Differential Recognition of Old World and New World ArenavirusEnvelope Glycoproteins by Subtilisin Kexin Isozyme 1 (SKI-1)/Site 1Protease (S1P)

Dominique J. Burri,a Joel Ramos da Palma,a Nabil G. Seidah,b Giuseppe Zanotti,c Laura Cendron,d Antonella Pasquato,a Stefan Kunza

Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerlanda; Laboratory of Biochemical Neuroendocrinology, ClinicalResearch Institute of Montreal (affiliated with the University of Montreal), Montreal, Canadab; Department of Biomedical Sciences, University of Padua, Padua, Italyc;Department of Biology, University of Padua, Padua, Italyd

The arenaviruses are an important family of emerging viruses that includes several causative agents of severe hemorrhagic feversin humans that represent serious public health problems. A crucial step of the arenavirus life cycle is maturation of the envelopeglycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P). Comparison of the cur-rently known sequences of arenavirus GPCs revealed the presence of a highly conserved aromatic residue at position P7 relativeto the SKI-1/S1P cleavage side in Old World and clade C New World arenaviruses but not in New World viruses of clades A andB or cellular substrates of SKI-1/S1P. Using a combination of molecular modeling and structure-function analysis, we found thatresidueY285 of SKI-1/S1P, distal from the catalytic triad, is implicated in the molecular recognition of the aromatic “signatureresidue” at P7 in the GPC of Old World Lassa virus. Using a quantitative biochemical approach, we show that Y285 of SKI-1/S1Pis crucial for the efficient processing of peptides derived from Old World and clade C New World arenavirus GPCs but not ofthose from clade A and B New World arenavirus GPCs. The data suggest that during coevolution with their mammalian hosts,GPCs of Old World and clade C New World viruses expanded the molecular contacts with SKI-1/S1P beyond the classical four-amino-acid recognition sequences and currently occupy an extended binding pocket.

The arenaviruses are a diverse family of emerging viruses thatincludes several important human pathogens. On the basis of

serological and genetic evidence, the arenaviruses are classicallysubdivided into the Old World (OW) and New World (NW)groups (1). The OW arenaviruses include the prototypic lympho-cytic choriomeningitis virus (LCMV) and the highly pathogenicLassa virus (LASV). The NW arenaviruses are found in the Amer-icas and comprise clades A, B, and C. All human pathogens in theNW family belong to clade B and include Junin virus (JUNV),Machupo virus, Guanarito virus, Sabia virus (2), and the recentlydiscovered Chapare virus (3). Among the arenaviruses, LASV isthe most prevalent pathogen, causing several hundred thousandinfections per year with a high mortality rate among hospitalizedpatients (4). A highly predictive factor for disease outcome inhuman arenavirus infection is the viral load (5), indicating closecompetition among viral spread, viral replication, and the pa-tient’s immune system. Antiviral drugs that limit viral replicationand spread may provide the patient’s immune system a window ofopportunity to develop antiviral immune responses to controland ultimately clear the virus.

Arenaviruses are enveloped negative-strand RNA viruses witha nonlytic life cycle (6). Their genome consists of two single-stranded RNA species, (i) a large segment encoding the virus poly-merase (L) and a small zinc finger motif protein (Z) and (ii) asmall segment encoding the virus nucleoprotein and glycoprotein(GP) precursor (GPC) (7). The GPC is synthesized as a singlepolypeptide chain. Upon processing by cellular signal peptidases,the unusually stable signal peptide (SSP) remains associated withthe GP and plays a role in subsequent maturation and transport(8, 9). The GPC is posttranslationally processed into GP1 and GP2by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease(S1P) (10–12), a member of the proprotein convertase (PC) fam-

ily involved in the regulation of cholesterol homeostasis, the en-doplasmic reticulum (ER) stress response, and lysosome biogen-esis (13, 14). The resulting tripartite SSP/GP1/GP2 complexrepresents the functional unit of host cell attachment and fusion.The GP1 part is located at the tip of the mature virion spike andundergoes interactions with cellular receptors. TransmembraneGP2 contains the machinery for membrane fusion and resemblesfusion-active GPs of other enveloped viruses (15). Maturation ofthe GPC by SKI-1/S1P is strictly required for the production ofinfectious particles and viral spread (10–12, 16). Proof-of-conceptstudies with protein- and peptide-based SKI-1/S1P inhibitors re-vealed that targeting of GPC maturation represents a novel andpromising antiviral strategy (17, 18). More recently, the small-molecule SKI-1/S1P inhibitor PF-429242 (19, 20) was shown toefficiently block arenavirus cell-to-cell propagation (21, 22) andwas able to clear the virus from persistently infected cells (21).However, considering the important role of SKI-1/S1P in normalphysiology, general inhibitors of SKI-1/S1P catalytic activity arelikely to cause unwanted side effects (21). A major thrust of ourcurrent research is therefore the development of inhibitors thatspecifically interfere with SKI-1/S1P processing of viral GPCs butnot with the activation of cellular substrates.

Processing of arenavirus GPCs by SKI-1/S1P relies on the RX-

Received 8 January 2013 Accepted 21 March 2013

Published ahead of print 27 March 2013

Address correspondence to Antonella Pasquato, Antonella.Pasquato@chuv.ch, orStefan Kunz, Stefan.Kunz@chuv.ch.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00072-13

6406 jvi.asm.org Journal of Virology p. 6406–6414 June 2013 Volume 87 Number 11

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

(hydrophobic)X motif, which is conserved among all arenavirusesand represents the minimal and sufficient requirement for recog-nition (13, 23). Although the four P1-to-P4 residues at the cleav-age site are generally believed to determine protease specificity,efficiency of GPC processing can be modulated by flanking resi-dues (24). Indeed, in vitro investigations of synthetic peptidesmimicking the LASV GPC-processing site showed that aromaticresidues at position P7 strongly enhance cleavage by SKI-1/S1P(23). Similarly, F259A replacement at P7 of the LCMV GPC im-paired maturation (10). On the basis of this indirect evidence, ithas been hypothesized that the catalytic pocket of SKI-1/S1P mayinteract with additional substrate residues distal from the actualcleavage site (23). In the present study, we further investigated thisaspect. Comparison of the currently known sequences of arenavi-rus GPCs revealed the presence of a conserved aromatic residue atposition P7 relative to the SKI-1/S1P cleavage side in OW andclade C NW arenaviruses but not in NW viruses of clades A and Bor cellular substrates of SKI-1/S1P. Combining molecular model-ing and biochemical analysis, we found that residueY285 of SKI-1/S1P, located distal from the catalytic triad, is implicated in themolecular recognition of the aromatic “signature residue” at P7and is crucial for efficient processing of OW and clade C NWarenavirus GPCs.

MATERIALS AND METHODSCell lines and viruses. Chinese hamster ovary K1 (CHOK1) cells werecultivated in Dulbecco’s modified Eagle’s medium (DMEM)-Ham’s F12at 1:1 (Biochrom AG, Berlin, Germany) supplemented with 10% fetalbovine serum and penicillin-streptomycin. SKI-1/S1P-deficient CHOK1cells (SRD12B) (25) were supplemented with 5 �g/ml cholesterol(Sigma), 20 �M sodium oleate (Sigma), and 1 mM sodium mevalonate(Sigma). All cell lines were grown at 37°C in 5% CO2.

Antibodies. An anti-V5 monoclonal antibody (MAb) was purchasedfrom Invitrogen. An anti-5xHis MAb was used to detect a soluble SKI-1/S1P form truncated before the transmembrane domain (SKI-1/S1PBTMD) (Qiagen, Chatsworth CA). Anti-FLAG MAb M2 was from Sigma-Aldrich. A polyclonal rabbit anti-mouse secondary antibody conjugatedto horseradish peroxidase (HRP; Dako, Glostrup, Denmark) was alsoused.

In silico modeling of SKI-1/S1P. The full-length human SKI-1/S1Pamino acid sequence was submitted to the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (26, 27). The crystal struc-ture of the PC furin (Protein Data Bank code 1P8J) was used to assigntemplate constraints. The prodomain of preproSKI-1/S1P was manuallyremoved with PyMol. The resulting structure was submitted to the energyminimization server ModRefiner (http://zhanglab.ccmb.med.umich.edu/ModRefiner/) (28). Peptide docking was achieved by replacement of theamino acids that make up the prodomain segment covering the catalyticsite in the original model (nonrefined) with the IYISRRLL sequence, fol-lowed by energy minimization. All pictures were produced by usingPyMol (http://www.pymol.org/).

Plasmids and transfections. Plasmids coding for wild-type (WT) andmutant SKI-1/S1P (pIR SKI-1/S1P FL WT, pIR SKI-1/S1P H249A, andpIR SKI-1/S1P BTMD) were previously reported (13, 29). All of the con-structs were cloned into a pIRES-EGFP vector (Invitrogen), and a V5 tagwas added to the C terminus, except for SKI-1/S1P BTMD (6�His tag).The full-length Y285A mutant SKI-1/S1P and Y285A mutant SKI-1/S1PBTMD constructs were generated by site-directed mutagenesis (Strat-agene) according to the manufacturer’s protocol. Forward primer 5=-CCAATAATCAGGTATCTGCCACATCTTGGTTTTTGG-3= and reverseprimer 5=-CCAAAAACCAAGATGTGGCAGATACCTGATTATTGG-3=were used. The cDNAs coding for LCMV GP and LASV GP are describedelsewhere (18). All transfections were performed with Lipofectamine 2000

(Invitrogen) according to the manufacturer’s instructions. Briefly, 2 �105

cells were seeded into the wells of a 24-well plate 24 h prior transfection.Plasmid DNA (0.8 �g) was dissolved in OPTI-MEM medium (GibcoBRL, New York, NY), mixed with OPTI-MEM–Lipofectamine 2000 (2�l/transfection), incubated for 15 min at room temperature, and added tothe cells. After 4 h, transfection solutions were replaced with fresh me-dium.

Western blotting. Cells were washed twice with cold phosphate-buff-ered saline (PBS) and lysed in SDS-PAGE sample buffer (62.5 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 10% dithiothreitol) supplementedwith cOmplete Mini Protease Inhibitor Cocktail (Roche). Samples wereboiled for 10 min at 95°C and centrifuged for 5 min at 13,000 rpm prior toloading. Proteins were separated on Tris-HCl ready precast 10% poly-acrylamide gel (Bio-Rad) and blotted onto nitrocellulose membrane. Un-specific binding sites were blocked for 1 h in blocking solution (PBS, 0.2%[wt/vol] Tween 20, 3% [wt/vol] nonfat milk) at room temperature. Mem-branes were incubated with primary antibody (anti-FLAG MAb at1:1,000) at 4°C with constant agitation. After overnight incubation, mem-branes were washed three times in washing solution (PBS, 0.2% [wt/vol]Tween 20) and incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (1:3,000). After three washes, membraneswere developed with the LiteABlot kit (EuroClone, Pero, Italy).

Real-time quantitative PCR (RT-qPCR). Total RNA was isolated withthe Qiagen RNeasy Mini Kit. The lysate was homogenized with aQIAshredder kit (Qiagen). The RT reaction was performed with the Qia-gen QuantiTect reverse transcription kit with 1 �g of template RNA. PCRwas done with SYBR green reagents. Specific primers were used to deter-mine the levels of expression of genes downstream of the sterol regulatoryelement binding protein (SREBP) and activation transcription factor(ATF6). TaqMan probes specific for 70-kDa heat shock protein 5(HSPA5; Hs99999174_m1), 3-hydroxy-3-methylglutaryl– coenzyme Asynthase (HMGCS1; Hs00940429_m), the low-density lipoprotein recep-tor (Hs00181192_m1), and glyceraldehyde-3-phosphate dehydrogenase(Hs99999905_m1) were obtained from Applied Biosystems (Foster City,CA). Real-time PCR analyses were performed in triplicate (ABI Prism7000 real-time PCR sequence detection system; Applied Biosystems) byusing the following protocol: 10 min at 95°C (one cycle), followed by 15 sat 95°C and 30 s at 58°C (40 cycles).

Pharmacological treatment. Induction of genes regulated by SREBPwas triggered by culturing CHOK1 cells under cholesterol deprivation inHam’s F-12 and DMEM (1:1)–lipoprotein-deficient serum–50 �M so-dium mevalonate–50 �M mevastatin (Enzo Life Sciences) for 18 h asreported previously (30). The induction of genes downstream of ATF6was triggered by the addition of 5 �g/ml tunicamycin (Sigma-Aldrich) for4 h before lysis as described previously (31). Dimethyl sulfoxide (DMSO;Sigma-Aldrich) was used as the vehicle control.

Determination of in vitro activities of soluble SKI-1/S1P variants.To assess the in vitro activities of WT and Y285A mutant SKI-1/S1PBTMD, constructs were expressed in HEK293T cells and enzymatic activ-ity was detected in concentrated conditioned medium with a reliable en-zymatic assay as described previously (32). Briefly, enzyme and the SKI-1/S1P substrate Ac-RRLL-MCA (custom synthesis; GenScript) (32) or alibrary of custom synthesized peptides were mixed. The reactions werecarried out at room temperature in buffer solution (25 mM Tris-HCl [pH7.5], 25 mM morpholinepropanesulfonic acid [MES], 1 mM CaCl2), andfluorescence was detected in a TriStar LB 941 multimode microplate spec-trofluorometer (Berthold Technologies, Bad Wildbad, Germany). Thepeptides Ac-YISRRLL-MCA, Ac-FISRRLL-MCA, and Ac-AISRRLL-MCA were purchased from GenScript Co. at �92% high-performanceliquid chromatography purity. Soluble WT and Y285A mutant SKI-1/S1Pwere produced as described above. Peptides at 25 �M were incubated with10 �l of HEK293T cell-conditioned medium containing either WT orY285A mutant SKI-1/S1P soluble enzyme in a final 100-�l volume con-taining 2 mM CaCl2, 25 mM MES, and 25 mM Tris at pH 7.5. Cleavage ofsubstrates was monitored by measuring fluorescence as described above.

Arenavirus Glycoprotein Processing

June 2013 Volume 87 Number 11 jvi.asm.org 6407

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

RESULTSAromatic residues at position P7 are conserved in GPCs of OWand NW clade C arenaviruses. Although the four amino acids atpositions P1 to P4 of the SKI-1/S1P processing site of arenavirusGPCs generally determine enzyme specificity, earlier studies pro-vided the first evidence that flanking residues can modulate sub-strate cleavability (23), affecting the energy levels of the transientenzyme-substrate complex. In order to identify further amino ac-ids potentially implicated in the recognition of arenavirus GPCsby SKI-1/S1P, we aligned the sequences of various GPCs includingthe processing site (P1 to P4) and the five amino acids upstream(P5 to P9) (Fig. 1). Sequences were grouped on the basis of theirclassification within the OW and NW subgroups and the relativeclades. Our sequence comparison revealed that R at P4 is abso-lutely conserved in all of the sequences and that R at position P3 isthe only amino acid that is accepted in OW and NW clade C GPCs.In contrast, NW clade A and B GPCs can accommodate differentresidues in that position, although the requirement for a hydro-philic side chain seems to be conserved (Fig. 1). This corroboratesearlier work that revealed that OW arenaviruses GPCs mimic SKI-

1/S1P autoprocessing site C (RRLL2) while NW arenavirusesGPCs resemble SKI-1/S1P autoprocessing site B (RSLK2) (32).At position P5, S or T is frequent among all arenavirus GPCs (Fig.1). The hydroxyl side chain at P5 may confer some advantage,since it is also found in all cellular substrates, with the exception ofATF6 (33). Our alignment further revealed that all currentlyknown GPCs of OW and NW clade C arenaviruses share a highlyconserved aromatic residue at position P7, which is absent fromclade A and B NW arenavirus GPCs, as well as cellular substrates(Fig. 1). The remarkable conservation of the aromatic signatureresidue at P7 in all known OW and clade C NW viruses warrantedfurther investigation.

Modeling of the SKI-1/S1P catalytic pocket identified Y285as a residue that potentially interacts with LASV GPC Y253. Togain insight into the nature of the residues within and sur-rounding the catalytic pocket of SKI-1/S1P, we used an in silicomodeling approach, as the structure of SKI-1/S1P has not yetbeen resolved. The amino acid sequence of human SKI-1/S1P(UniProt entry Q14703) was submitted to the I-TASSER server(27), which generated a series of predicted models by multiplealignments with already known three-dimensional structures.Furin, a closely related PC whose catalytic domain shares morethan 30% identity with SKI-1/S1P, was used as the referencetemplate. Each model was then manually investigated to assessits accuracy and reliability. On the basis of the relative positionof the catalytic triad (D218, H249, and S414), only one of thethree models generated showed a conformation compatiblewith a catalytic function. This model was then edited to removethe prodomain to highlight the residues within and surround-ing the catalytic pocket and refined with ModRefiner (http://zhanglab.ccmb.med.umich.edu/ModRefiner/) and FG-MD(34). Since the available data suggested that aromatic residuesat P7 are preferred over hydrophobic ones (23), we hypothe-sized that the side chain of an aromatic amino acid exposed inthe SKI-1/S1P catalytic groove (S7 position) may be engaged inan interaction with the corresponding residue at P7, as, amongnoncovalent bonds, aromatic-aromatic interactions involvingthe �-electron systems are energetically favored. We thusscanned the surface of the SKI-1/S1P catalytic groove and iden-tified Y at position 285 as a possible candidate contact residue,being exposed, free from further interactions, and located in aregion accessible to the substrate’s P7 residue (Fig. 2A). Toconfirm the accessibility of the substrate at P7 to Y285, a pep-tide-docking analysis was performed. The LASV GPC-derivedpeptide IYISRRLL was virtually docked into the catalyticgroove of the SKI-1/S1P model. The results suggested that thearomatic residue in P7 of GPC may indeed interact with Y285of SKI-1/S1P (Fig. 2A).

Next, we generated Y285A mutant SKI-1/S1P by site-directedmutagenesis (Fig. 2B). Protein expression and maturation wereassessed by transient transfection in SKI-1/S1P-deficient SRD12Bcells. Wild-type SKI-1/S1P and the catalytically dead H249A mu-tant protein were used as positive and negative controls, respec-tively. In cell lysates, the expression levels of Y285A mutant SKI-1/S1P were comparable to those of the WT, as assessed by Westernblotting. Interestingly, autoprocessing at site C of Y285A mutantSKI-1/S1P was less efficient, with a consequent accumulation ofthe B=B intermediate form (Fig. 2C). In line with published data,the control mutant H249A showed no autoprocessing (Fig. 2C).

In order to characterize the catalytic activity of Y285A mutant

FIG 1 Alignment of SKI-1/S1P recognition motifs within arenavirus GPCs.Amino acid sequences of NW and OW arenavirus GPCs were aligned withClustalW. The aligned sequences were then submitted to the WebLogo server(41). The size of the letter representing an amino acid represents the frequencyof its occurrence at that position in the alignment. Aromatic amino acids atposition P7 of OW and clade C of NW arenavirus GPC are shaded light gray.The vertical arrow indicates the location of the cleavage site. Positions up-stream of the cleavage site (P1 to P9) are also indicated.

Burri et al.

6408 jvi.asm.org Journal of Virology

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

SKI-1/S1P, we tested its ability to process the transcription factorSREBP2, a cellular substrate of SKI-1/S1P. Upon consecutivecleavage by SKI-1/S1P in the Golgi lumen and the protease site 2protease (S2P) at the cytoplasmic face, the cytosolic part of SREBPtranslocates into the nucleus, where it activates the transcriptionof target genes, whose expression can be quantified by RT-qPCR.Following transfection of WT SKI-1/S1P (positive control) orY285A mutant SKI-1/S1P into SKI-1/S1P-deficient SRD12B cells

(25), processing of SREBP2 was induced by sterol depletion andRT-qPCR was performed to monitor the induction of the down-stream gene for HMGCS1. No significant differences between WTand Y285A mutant SKI-1/S1P were observed (Fig. 2D). In addi-tion, we tested the ability of Y285A mutant SKI-1/S1P to processanother cellular substrate, ATF6, involved in the cellular ER stressresponse. Upon the induction of ER stress, e.g., by tunicamycin,an inhibitor of protein N-glycosylation, ATF6 is cleaved by SKI-

FIG 2 Molecular modeling and characterization of Y285 mutant SKI-1/S1P. (A, left side) Cartoon representation of the structural model of SKI-1/S1P.The positions of catalytic residues D212, H249, and S414 are highlighted in red. The position of Y285 in the extended SKI-1/S1P catalytic groove is shown.(A, right side) Surface representation of the SKI-1/S1P model. The positions of catalytic residues H249 and S414 are in red. The modeled position of Y285is in orange. The in silico docking result shows the conformation of LASV GPC-derived peptide IYISRRLL in yellow. (B) Schematic representation of WTand Y285A mutant SKI-1/S1P. The prodomain is in dark gray, and the catalytic domain is in light gray, with critical residues of the catalytic triadhighlighted. The transmembrane domain is represented by a gray box with an arrow, followed by the cytoplasmic tail. The signal peptidase processing site(site A) and autoprocessing sites B/B= and C are highlighted. The C-terminal V5 tag is shown, and the residues at position 285 are highlighted in red. (C)Expression and autoprocessing of SKI-1/S1P variants. WT and Y285A and H249A mutant SKI-1/S1P were transfected into SKI-1/S1P-deficient SRD12Bcells. Expression and autoprocessing were assessed by Western blotting. H249A mutant SKI-1/S1P, an inactive mutant, was used as a negative control. Thepositions of the SKI-1/S1P precursor (pSKI-1/S1P) and the BB= and C forms of the enzyme are indicated. (D) Normal processing of the cellular substratesSREBP2 and ATF6 by Y285A mutant SKI-1/S1P. Cells were subjected to cholesterol depletion to monitor the SREBP2-mediated upregulation of the genesfor HMGCS1 and the ER stress response to assess the ATF6-mediated induction of HSPA5. SRD12B cells were transfected with WT or Y285A mutantSKI-1/S1P or the vector only. After 48 h, genes downstream of that for SREBP2 were induced by treatment with 50 �M mevastatin (Mev). To induce genesdownstream of ATF6, cells were treated with 5 �g/ml tunicamycin (Tun) for 4 h. CHOK1 cells were used as a positive control. After treatment, cells werewashed twice with PBS and total RNA was isolated for RT-qPCR analyses as described in Materials and Methods. Data were normalized by using thecalibrator gene for hydroxymethylbilane synthase. Data are presented as n-fold induction above the level of mock (DMSO)-treated cells (means �standard deviations; n � 3). Data were analyzed by two-way analysis of variance, followed by a Bonferroni posttest with GraphPad Prism. The lack of astatistically significant difference between WT and Y285A mutant SKI-1/S1P (NS) is indicated.

Arenavirus Glycoprotein Processing

June 2013 Volume 87 Number 11 jvi.asm.org 6409

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

1/S1P and S2P. The released ATF6 translocates to the nucleus,where it activate ER stress response genes, including HSPA5. Sim-ilar levels of HSPA5 induction were detected in Y285A mutant andWT SKI-1/S1P-transfected cells after exposure to tunicamycin(Fig. 2D). Taken together, these results show that replacement ofY285 of SKI-1/S1P with A, although it reduces the efficacy of zy-mogen processing, still generates enough of a fully functional en-zyme against key cellular substrates.

SKI-1/S1P Y285 is crucial for recognition of the aromatic sig-nature residue at P7 in arenavirus GPCs. The modeling data athand suggested that residue Y285 of SKI-1/S1P may contribute tothe molecular interaction of aromatic residues at position P7 ofsome arenavirus GPCs with the catalytic pocket of the enzyme,thereby promoting substrate processing. To validate our predic-tion experimentally, we generated FLAG-tagged variants of LASVGPC that contained Y, F, or A at position P7. The LASV GPCvariants were cotransfected with Y285A mutant or WT SKI-1/S1Pinto SKI-1/S1P-null SRD12B cells. As a control, we used the GPCof the clade B NW virus JUNV, which lacks an aromatic residue atP7 (Fig. 1). To mimic the likely molar excess of GPC over SKI-1/S1P in infected cells, expression plasmids were used at a ratio of 3:1in favor of the GPC. At 48 h posttransfection, cells were lysed andGPC processing was examined by Western blotting with an anti-FLAG antibody. In line with previous results, the Y253A mutationin LASV GPC resulted in reduced cleavage by WT SKI-1/S1P (Fig.3). In contrast, replacement of LASV GPC Y253 with F did notaffect SKI-1/S1P processing, emphasizing the importance of thearomatic nature of the residue at P7, but not Y per se. Consistentwith our modeling-based predictions, processing of WT LASVGPC by Y285A mutant SKI-1/S1P was impaired (Fig. 3). As ex-pected, processing of Y253A mutant LASV GPC by Y285A mutantSKI-1/S1P was also markedly reduced. In contrast, the processingof JUNV GPC that contains an L in position P7 was not affected bythe Y285A mutation in SKI-1/S1P (Fig. 3). These data providedthe first evidence of a possible interaction between Y285 of SKI-1/S1P and aromatic residues at P7 of a GPC substrate.

In order to quantify the enzymatic parameters of Y285A mu-

tant SKI-1/S1P toward different viral and cellular substrates, weemployed a robust and reliable biochemical assay with a solublerecombinant form of SKI-1/S1P. To this end, we introduced theY285A mutation into SKI-1/S1P BTMD by site-directed mu-tagenesis (Fig. 4A). Y285A mutant and WT SKI-1/S1P BTMDwere transiently transfected into HEK293T cells, followed by theexpression of recombinant soluble proteins in serum-free me-dium. Detection of the soluble SKI-1/S1P variants in the cell su-pernatant by Western blotting revealed the secretion of signifi-cantly less Y285A mutant than WT SKI-1/S1P BTMD (Fig. 4B).This was expected, considering the previously observed impairedprocessing of the B/B= form of full-length Y285A mutant SKI-1/S1P into the mature C form (Fig. 2C). The specific activity ofSKI-1/S1P BTMD present in conditioned supernatants was mea-sured with the well-characterized peptide substrate Ac-RRLL-MCA, which contains the critical four-amino-acid SKI-1/S1P rec-ognition sequence RRLL present at the C autoprocessing site andLASV GPC (32), as described in Materials and Methods. Despitethe apparently lower concentration of Y285A mutant SKI-1/S1PBTMD in conditioned supernatants (Fig. 4B), the enzymatic ac-tivity detected in our peptide-based assay seemed higher overall,indicating a robust specific activity of Y285A mutant SKI-1/S1PBTMD against the basic substrate sequence RRLL (Fig. 4C).

Next, we compared the activities of Y285A mutant and WTSKI-1/S1P BTMD against LASV GPC-derived peptide Ac-IYISRLL-MCA, peptide Ac-FYISRLL-MCA derived from theGPC of the OW Mopeia virus (MOPV), and peptide Ac-QKSIA-VGRTLK-MCA from the GPC of the clade B NW Tacaribe virus(TACV). The TACV-derived peptide was chosen over the JUNVpeptide because of its superior properties as a substrate in our invitro enzymatic assay (32). Briefly, Y285A mutant and WT SKI-1/S1P BTMD were incubated with increasing concentrations of thesubstrates and enzymatic activities were assessed by monitoringthe release of 7-amino-4-methyl-coumaride (AMC) fluorescenceover time. Data were plotted as the initial rate (relative fluores-cence units [RFU] per second) versus the substrate concentration.The Michaelis-Menten curves for the LASV and MOPV GPC-derived peptides showed a dramatic reduction of the reactionvelocity when the substrate was digested with Y285A mutant SKI-1/S1P BTMD (Vmax-YA) rather than the WT enzyme (Vmax-WT)(Fig. 4D; Table 1). In contrast, the Vmax of the TACV GPC-derivedpeptide reached similar values with WT and Y285A mutant SKI-1/S1P BTMD (Fig. 4D; Table 1). To highlight the specific contri-bution of the aromatic residue in P7, we compared LASV GPC-derived peptides of different lengths. Processing by Y285A mutantSKI-1/S1P BTMD revealed lower processing of longer peptidesincluding the P7 position than the four-amino-acid substrate pep-tide RRLL (Fig. 4E; Table 1). As expected, the length of the TACVGPC-derived peptides did not affect the efficiency of processing.

To extend our findings, we examined the processing of pep-tides derived from the GPCs of the clade A Pichinde virus (PICV)and the clade C Latino virus (LATV) by Y285A mutant and WTSKI-1/S1P BTMD. As shown in Fig. 5, the Y285A mutant showeda markedly reduced reaction velocity for substrates derived fromthe clade C virus LATV containing an aromatic residue at positionP7, while peptides derived from the clade A virus PICV, lackingaromatic residues at P7, were only mildly affected by the mutation(Fig. 5; Table 1).

Lastly, we sought to confirm the biochemical interaction be-tween the aromatic signature residue at P7 of the GPC with Y285

FIG 3 The Y285A mutation in SKI-1/S1P affects the processing of LASV GPCbut not that of JUNV GPC. SRD12B cells were cotransfected with the indicatedFLAG-tagged LASV GPC and V5-tagged SKI-1/S1P variants with a 3-fold ex-cess of plasmids encoding the GPCs. As a negative control, FLAG-taggedJUNV GPC was used. After 48 h, cells were lysed and subjected to SDS-PAGE,followed by Western blotting. GPC and GP2 were detected with anti-FLAGantibodies. Processing efficiency was defined as the ratio of GP2 to GPC andnormalized to the efficiencies of processing of the WT LASV and JUNV GPCsby WT SKI-1/S1P, which were set at 100.

Burri et al.

6410 jvi.asm.org Journal of Virology

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

of SKI-1/S1P in a complementary manner with 7mer peptidesmimicking the cleavage site of LASV GPC (P1 to P6) and carryingeither WT Y or F/A at P7, coupled to the fluorogenic MCA groupat the C terminus (Ac-YISRRLL-MCA, Ac-FISRRLL-MCA, Ac-

AISRRLL-MCA). Peptides at a 25 �M concentration were di-gested in vitro with soluble WT and Y285A mutant SKI-1/S1P inbuffer solution at pH 7.5, and processing was evaluated by moni-toring the release of AMC over time. For each peptide, fluores-

FIG 4 The Y285A mutation in SKI-1/S1P specifically affects the processing of peptides derived from OW and clade C NW viruses. (A) Schematic representationof the soluble form of Y285A mutant SKI-1/S1P. Y285A mutant SKI-1/S1P BTMD was generated by removing the transmembrane domain and cytosolic tail offull-length Y285A mutant SKI-1/S1P. (B) Expression of WT and Y285A mutant SKI-1/S1P BTMD. HEK293T cells were transfected with either WT or Y285Amutant SKI-1/S1P BTMD. The medium was then replaced with serum-free medium. Expression of the soluble SKI-1/S1P variants in the medium was detectedby Western blotting. The position of the active enzyme is indicated by the arrowhead. C, control. (C) Enzymatic activities of WT and Y285A mutant SKI-1/S1PBTMD. The cell culture medium used in the experiment shown in panel B was used for in vitro determination of SKI-1/S1P enzymatic activity. Samples wereincubated with 10 �M Ac-RRLL-MCA in pH 7.5 buffer solution, and the release of fluorescent AMC (RFU) was monitored for 6 h. Data are triplicates � standarddeviations. (D) Processing of arenavirus GPC-derived peptides by Y285A mutant and WT SKI-1/S1P. LASV GPC-derived peptide Ac-IYISRRLL-MCA, MOPV-derived peptide Ac-FYISRRLL-MCA, or TACV GPC-derived peptide Ac-QKSIAVGRTLK-MCA was incubated with a normalized amount of either WT orY285A mutant SKI-1/S1P BTMD. Fluorescence was monitored and analyzed as described in Materials and Methods. Michaelis-Menten curves show initial rates(Vi) as a function of the substrate concentration (�M). One out of three representative experiments is shown. (E) Impaired processing of extended LASVGPC-derived peptides by Y285A mutant SKI-1/S1P. The indicated peptides were digested with Y285A mutant and WT SKI-1/S1P as described for panel D. Thepercentages of Vmax SKI-1/S1P Y285A/Vmax SKI-1/S1P WT for the different substrates were then calculated (means � standard deviations; n � 3).

Arenavirus Glycoprotein Processing

June 2013 Volume 87 Number 11 jvi.asm.org 6411

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

cence values were extrapolated after 3 h of digestion. In line withprevious reports (23), Ala at P7 affected the interaction with thewild-type enzyme, resulting in decreased peptide processing com-pared to Ac-YISRRLL-MCA and Ac-FISRRLL-MCA (Fig. 6; Table1). The opposite was observed when the three peptides were di-gested by Y285A mutant SKI-1/S1P; the efficiency of cleavage ofboth Ac-YISRRLL-MCA and Ac-FISRRLL-MCA was greatly af-fected, resulting in a significant decrease in fluorescence detection.In contrast, the digestion of Ac-AISRRLL-MCA was unaffectedFig. 6; Table 1), supporting the hypothesis that Y285 of SKI-1/S1Pdoes not represent a key contact residue for the recognition ofsubstrates lacking an aromatic residue at position P7. Corroborat-ing our molecular modeling, the experimental data indicate thatresidueY285 of SKI-1/S1P, distal from the catalytic triad, is likely

implicated in the molecular recognition of the aromatic signatureresidue at P7 and is crucial for efficient processing of OW andclade C NW arenavirus GPCs.

DISCUSSION

Using a combination of molecular modeling, mutagenesis, andbiochemical assays, we investigated the molecular recognition ofarenavirus GPCs by SKI-1/S1P and reached the following conclu-sions. (i) The GPCs of OW and clade C NW arenaviruses containa conserved aromatic residue at position P7 of the SKI-1/S1Pcleavage site, which is absent from other cellular and viral sub-strates. (ii) In docking experiments, residueY285 of SKI-1/S1P,located distal from the catalytic triad, likely contributes to themolecular recognition of the aromatic residue at P7 of LASV GPC.

TABLE 1 Kinetic parameters of peptide processing by WT and Y285A mutant SKI-1/S1P

Substratea

Mean Km(app) [�M] � SD Mean Vmax(app) [RFU min�1] � SD

WT Y285A WT Y285A

IYISRRLL 4.7 � 0.2 7.4 � 0.5 1,507.0 � 25.4 386.0 � 8.6YISRRLL 2.8 � 0.1 5.6 � 0.3 674.5 � 11.1 111.6 � 2.21RRLL 15.3 � 1.6 22.8 � 1.1 1,032.0 � 26.3 462.6 � 6.3QKSIAVGRTLK 2.5 � 0.4 15.6 � 4.3 1,227.0 � 53.7 1,466.0 � 164.5IAVGRTLK 22.6 � 1.8 84.6 � 21.6 2,848.0 � 71.5 1,904.0 � 233.7RTLK 96.7 � 17.8 216.7 � 113.9 5,157.0 � 479.9 3,906.0 � 1,359.0SFITRRLQ 44.3 � 7.9 78.4 � 19.1 305.1 � 18.0 151.4 � 15.1RRLQ 97.1 � 24.38 226.3 � 34.2 311.8 � 35.0 301.3 � 34.2YSSVSRKLL 21.2 � 3.0 28.1 � 3.6 1,074.0 � 41.2 653.6 � 24.1RKLL 27.4 � 5.6 56.4 � 12.6 1,572.0 � 91.1 1,215.0 � 98.2YISRRLL 5.0 � 0.9 5.7 � 1.2 574.7 � 24.8 154.3 � 8.2FISRRLL 12.3 � 0.7 15.0 � 1.1 575.2 � 9.8 191.8 � 4.5AISRRLL 50.4 � 1.1 67.0 � 3.3 270.2 � 2.8 188.0 � 4.9a Underlining indicates permutations at P7 residues of LASV GPC-derived peptides.

FIG 5 Processing of peptides derived from clade A and C NW arenaviruses. LATV GPC-derived peptides (Ac-SFITRRLQ-MCA and Ac-RRLQ-MCA) andPICV-derived peptides Ac-YSSVSRKLL-MCA and Ac-RKLL-MCA were incubated with normalized amounts of either WT or Y285A mutant SKI-1/S1P BTMDand analyzed as described in the legend to Fig. 4D. Fluorescence was monitored, and Michaelis-Menten curves were recorded. The data shown are initial rates (Vi)as a function of the substrate concentration (�M). One experiment representative of the three performed is shown.

Burri et al.

6412 jvi.asm.org Journal of Virology

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

(iii) Site-directed mutagenesis of SKI-1/S1P and biochemical as-says provided evidence that Y285 is crucial for efficient processingof OW and clade C NW arenavirus GPC sequences containing anaromatic signature residue at P7.

Substrate recognition by cellular PCs is generally thought to bedependent on the amino acid residues that make up the consensussequence (14, 35). In substrates for basic amino acid-specific PCs,residues flanking the basic amino acids at positions P1 and P4 ofthe recognition sequences can influence the specificity for the pro-tease (36). Classical examples in viral PC substrates are mutationsnear the furin-processing site of the hemagglutinin of the HongKong and H5N1 strains of avian influenza virus that can acceleratethe rate of cleavage and increase the infectivity of virus particles(37, 38). Several examples of critical residues beyond the P1-to-P4positions of cellular PC substrates have also been reported. In thesubstrate proGdf11, the presence of an N at position P1= after thescissile bond markedly reduces cleavage by PACE4, furin, and PC7while being selectively permissive to PC5/6 (39). Processing ofnodal, a regulator of the fate of pluripotent cells, is dramaticallyenhanced by L and G at the P2= and P3= positions, respectively,providing further support for the concept that residues distal fromthe cleavage site can modulate processing efficiency (40).

Sequence alignments of all currently known arenavirus GPCsrevealed that GPCs of OW and clade C NW viruses contain con-served aromatic residue Y or F at position P7, which is absent fromother NW arenavirus GPCs or cellular substrates. The extent ofconservation indicated a strong selective pressure at work andsuggested that the RRLL motif alone may be suboptimal in termsof binding to the catalytic pocket of SKI-1/S1P and may need anaromatic residue at P7 for stabilization and optimal cleavage.

Starting from the hypothesis that OW and clade C NW arena-virus GPCs may have evolved to fit into an extended pocket ofSKI-1/S1P, we performed molecular modeling of the catalytic do-main and performed docking experiments with LASV GPC-de-

rived peptides. These molecular modeling studies pinpointedY285 of SKI-1/S1P as a putative contact residue involved in therecognition of the aromatic signature residue at P7 of the viralGPC. SKI-1/S1P sequence alignments revealed a high degree ofconservation of aromatic residues in positions corresponding to285 of the human form, including in rodent species that are nat-ural hosts for arenaviruses. Replacing Y285 of SKI-1/S1P with anA selectively reduced the processing of LASV GPC but not that ofJUNV GPC, which contains an L at P7. Using a robust and reliablebiochemical assay, we found that Y285A mutant SKI-1/S1P wasspecifically and markedly impaired in the processing of peptidesderived from OW and clade C NW arenavirus GPCs but not inthat of peptides from clade A and B NW viruses or the SKI-1/S1PB autoprocessing site (32). In sum, our study provides evidencethat during coevolution with their mammalian hosts, OW andclade C NW arenaviruses expanded the molecular contacts be-tween their GPCs with SKI-1/S1P beyond the classical four-ami-no-acid recognition sequence and currently occupy an extendedbinding pocket including residue Y285. The specificity of the in-teraction between Y285 of SKI-1/S1P and aromatic P7 residues forLASV GPC processing, but not cleavage of cellular substrates,makes this interaction further a promising target for the develop-ment of specific antiviral drugs.

ACKNOWLEDGMENTS

We thank Michael S. Brown and Joseph L. Goldstein (University of TexasSouthwestern Medical Center, Dallas, TX) for S1P-deficient SRD12Bcells.

This research was supported by Swiss National Science Foundationgrant FN 31003A-135536 (S.K.), funds from the University of Lausanne(S.K.), and a Canada Research Chair grant 216684 (N.G.S.).

REFERENCES1. Emonet SF, de la Torre JC, Domingo E, Sevilla N. 2009. Arenavirus

genetic diversity and its biological implications. Infect. Genet. Evol.9:417– 429.

2. Borio L, Inglesby T, Peters CJ, Schmaljohn AL, Hughes JM, JahrlingPB, Ksiazek T, Johnson KM, Meyerhoff A, O’Toole T, Ascher MS,Bartlett J, Breman JG, Eitzen EM, Jr, Hamburg M, Hauer J, HendersonDA, Johnson RT, Kwik G, Layton M, Lillibridge S, Nabel GJ, OsterholmMT, Perl TM, Russell P, Tonat K. 2002. Hemorrhagic fever viruses asbiological weapons: medical and public health management. JAMA 287:2391–2405.

3. Delgado S, Erickson BR, Agudo R, Blair PJ, Vallejo E, Albarino CG,Vargas J, Comer JA, Rollin PE, Ksiazek TG, Olson JG, Nichol ST. 2008.Chapare virus, a newly discovered arenavirus isolated from a fatal hemor-rhagic fever case in Bolivia. PLoS Pathog. 4:e1000047. doi:10.1371/journal.ppat.1000047.

4. McCormick JB, Fisher-Hoch SP. 2002. Lassa fever. Curr. Top Microbiol.Immunol. 262:75–109.

5. Johnson KM, McCormick JB, Webb PA, Smith ES, Elliott LH, King IJ.1987. Clinical virology of Lassa fever in hospitalized patients. J. Infect. Dis.155:456 – 464.

6. Buchmeier MJ, de la Torre JC, Peters CJ. 2007. Arenaviridae: the virusesand their replication, p 1791–1828. In Knipe DL, Howley PM (ed), Fieldsvirology, 4th ed. Lippincott-Raven, Philadelphia, PA.

7. de la Torre JC. 2009. Molecular and cell biology of the prototypic arena-virus LCMV: implications for understanding and combating hemorrhagicfever arenaviruses. Ann. N. Y. Acad. Sci. 1171(Suppl. 1):E57–E64.

8. Eichler R, Lenz O, Strecker T, Eickmann M, Klenk HD, Garten W.2003. Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor. EMBO Rep. 4:1084 –1088.

9. York J, Romanowski V, Lu M, Nunberg JH. 2004. The signal peptide ofthe Junin arenavirus envelope glycoprotein is myristoylated and forms anessential subunit of the mature G1-G2 complex. J. Virol. 78:10783–10792.

10. Beyer WR, Popplau D, Garten W, von Laer D, Lenz O. 2003. Endo-

FIG 6 Processing of mutant LASV GPC-derived peptides by WT and Y285Amutant SKI-1/S1P. Peptides Ac-YISRRLL-MCA, Ac-AISRRLL-MCA, and Ac-FISRRLL-MCA were subjected to in vitro digestion by soluble SKI-1/S1P andSKI-1/S1P Y285A. The peptides (25 �M) were incubated in vitro with 10 �l ofenzyme in buffer pH 7.5, relative fluorescence was measured at 3 h as describedin the legend to Fig. 4, and RFU values were assessed. Data were analyzed bytwo-way analysis of variance as described in the legend to Fig. 2D. YISRRLL-MCA comparison, P � 0.0003 (***); AISRRLL-MCA comparison, P � 0.9899(not significant [NS]); FISRRLL-MCA comparison, P � 0.0009 (***).

Arenavirus Glycoprotein Processing

June 2013 Volume 87 Number 11 jvi.asm.org 6413

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from

proteolytic processing of the lymphocytic choriomeningitis virus glyco-protein by the subtilase SKI-1/S1P. J. Virol. 77:2866 –2872.

11. Lenz O, ter Meulen J, Klenk HD, Seidah NG, Garten W. 2001. The Lassavirus glycoprotein precursor GP-C is proteolytically processed by subti-lase SKI-1/S1P. Proc. Natl. Acad. Sci. U. S. A. 98:12701–12705.

12. Rojek JM, Lee AM, Nguyen N, Spiropoulou CF, Kunz S. 2008. Site 1protease is required for proteolytic processing of the glycoproteins of theSouth American hemorrhagic fever viruses Junin, Machupo, and Gua-narito. J. Virol. 82:6045– 6051.

13. Touré BB, Munzer JS, Basak A, Benjannet S, Rochemont J, Lazure C,Chretien M, Seidah NG. 2000. Biosynthesis and enzymatic characteriza-tion of human SKI-1/S1P and the processing of its inhibitory prosegment.J. Biol. Chem. 275:2349 –2358.

14. Seidah NG, Prat A. 2012. The biology and therapeutic targeting of theproprotein convertases. Nat. Rev. Drug Discov. 11:367–383.

15. Igonet S, Vaney MC, Vonhrein C, Bricogne G, Stura EA, Hengartner H,Eschli B, Rey FA. 2011. X-ray structure of the arenavirus glycoproteinGP2 in its postfusion hairpin conformation. Proc. Natl. Acad. Sci. U. S. A.108:19967–19972.

16. Kunz S, Edelmann KH, de la Torre J-C, Gorney R, Oldstone MBA.2003. Mechanisms for lymphocytic choriomeningitis virus glycoproteincleavage, transport, and incorporation into virions. Virology 314:168 –178.

17. Maisa A, Stroher U, Klenk HD, Garten W, Strecker T. 2009. Inhibitionof Lassa virus glycoprotein cleavage and multicycle replication by site 1protease-adapted alpha(1)-antitrypsin variants. PLoS Negl. Trop. Dis.3:e446. doi:10.1371/journal.pntd.0000446.

18. Rojek JM, Pasqual G, Sanchez AB, Nguyen NT, de la Torre JC, Kunz S.2010. Targeting the proteolytic processing of the viral glycoprotein pre-cursor is a promising novel antiviral strategy against arenaviruses. J. Virol.84:573–584.

19. Hawkins JL, Robbins MD, Warren LC, Xia D, Petras SF, Valentine JJ,Varghese AH, Wang IK, Subashi TA, Shelly LD, Hay BA, LandschulzKT, Geoghegan KF, Harwood HJ, Jr. 2008. Pharmacologic inhibition ofsite 1 protease activity inhibits sterol regulatory element-binding proteinprocessing and reduces lipogenic enzyme gene expression and lipid syn-thesis in cultured cells and experimental animals. J. Pharmacol. Exp. Ther.326:801– 808.

20. Hay BA, Abrams B, Zumbrunn AY, Valentine JJ, Warren LC, Petras SF,Shelly LD, Xia A, Varghese AH, Hawkins JL, Van Camp JA, RobbinsMD, Landschulz K, Harwood HJ, Jr. 2007. Aminopyrrolidineamideinhibitors of site-1 protease. Bioorg. Med. Chem. Lett. 17:4411– 4414.

21. Pasquato A, Rochat C, Burri DJ, Pasqual G, de la Torre JC, Kunz S.2012. Evaluation of the anti-arenaviral activity of the subtilisin kexinisozyme-1/site-1 protease inhibitor PF-429242. Virology 423:14 –22.

22. Urata S, Yun N, Pasquato A, Paessler S, Kunz S, de la Torre JC. 2011.Antiviral activity of a small-molecule inhibitor of arenavirus glycoproteinprocessing by the cellular site 1 protease. J. Virol. 85:795– 803.

23. Pasquato A, Pullikotil P, Asselin MC, Vacatello M, Paolillo L, GhezzoF, Basso F, Di Bello C, Dettin M, Seidah NG. 2006. The proproteinconvertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derivedsubstrates and ex vivo validation of irreversible peptide inhibitors. J. Biol.Chem. 281:23471–23481.

24. Zhang Y, Sun Y, Sun H, Pu J, Bi Y, Shi Y, Lu X, Li J, Zhu Q, Gao GF,Yang H, Liu J. 2012. A single amino acid at the hemagglutinin cleavagesite contributes to the pathogenicity and neurovirulence of H5N1 influ-enza virus in mice. J. Virol. 86:6924 – 6931.

25. Rawson RB, Cheng D, Brown MS, Goldstein JL. 1998. Isolation ofcholesterol-requiring mutant Chinese hamster ovary cells with defects in

cleavage of sterol regulatory element-binding proteins at site 1. J. Biol.Chem. 273:28261–28269.

26. Roy A, Kucukural A, Zhang Y. 2010. I-TASSER: a unified platform forautomated protein structure and function prediction. Nat. Protoc. 5:725–738.

27. Zhang Y. 2008. I-TASSER server for protein 3D structure prediction.BMC Bioinformatics 9:40. doi:10.1186/1471-2105-9-40.

28. Xu D, Zhang Y. 2011. Improving the physical realism and structuralaccuracy of protein models by a two-step atomic-level energy minimiza-tion. Biophys. J. 101:2525–2534.

29. Seidah NG, Mowla SJ, Hamelin J, Mamarbachi AM, Benjannet S, TouréBB, Basak A, Munzer JS, Marcinkiewicz J, Zhong M, Barale JC, LazureC, Murphy RA, Chretien M, Marcinkiewicz M. 1999. Mammalian sub-tilisin/kexin isozyme SKI-1: A widely expressed proprotein convertasewith a unique cleavage specificity and cellular localization. Proc. Natl.Acad. Sci. U. S. A. 96:1321–1326.

30. Pullikotil P, Vincent M, Nichol ST, Seidah NG. 2004. Development ofprotein-based inhibitors of the proprotein of convertase SKI-1/S1P: pro-cessing of SREBP-2, ATF6, and a viral glycoprotein. J. Biol. Chem. 279:17338 –17347.

31. Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, Brown MS,Goldstein JL. 2000. ER stress induces cleavage of membrane-bound ATF6by the same proteases that process SREBPs. Mol. Cell 6:1355–1364.

32. Pasquato A, Burri DJ, Traba EG, Hanna-El-Daher L, Seidah NG, KunzS. 2011. Arenavirus envelope glycoproteins mimic autoprocessing sites ofthe cellular proprotein convertase subtilisin kexin isozyme-1/site-1 pro-tease. Virology 417:18 –26.

33. Stirling J, O’Hare P. 2006. CREB4, a transmembrane bZip transcriptionfactor and potential new substrate for regulation and cleavage by S1P.Mol. Biol. Cell 17:413– 426.

34. Zhang J, Liang Y, Zhang Y. 2011. Atomic-level protein structure refine-ment using fragment-guided molecular dynamics conformation sam-pling. Structure 19:1784 –1795.

35. Seidah NG, Chretien M. 1999. Proprotein and prohormone convertases:a family of subtilases generating diverse bioactive polypeptides. Brain Res.848:45– 62.

36. Holyoak T, Kettner CA, Petsko GA, Fuller RS, Ringe D. 2004. Structuralbasis for differences in substrate selectivity in Kex2 and furin protein con-vertases. Biochemistry 43:2412–2421.

37. Basak A, Zhong M, Munzer JS, Chretien M, Seidah NG. 2001. Impli-cation of the proprotein convertases furin, PC5 and PC7 in the cleavage ofsurface glycoproteins of Hong Kong, Ebola and respiratory syncytial vi-ruses: a comparative analysis with fluorogenic peptides. Biochem. J. 353:537–545.

38. Remacle AG, Shiryaev SA, Oh ES, Cieplak P, Srinivasan A, Wei G,Liddington RC, Ratnikov BI, Parent A, Desjardins R, Day R, Smith JW,Lebl M, Strongin AY. 2008. Substrate cleavage analysis of furin andrelated proprotein convertases. A comparative study. J. Biol. Chem. 283:20897–20906.

39. Essalmani R, Zaid A, Marcinkiewicz J, Chamberland A, Pasquato A,Seidah NG, Prat A. 2008. In vivo functions of the proprotein convertasePC5/6 during mouse development: Gdf11 is a likely substrate. Proc. Natl.Acad. Sci. U. S. A. 105:5750 –5755.

40. Constam DB, Robertson EJ. 1999. Regulation of bone morphogeneticprotein activity by pro domains and proprotein convertases. J. Cell Biol.144:139 –149.

41. Crooks GE, Hon G, Chandonia JM, Brenner SE. 2004. WebLogo: asequence logo generator. Genome Res. 14:1188 –1190.

Burri et al.

6414 jvi.asm.org Journal of Virology

on April 30, 2016 by guest

http://jvi.asm.org/

Dow

nloaded from