (means + SD; n = 3). 582

583

FIG. 5. Processing of peptides derived from Clade A and C NW arenaviruses. The indicated 584

LATV GPC-derived peptides (Ac-SFITRRLQ-MCA, Ac-RRLQ-MCA) and the PICV-derived 585

peptides (Ac-YSSVSRKLL-MCA, Ac-RKLL-MCA) were incubated with normalized amounts 586

of either SKI-1/S1P-BTMD WT or Y285A and in 4D. Fluorescence was monitored and 587

Michaelis-Menten curves recorded. Data shown are initial rates (Vi) in function of substrate 588

concentration (in μM). One out of three representative experiments is shown. 589

590

FIG.6. Processing of mutant LASV GPC-derived peptides by SKI-1/S1P and SKI-1/S1P 591

Y285A. The peptides Ac-YISRRLL-MCA, Ac-AISRRLL-MCA, and Ac-FISRRLL-MCA were 592

subjected to in vitro digestion by soluble SKI-1/S1P and SKI-1/S1P Y285A. The peptides (25 593

μM) were incubated in vitro with 10μl of enzyme in buffer pH 7.5 and relative fluorescence 594

measured at 3 hrs as in Fig. 4 and RFU values assessed. Data were analyzed using two-way 595

ANOVA as in 2D. YISRRLL-MCA comparison *** p=0.0003, AISRRLL-MCA comparison 596

not significant p=0.9899, FISRRLL-MCA comparison *** p=0.0009. 597

598

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Table 1. Kinetic parameters processing of peptides by SKI-1/S1P WT and Y285A.

Shown are Km(app) [μM] and Vmax(app) [RFU min−1]. Data are means + SD, n = 3.

Km(app) [μM] Vmax(app) [RFU min−1]

SKI-1/S1P: WT Y285A WT Y285A

Substrates

IYISRRLL 4.7 ± 0.2 7.4 ± 0.5 1507.0 ± 25.4 386.0 ± 8.6

YISRRLL 2.8 ± 0.1 5.6 ± 0.3 674.5 ± 11.1 111.6 ± 2.21

RRLL 15.3 ± 1.6 22.8 ± 1.1 1032.0 ± 26.3 462.6 ± 6.3

QKSIAVGRTLK 2.5 ± 0.4 15.6 ± 4.3 1227.0 ± 53.7 1466.0 ± 164.5

IAVGRTLK 22.6 ± 1.8 84.6 ± 21.6 2848.0 ± 71.5 1904.0 ± 233.7

RTLK 96.7 ± 17.8 216.7 ± 113.9 5157.0 ± 479.9 3906.0 ± 1359.0

SFITRRLQ 44.3 ± 7.9 78.4 ± 19.1 305.1 ± 18.0 151.4 ± 15.1

RRLQ 97.1 ± 24.38 226.3 ± 34.2 311.8 ± 35.0 301.3 ± 34.2

YSSVSRKLL 21.2 ± 3.0 28.1 ± 3.6 1074.0 ± 41.2 653.6 ± 24.1

RKLL 27.4 ± 5.6 56.4 ± 12.6 1572.0 ± 91.1 1215.0 ± 98.2

YISRRLL 5.0 ± 0.9 5.7 ± 1.2 574.7 ± 24.8 154.3 ± 8.2

FISRRLL 12.3 ± 0.7 15.0 ± 1.1 575.2 ± 9.8 191.8 ± 4.5

AISRRLL 50.4 ± 1.1 67.0 ± 3.3 270.2 ± 2.8 188.0 ± 4.9

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