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Virus Research 131 (2008) 260–270

A wide range of NS3/4A protease catalytic efficienciesin HCV-infected individuals

Sandra Franco, Bonaventura Clotet, Miguel Angel Martınez ∗Fundacio irsiCaixa, Universitat Autonoma de Barcelona (UAB), Spain

Received 11 July 2007; received in revised form 29 September 2007; accepted 8 October 2007Available online 26 November 2007

bstract

The hepatitis C virus (HCV) NS3/4A protease acts as an antagonist of virus-induced interferon (IFN) regulatory factor (IRF)-3 activationnd IFN-� expression. The NS3/4A protease performs this function by cleaving Cardif and TRIF proteins to block retinoic-acid-inducible gen(RIG-I) and toll-like receptor (TLR)-3 signaling, respectively. NS3/4A protease inhibition can prevent Cardif and/or TRIF inactivation duringCV infection, thereby maintaining the innate immune response. Thus, differences in NS3/4A protease catalytic efficiency could be related toiral pathogenicity. In this study, we analyzed the catalytic efficiency of the most abundant NS3/4A protease isolated from each of 12 individualsnfected with HCV genotypes 1b, 1a, 3a, 4a or 4d. A diversity of NS3/4A protease catalytic efficiencies (up to a six-fold difference) was foundn the analyzed samples. The genotype 1b NS3/4A proteases displayed the highest catalytic efficiencies. However, within this genotype up tohree-fold differences were observed. Cross-genotypic interactions between the NS3 protease domain and the NS4A cofactor peptide were alsonvestigated. Overall, catalytic efficiencies of NS3 proteases cross-interacting with NS4A cofactors from heterologous genotypes were as efficients the homologous NS3/4A interactions. Of the 28 heterologous interactions tested, only 6 resulted in deleterious or nonfunctional enzymes.

onfunctional interactions were not genotype-specific, suggesting that enhancement of NS3 catalytic efficiency by the NS4A cofactor dependsn a few specific amino acid residues. Characterization of the proteolytic activities of individual NS3/4A proteases should provide clues fornderstanding HCV-host interactions, as well as assisting in the development of new classes of NS3/4A protease inhibitors.

2007 Elsevier B.V. All rights reserved.

at(atc(tca

eywords: HCV; NS3 protease; Genotype; Catalytic efficiency

. Introduction

The hepatitis C virus (HCV), a positive-stranded RNA virus,s the causal agent of a chronic liver infection afflicting more than70 million people worldwide. The infection is usually persis-ent, and after an asymptomatic period often lasting years, manyatients develop chronic liver disease, including cirrhosis andepatocellular carcinoma (Alter, 1995; Choo et al., 1991). TheCV genome is ∼9.6 kb in length and encodes a polyproteinf ∼3000 amino acid residues. This polyprotein is processed

nto structural and non-structural proteins by host signal pep-idases as well as by two viral proteases, NS2/3 and NS3/4AAppel et al., 2006; Moradpour et al., 2007; Tellinghuisen et

∗ Corresponding author at: Fundacio irsiCaixa, Laboratori de Retrovirologia,ospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain.el.: +34 934656374; fax: +34 934653968.

E-mail address: mmartinez@irsicaixa.es (M.A. Martınez).

NFteT((c

168-1702/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2007.10.003

l., 2007). The role of the NS2/3 protease appears to be limitedo the autoproteolytic cleavage in cis of the NS2-NS3 junctionLorenz et al., 2006; Reed et al., 1995). The 181 amino-terminalmino acid residues of the NS3 protein encode a serine pro-ease that cleaves at the NS3/4A junction in cis followed byleavage at the NS4A/B, NS4B/5A, and NS5A/B sites in transHijikata et al., 1993; Tomei et al., 1993). The NS3 serine pro-ease requires an accessory viral protein, NS4A, for optimalleavage activity. The cofactor activity has been mapped to ∼12mino acids in the central region of NS4A that interact with the-terminus of the NS3 protease (Bartenschlager et al., 1994;ailla et al., 1994; Lin et al., 1995). The contribution of NS4A

o NS3 protease activity can be mimicked by a synthetic peptidencompassing residues 21–34 of NS4A (Tomei et al., 1996).

he three-dimensional structure of the NS3 protease domain

residues 1–181) complexed with a synthetic NS4A cofactorresidues 21–34) reveals that the NS4A peptide is an integralomponent of the NS3 protease structure (Kim et al., 1996).

S. Franco et al. / Virus Research 131 (2008) 260–270 261

Table 1Clinical and virological characteristics of the 12 HCV-infected individuals in the study

Sample HCVgenotype

HCV viralload (IU/ml)

ALT/AST(U/l)

HIV-1 viral load(RNA copies/ml)

HIV-1 treatment CD4/CD8(cells/�l)

HCV treatmentresponse

A 1b 2364 20/13 <80 ddI, AMP, KLTR 734/1788 RB 1b 109,142 24/35 <80 d4T, 3TC, NFV 992/718 NRC 1b 1,300,000 814/Nd – – Nd/Nd Nt1 1b 23,300,000 100/74 116,984 Nt 390/1270 NtD 1a 1,981,682 87/55 – – Nd/Nd Drop outE 1a 166,923 45/93 <80 ddI, d4T, NVP 881/801 NRG 3a 650,060 157/239 <80 d4t, 3TC, NFV 374/Nd Drop outH 3a 245,849 14/9 23,000 TDF, 3TC, ddI, KLTR 275/367 RI 3a 1,147,565 137/124 <80 TDF, ddI, EFV 1182/989 NRJ 3a 16,852 61/46 19,000 d4T, 3TC, IDV 755/954 R24 4d 1,319,491 204/122 12,000 NT 339/580 NR2

A ; Nd,

padMI(FeiptishffvwlilPct(Pe

itct1bticPa

ol

2

2

DHuH(swTINwtlMifKtaTivCT

2o

5 4a 268,592 59/34 <80

LT, alanine aminotransferase; AST, aspartate aminotransferase; Nt, not treated

The HCV NS3/4A serine protease targets two adaptorroteins, TRIF (toll/interleukin-1 receptor domain-containingdaptor inducing IFN) and Cardif [caspase and recruitmentomain (CARD) adaptor inducing IFN-�, also termed VISA,AVS or IPS-1], for proteolysis, thereby disrupting host

FN induction initiated through the TLR-3 or RIG-I pathwayBreiman et al., 2005; Cheng et al., 2006; Ferreon et al., 2005;oy et al., 2003; Johnson et al., 2007; Loo et al., 2006; Meylant al., 2005; Saito et al., 2007). Thus, viral control of IFNnduction may contribute to the systemic immune defects andermissiveness observed during HCV infection. NS3/4A pro-ease activity is an ideal target for antiviral therapy because itsnhibition is expected to block HCV replication both by directuppression of viral protein production and by restoration ofost responsiveness to IFN. Combination therapy of peginter-eron (peg-IFN) plus ribavirin is the current standard of careor HCV-infected individuals. However, more than 50% of indi-iduals infected with HCV genotypes 1 or 4 who are treatedith peg-IFN plus ribavirin fail to achieve a sustained viro-

ogical response. In contrast, more than 75% of individualsnfected with HCV genotypes 2 or 3 have a sustained viro-ogical response to IFN-based therapies (Carrat et al., 2004;oynard et al., 2003). Cardif cleavage by the NS3/4A proteaseould limit the efficacy of IFN-based therapies, suggesting thathe inhibition of the NS3/4A protease by protease inhibitorsPIs) may enhance IFN actions. Moreover, in the absence ofIs, a less efficient NS3/4A protease may also enhance IFNffectiveness.

Because the HCV NS3/4A protease may play important rolesn various aspects of the viral life cycle including virus responseo current IFN-based therapies, the present study aimed toharacterize the catalytic efficiencies of the master NS3/4A pro-eases isolated from 12 individuals infected with HCV genotypesb, 1a, 3a, 4a or 4d. In addition, cross-genotypic interactionsetween the NS3 protease domain and the NS4A cofactor pep-ide were investigated. Several reports have analyzed intra- or

nter-individual genetic diversity in the HCV NS3 protease-oding region (Holland-Staley et al., 2002; Lodrini et al., 2003;awlotsky, 2006; Quer et al., 2005; Vallet et al., 2005; Winters etl., 2006). Nevertheless, data comparing the enzymatic activities

aar

TDF, 3TC, ABV, DDI 519/942 R

not determined; R, responders; NR, non-responders.

f different HCV NS3/4A proteases from different genotypes areacking.

. Materials and methods

.1. Individuals

Twelve HCV-infected individuals (designated A, B, C, 1,, E, G, H, I, J, 25, and 24) were chosen for this study. TheCV genotype of each sample is shown in Table 1. Individ-als A, B, 1, E, G, H, I, J, 25 and 24 were coinfected withIV-1, whereas individuals C and D were HCV monoinfected

Table 1). Samples A, B and C were selected from a previoustudy (Franco et al., 2007a). Likewise, individuals 1, 25 and 24ere selected from another previous study (Franco et al., 2007b).he HCV genotype was determined by the INNO-LIPA HCV

I assay (Innogenetics NU, Ghent, Belgium) and confirmed byS3 sequence analysis (see below). The HCV RNA viral loadas quantified by the Amplicor Monitor v2.0 (Roche Diagnos-

ics Systems, Inc., Branchburg, NJ, USA). The HIV-1 RNA viraload was measured by NASBA (Nuclisens HIV-QT, Biomerieux,

adrid, Spain). After submitting the samples used in this study,ndividuals A, B, D, E, G, H, I, J, 25 and 24 were treatedor 48 weeks with peg-IFN-�-2b (Peg-Intron; Shering-Plough,enilworth, NJ, USA) in combination with ribavirin (Rebe-

ol; Schering-Plough), as previously described (Ballesteros etl., 2004). Responses to HCV infection therapy are shown inable 1. The sample from individual C was taken during acute

nfection; afterwards, HCV infection was resolved in this indi-idual. Individual 1 did not receive HCV infection therapy.linical characteristics of the study individuals are shown inable 1.

.2. Genetic screen for determining the catalytic efficiencyf HCV NS3/4A protease

HCV RNA extraction and amplification were performeds previously described (Franco et al., 2007a,b; Martineznd Clotet, 2003). After viral RNA was isolated, 10 �l ofesuspended RNA was reverse transcribed at 42 ◦C by using the

2 esear

atGor(oGaAttpgHrTfGJCaGsg(lasTActrcGA4fCTCNGoGTGoG3AGA3GA

fGG3(Gaw4c(pvpT(ohcpp

susorsc(ktS

gp

pb2JCSpocdNitaaA

62 S. Franco et al. / Virus R

vian myeloblastosis virus reverse transcriptase (Promega) andhe genotype 1b oligonucleotide HCVproR1 (antisense) (5′-GATGAGTTGTCTGTGAAGACC-3′, residues 3953–3974f the HCV-J strain). RNA from genotypes 1a, 3a and 4 waseverse transcribed with oligonucleotides NS3P1A2 (antisense)5′-AGAGGAGTTGTCCGTGAACAC-3′, residues 3954–3974f the HCV-J strain), NS3P32 (antisense) (5′-GCAGGAGGA-TTGAATTGTC-3′, residues 3963–3982 of the HCV-J strain),

nd HCVPROTR1–4 (antisense) (5′-GGGTGTTGAGTTGTC-GTGAA-3′, residues 3957–3976 of the HCV-J strain), respec-

ively. Five microliters of the reverse transcriptase reaction washen used in the first PCR amplification with 0.04 U/�l of theroofreading Platinum Taq DNA polymerase (Invitrogen). Theenotype 1b oligonucleotides used for this amplification wereCVproL1 (sense) (5′-GCAAGGGTGGCGACTCCTTGC-3′,

esidues 3389–3409 of the HCV-J strain) and HCVproR1.he amplification of genotype 1a, 3a and 4 DNA was per-

ormed with oligonucleotides NS3P1A1 (sense) (5′-CAAGG-GTGGAGGTTGCTGGC-3′, residues 3389–3409 of the HCV-strain) and NS3P1A2, NS3P31 (sense) (5′-CTGATGACTAT-GGGAGATGG-3′, residues 3373–3393 of the HCV-J strain)nd NS3P32, and HCVPROTS1–4 (sense) (5′-AAAGGGGTG-AGACTCCTTGC-3′, residues 3389–3409 of the HCV-J

train) and HCVPROTR1–4, respectively. Nested PCR ofenotype 1b DNA was then performed. The 5′ oligonucleotideHCVproL2) for nested PCR encoded an EcoRI site (under-ined), residues 21–34 of NS4A, a dipeptide linker (Gly-Gly),nd residues 2–8 of NS3 (residues 3411–3431 of the HCV-Jtrain) (5′-GGGTTGAATTCTATGGCTCCTATTGGATCTG-TGTTATTGTTGG AAGAATTATTTTGTCTGGAAGAGG-GGACCTATCACGGCCTACTCCCAA-3′). The 3′ oligonu-leotide (HCV proR) for nested PCR was complementaryo residues 3930–3950 of the HCV-J strain (amino acidesidues 175–181 of NS3) and encoded an in-frame stopodon (bold face) next to a XhoI site (underlined) (5′-GGAGGGGCTCGAGTCAAGACCGCATAGTAGTTTCC-T-3′). Nested PCR amplification of DNA genotypes 1a, 3a,a and 4d was performed with the following oligonucleotides:or 1a, NS31aNS4A1a (sense) (5′-GGGTTGAATTCTATGG-TCCTATTGGCTGCGTGGTCATAGTGGGCAGGATTGT-TTG TCCGGGAAGGGAGGACCCATCACGGCGTACGC-CAG-3′, residues 3411–3431 of the HCV-J strain) andS3P1A4 (antisense) (5′-GGGAGGGGCTCGAGTCA-GAYCTCATGGTTGTCYCTAG-3′, residues 3930–3950f the HCV-J strain); for 3a, NS33aNS4A3a (sense) (5′-GGTTGAATTCTATGGCTCCTATTGGCTGCGTTGTGAT-GTGGGCCATATTGAGCTGGGGGGCAAGGGAGGACC-ATYACAGCATAYGCCCAG-3′, residues 3411–3431-f the HCV-J strain) and NS3P34 (antisense) (5′-GGGAG-GGCTCGAGTCAAGACCTAGCCTGTGTACTVAGGGT-′, residues 3927–3950 of the HCV-J strain); for 4a, NS34aNS4-4a (sense) (5′-GGGTTGAATTCTATGGCTCCTATTGGCA-CGTGGTGATTGTCGGGAGAGTTGTCCTGTCGGGCC-

AGGAGGACCCATCACAGCATACGC-3′, residues 3411–-427 of the HCV-J strain) and HCVPROTR2–4 (antisense) (5′-GGAGGGGCTCGAGTCATGATCTCATGGTAGTTTCA-GAGA-3′, residues 3927–3950 of the HCV-J strain); and

mB6i

ch 131 (2008) 260–270

or 4d, NS34dNS4A4d (sense) (5′-GGGTTGAATTCTAT-GCTCCTATTGGCAGCGTGGTGATTGTCGGGAGGGTC-TTATATCTGGCCAAGGAGGACCCATCACAGCATACGC-′, residues 3414–3434 of the HCV-J strain) and NS3p4-R2antisense) (5′-GGGAGGGGGGGCCCTCATGATCTCATG-TAGTTTCAAG-3′, residues 3927–3947 of the HCV-J strain,

n ApaI restriction site is underlined). The PCR productsere digested with EcoRI and XhoI (or ApaI in genotyped) and ligated to pBSK-(Stratagene) to generate a singlehain �gal-HCV NS32–181/421–34 protease fusion proteinpHCVNS32–181/421–34 protease). The HCVNS32–181/421–34rotease construct contains NS4 residues 21–34 fused in-frameia a short linker (GG) to the amino terminus of the NS3rotease domain (residues 2–181) (Martinez and Clotet, 2003).his strategy was similar to that described by Dimasi et al.

Dimasi et al., 1998). This construction was identical for eachf the samples cloned and analyzed. Recent ex vivo experimentsave demonstrated that single chain protease, lacking theomplete helicase domain of NS3, mediated cleavage of viralolyprotein and cellular Cardif similar to wild-type NS3/4Arotease (Johnson et al., 2007).

To select the master (most abundant) NS3 proteaseequence in each sample, a minimum of five individ-al plasmid clones were obtained and sequenced for eachample. To avoid PCR errors entering into the analysis,ne clone for each of the samples identical to the cor-esponding sample amino acid consensus sequence waselected. Sequencing was performed with the flanking oligonu-leotides T3 (5′-ATTAACCCTCACTAAAGGGA-3′) and T75′-TAATACGACTCACTATAGGG-3′) and the Big Dye v3.1it in the 3100 DNA sequencing system (Applied Biosys-ems). Sequence alignment and editing were performed withequencerTM version 4.1 (GeneCodes) software.

Nested sense oligonucleotides used to construct heterolo-ous NS3/4A proteases (i.e., NS3 proteases with NS4A cofactoreptides from other genotypes) are shown in Table 2.

The catalytic efficiencies of the different HCV NS3/4Aroteases were determined using a bacteriophage lambda (�)-ased genetic screen as previously described (Franco et al.,007a,b; Martinez and Clotet, 2003). Briefly, Escherichia coliM109 cells containing plasmid pcI.HCVcro (Martinez andlotet, 2003) that included the NS5A/NS5B (ASEDVVCC-MSYTWTGA) cleavage site were transformed with plasmidHCVNS32–181/421–34 protease. The resulting cells were grownvernight at 30 ◦C in the presence of 0.2% maltose, harvested byentrifugation, and resuspended in 10 mM MgSO4 to an opticalensity at 600 nm of 2.0/ml. To induce the expression of HCVS32–181/421–34 protease, cells (200 �l) were incubated for 1 h

n 1 ml of Luria-Bertani (LB) medium containing 12.5 �g ofetracycline, 20 �g of ampicillin, 0.2% maltose, 10 mM MgSO4,nd 1 mM isopropyl-�-d-thiogalactopyranoside (IPTG). There-fter, cell cultures were infected with 107 PFU of � phage.fter 3 h at 37 ◦C, the titer of the resulting phage was deter-ined by coplating the cultures with 200 �l of E. coli XL-1

lue cells (adjusted in 10 mM MgSO4 to an optical density at00 nm of 2.0/ml) on LB plates using 3 ml of top agar contain-ng 12.5 �g/ml tetracycline, 0.2% maltose, and 0.1 mM IPTG.

S.Franco

etal./VirusR

esearch131

(2008)260–270

263

Table 2Sequences of PCR oligonucleotides used to construct NS3 proteases with NS4A cofactor peptides from heterologous genotypes

Namea Sequence (5′ to 3′)b,c

NS31bNS4A1a GGG TTG AAT TCT ATG GCT CCT ATT GGC TGC GTG GTC ATA GTG GGC AGG ATT GTT TTG TCC GGG AAG GGA GGA CCT ATC ACG GCC TAC TCC CAA

NS31bNS4A3a GGG TTG AAT TCT ATG GCT CCT ATT GGC TGC GTT GTG ATT GTG GGC CAT ATT GAG CTG GGG GGC AAG GGA GGA CCT ATC ACG GCC TAC TCC CAA

NS31bNS4A4a GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGA GTT GTC CTG TCG GGC CAA GGA GGA CCT ATC ACG GCC TAC TCC CAA

NS31bNS4A4d GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGG GTC GTT ATA TCT GGC CAA GGA GGA CCT ATC ACG GCC TAC TCC CAA

NS31aNS4A1b GGG TTG AAT TCT ATG GCT CCT ATT GGA TCT GTT GTT ATT GTT GGA AGA ATT ATT TTG TCT GGA AGA GGA GGA CCC ATC ACG GCG TAC GCC CAG

NS31aNS4A3a GGG TTG AAT TCT ATG GCT CCT ATT GGC TGC GTT GTG ATT GTG GGC CAT ATT GAG CTG GGG GGC AAG GGA GGA CCC ATC ACG GCG TAC GCC CAG

NS31aNS4A4a GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGA GTT GTC CTG TCG GGC CAA GGA GGA CCC ATC ACG GCG TAC GCC CAG

NS31aNS4A4d GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGG GTC GTT ATA TCT GGC CAA GGA GGA CCC ATC ACG GCG TAC GCC CAG

NS33aNS4A1b GGG TTG AAT TCT ATG GCT CCT ATT GGA TCT GTT GTT ATT GTT GGA AGA ATT ATT TTG TCT GGA AGA GGA GGA CCG ATY ACA GCA TAY GCC CAG

NS33aNS4A1a GGG TTG AAT TCT ATG GCT CCT ATT GGC TGC GTG GTC ATA GTG GGC AGG ATT GTT TTG TCC GGG AAG GGA GGA CCG ATY ACA GCA TAY GCC CAG

NS33aNS4A4a GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGA GTT GTC CTG TCG GGC CAA GGA GGA CCG ATY ACA GCA TAY GCC CAG

NS33aNS4A4d GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGG GTC GTT ATA TCT GGC CAA GGA GGA CCG ATY ACA GCA TAY GCC CAG

NS34dNS4A1a GGG TTG AAT TCT ATG GCT CCT ATT GGC TGC GTG GTC ATA GTG GGC AGG ATT GTT TTG TCC GGG AAG GGA GGA CCC ATC ACA GCA TAC GC

NS34dNS4A3a GGG TTG AAT TCT ATG GCT CCT ATT GGC TGC GTT GTG ATT GTG GGC CAT ATT GAG CTG GGG GGC AAG GGA GGA CCC ATC ACA GCA TAC GC

NS34dNS4A1b GGG TTG AAT TCT ATG GCT CCT ATT GGA TCT GTT GTT ATT GTT GGA AGA ATT ATT TTG TCT GGA AGA GGA GGA CCC ATC ACA GCA TAC GC

NS34dNS4A4a GGG TTG AAT TCT ATG GCT CCT ATT GGC AGC GTG GTG ATT GTC GGG AGA GTT GTC CTG TCG GGC CAA GGA GGA CCC ATC ACA GCA TAC GC

a Names include the NS3 and NS4A genotypes of the corresponding oligonucleotide.b Underlined residues correspond to an EcoRI restriction site. Bold-faced residues correspond to heterologous NS4A peptides.c Residue positions are 3411–3431 according to the reference sequence HCV-J (1b).

264 S. Franco et al. / Virus Research 131 (2008) 260–270

F d in tr ntity.

Awct2tscn

2

hsAEa

3

3

ppNt4

tvifAtsbstaAvat9t

b2aWbse

ig. 1. Amino acid sequence alignment of the 12 HCV NS3 proteases analyzeeplicon I389/NS3-3′ protease sequence. Dots indicate amino acid sequence ide

fter incubation at 37 ◦C for 6 h, the resulting phage plaquesere counted to score growth. In the experiments in which E.

oli cells expressed wild-type HCV I389/NS3-3 NS3/4A pro-ease obtained from the subgenomic replicon (Lohmann et al.,001, 1999), � phage replicated up to 6000-fold more efficientlyhan in cells that did not express the HCV NS32–181/421–34 con-truct. This value was similar to that obtained with a singlehain construct that included the NS3 helicase domain (dataot shown).

.3. Nucleotide sequence accession numbers

HCV NS3 sequences obtained and characterized in this studyave been submitted to the GenBank database under acces-ion numbers EF013788 (A), EF013887 (B), EF013984 (C),F510039 (1), EF363555 (D), EF363556 (E), EF363557 (G),F363558 (H), EF363559 (I), EF363560 (J), DQ516083 (24)nd DQ516084 (25).

. Results

.1. Catalytic efficiencies of HCV NS3/4A proteases

Individual plasmid clones carrying the HCV NS3/4Arotease-coding region were generated from a single time-point

lasma sample from 12 HCV-infected individuals (Table 1).S3/4A proteases from different HCV genotypes (four of geno-

ype 1b, two of genotype 1a, four of genotype 3a, one of genotypea and one of genotype 4d) were chosen for this study. To ensure

t(it

his study. Amino acid changes are indicated relative to the subgenomic HCV

hat the protease clones analyzed were representative of theirus populations in the 12 individuals, several clones from eachndividual were sequenced. The master (most abundant) clonerom each individual was used for enzymatic determinations.s shown in Fig. 1, there were no substitutions in residues of

he catalytic triad (H57, D81 and S139), residues involved inubstrate recognition (L135, F154, A157 and R161), or zinc-inding site residues (C97, C99, C145 and H149). In contrast,everal substitutions were detected at residues involved in con-acting the NS4A cofactor (the 30 N-terminal residues of NS3nd residues in the NS3 hydrophobic core) (Kim et al., 1996).lthough the HCV NS3 gene has limited natural amino acidariability, only 63% of NS3 protease residues were conservedmong the 12 samples. When sequences from the same geno-ype were compared, the percentages of conserved residues were5, 96, 97 and 91% for genotypes 1b, 1a, 3a and 4, respec-ively.

The enzymatic activities of the 12 proteases were determinedy using a bacteriophage �-based genetic screen (Cabana et al.,002; Fernandez et al., 2007; Franco et al., 2007a,b; Martinez etl., 2000; Martinez and Clotet, 2003; Parera et al., 2004, 2007).e evaluated the enzymatic activity of each variant protease

y engineering the HCV polyprotein NS5A/NS5B cleavageite into the cI � repressor (Martinez and Clotet, 2003). Thenzymatic activities were expressed relative to the activity of

he HCV subgenomic replicon I389/NS3-3 NS3/4A protease100%) (Lohmann et al., 2001, 1999). To rule out the possibil-ty that observed differences in protease activity could be dueo different efficiencies in processing various target cleavage

S. Franco et al. / Virus Research 131 (2008) 260–270 265

Fig. 2. Amino acid sequence alignment of the NS4A cofactor peptides from genotypes lb, 1a, 3a, 4a and 4d. Amino acid sequences of the different samples used inthis study were compared with the NS4A cofactor peptide included in our protease constructs. The NS4 sequence found in the 24 and 25 samples (genotypes 4d and4a, respectively) was incorporated in the protease construct. Dots indicate amino acid sequence identity.

Fig. 3. Relative catalytic efficiencies of the 12 HCV NS3/4A proteases targeting the NS5A/NS5B cleavage site. The catalytic efficiencies of individual NS3 proteasesw hownc peptid2 e repl

sdwat

Np4cacRetptav2dtoei

ci(

5165 ± 10% (C, 1b), 123.0 ± 11% (1, 1b), 38.4 ± 4.5% (D, 1a),58.8 ± 0.9% (E, 1a), 86.5 ± 1% (G, 3a), 39.1 ± 6% (H, 3a),72.6 ± 6.7% (I, 3a), 68.4 ± 18.4% (J, 3a), 23.5 ± 3% (25, 4a),and 70.6 ± 8% (24, 4d) (Fig. 3). Therefore, a broad range of

ere compared with wild-type I389/NS3-3 protease (100%). As previously sompromised in cells expressing an NS3 protease construct in which the NS4A5 and 29 of NS4A were replaced by A residues (mutated NS4A). At least thre

ites, genotype 1b NS3/4A proteases were also tested with aifferent target cleavage site, the NS4B/NS5A cleavage site,hich is targeted in trans by the NS3/4A protease (Franco et

l., 2007a). Overall, these results paralleled those observed withhe NS5A/NS5B cleavage site (data not shown).

Genotype-specific NS4A cofactors corresponding to theS4A central region (NS4A residues 21–34) were used in ourrotease constructs (see Section 2) (Fig. 2). Because no genotyped NS4A sequence was in the database, the NS4A cofactor-oding region from sample 24 was sequenced, and the identifiedmino acid sequence was used in the corresponding proteaseonstruct. Within the NS4 cofactor, a substitution at position34K was found in samples I389/NS3-3, A and B (Fig. 2); nev-rtheless, site-directed mutagenesis of this residue demonstratedhat this substitution did not affect the catalytic efficiency of ourrotease construct (data not shown). When the NS4A cofac-or sequence was deleted from the construct or the I residuest positions 25 and 29 of NS4A were replaced by A residues,ery little proteolytic activity was detected (Martinez and Clotet,003) (0.02 ± 0.001 and 0.005 ± 0.001%, respectively; Fig. 3),emonstrating the ability of the NS4A cofactor to enhance

he proteolytic activity of the NS3 protease. To verify that thebserved differences among the 12 proteases were due to differ-nces in the NS3 protease region and not to possible differencesn critical residues of the target NS5A/NS5B cleavage site, the

FscD

(Martinez and Clotet, 2003), the activity of the NS3 protease was severelye was absent (deleted NS4A) or a construct in which the I residues at positions

icates were analyzed for each sample.

onsensus amino acid sequence of the target NS5A/NS5B cod-ng regions was determined for each of the 12 viral samplesFig. 4).

The catalytic efficiencies of the 12 proteases were3.2 ± 10% (sample A, genotype 1b), 73.9 ± 5% (B, 1b),

ig. 4. Amino acid sequence alignment of the 5A/5B target cleavage siteequence of the 12 samples analyzed in this study. Amino acid changes are indi-ated relative to the subgenomic HCV replicon I389/NS3-3′ protease sequence.ots indicate amino acid sequence identity.

266 S. Franco et al. / Virus Research 131 (2008) 260–270

Fig. 5. Relative catalytic efficiencies of HCV NS3/4A proteases containing the NS4A cofactor from four heterologous genotypes and targeting the NS5A/NS5Bcleavage site. Heterologous NS4A cofactors were tested for their abilities to enhance the NS3 protease activities of different genotypes. Two samples of the NS3domain from genotypes 1b, 1a and 3a and one sample from genotype 4d were analyzed for their cross-interactions with the NS4A cofactor from four heterologousg activit(

c(ebddfpohtw(coap

3d

tNNtp4ftd

enotypes. The catalytic efficiency of each NS3 protease was compared to the100%). At least three replicates were analyzed for each sample.

atalytic efficiencies was detected. The least efficient protease25) showed only 14% of the activity of the most efficientnzyme (C) (Fig. 3). Within the same genotype, it was possi-le to distinguish different catalytic efficiencies. Genotype 1bisplayed the widest differences: the protease from sample Aisplayed 32 and 42% of the efficiency observed for proteasesrom samples C and 1, respectively. Overall, genotype 1b dis-layed the highest catalytic efficiencies when compared with thether three genotypes. Genotype 3a proteases displayed a moreomogeneous range of catalytic efficiencies when comparedo genotype 1b proteases. Notably, the genotype 3a proteasesere also the most homogeneous group at the amino acid level

Fig. 1). These results indicate that very different catalytic effi-

iencies can be found among NS3/4A proteases from differentr similar HCV genotypes. Nevertheless, some genotypes, suchs 3a, displayed homogeneous NS3 protease genotypes andhenotypes.

Orap

y of the NS3 protease domain interacting with its homologous NS4A cofactor

.2. Cross-genotypic interactions between the NS3 proteaseomain and NS4A cofactor

After the activities of proteases having homologous interac-ions with the NS4A cofactor were determined, heterologousS4A cofactors were tested for their abilities to enhance theS3 protease activities of different genotypes. Two samples of

he NS3 domain from each of the five genotypes studied in therevious set of experiments (only one NS3 protease of genotype) were analyzed for their interactions with NS4A cofactorsrom heterologous genotypes (Fig. 5). The enzymatic activi-ies were expressed relative to the activity of the NS3 proteaseomain interacting with its homologous NS4A cofactor (100%).

f the 28 heterologous interactions tested, only three (11%)

esulted in nonfunctional enzymes (Fig. 5). These lethal inter-ctions were not genotype-specific. The interaction of the NS3rotease domain from sample B (genotype 1b) with the genotype

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a NS4A cofactor generated a nonfunctional enzyme, but thenteraction of the NS3 protease domain from sample A (alsof genotype 1b) with the genotype 1a NS4A cofactor created aully functional enzyme (Fig. 5). Four amino acid differencesR26, I71, S147 and V170) were found between the proteasesrom individuals A and B (Fig. 1). Residues I71, S147 and V170re not directly involved in the hydrophobic NS3-NS4A contactKim et al., 1996). Only the substituted residue R26, locatedn the 30 N-terminal residues of NS3, may be involved in theS3-NS4A interaction. Similarly, the interaction of the NS3 pro-

ease domain from sample D (genotype 1a) with the genotypeb NS4A cofactor resulted in a nonfunctional protease, but thenteraction of the NS3 domain from sample E (also genotypea) with the genotype 1b NS4A cofactor produced a functionalrotease (Fig. 5). Six substituted residues (P67, Q80, A91, I114,143 and S174) were observed between D and E NS3 proteases

Fig. 1). Any of these six residues might be involved in theS3-NS4A contact (Kim et al., 1996).Intriguingly, seven of the eight interactions of genotype 1a

S3 domains with heterologous cofactors generated enzymesith better catalytic efficiencies than the wild-type enzyme

Fig. 5). Nevertheless, statistical significance was only reachedhen the sample E (genotype 1a) was expressed with the geno-

ype 1b, 3a, 4a and 4d NS4 cofactors (p < 0.05, paired t-test).hese results parallel those previously obtained for the heterolo-ous interactions of the genotype 2a NS3 domain with genotypesa, 1b and 3a/b NS4A cofactors (Wright-Minogue et al., 2000).ecause only one target cleavage site was evaluated here and

n other studies, it remains to be elucidated whether heterol-gous interactions are more efficient in processing other viralr cellular NS3/4A protease target cleavage sites. HeterologousS3-NS4A interactions may have lower efficiencies than theomologous interaction when tested with other target cleavageites. Homologous NS3-NS4A interactions could be optimizedor processing several different target cleavage sites with highfficiency. In contrast to the results obtained with genotype 3aS3 proteases, the interaction of the genotype 4d NS3 pro-

ease domain from sample 24 with heterologous NS4A cofactorslways resulted in nonfunctional or deleterious enzymes (Fig. 5).n short, these data show how a small peptide (Fig. 2) represent-ng the NS4A cofactor can exquisitely modulate the enzymaticctivity of the HCV NS3 protease domain. Moreover, this mod-lation appears to be dependent on the interactions of a smallumber of NS4A amino acid residues. Because some of theeterologous NS3-NS4A interactions resulted in nonfunctionalroteases, interference with NS4A binding represents a viabletrategy for the development of a new class of NS3 PIs.

. Discussion

The present study revealed a large range of NS3/4A proteaseatalytic efficiencies in HCV-infected individuals. Because theS3/4A protease not only liberates the nonstructural proteins

rom the HCV polyprotein during virus replication but also con-ributes to permissiveness for HCV infection (Cheng et al., 2006;erreon et al., 2005; Foy et al., 2003; Johnson et al., 2007; Loot al., 2006; Meylan et al., 2005; Saito et al., 2007), the rela-

co(2

ch 131 (2008) 260–270 267

ive efficiency of this enzyme may impact the cellular antiviralesponse. The role of the NS3/4A protease in ablating the signal-ng pathway involved in the production of IFN-�/� suggests aelationship between NS3/4A proteolytic activity and responseo IFN-based therapy. Interestingly, specific mutations leadingo enhanced replication in the HCV replicon system, some ofhem within the NS3 protease domain, were associated with alower decrease in HCV RNA concentration during IFN-basedherapy (Sarrazin et al., 2005; Wohnsland et al., 2007).

The cross-sectional nature of our study does not allowxtraction of any conclusion regarding the relationship betweennzyme activity and pathogenesis or treatment response. Nev-rtheless, we describe here a simple and accurate method toxamine the catalytic efficiency of the NS3/4A protease thatill allow the characterization of a large cohort of samples. Theigher catalytic efficiency displayed by genotype 1b NS3 pro-eases might be indicative of the poor response to IFN-basedherapies of individuals bearing genotype 1b viruses. Becausempairment of human immunodeficiency virus type 1 (HIV-1)rotease catalytic efficiency has been associated with a reducedirus replication capacity ex vivo and a less pathogenic coursef infection in vivo (Stoddart et al., 2001), characterization ofhe catalytic efficiencies of HCV NS3/4A proteases might reveallues about the interaction of the virus with its host.

In a recent study, we analyzed at a high resolution (∼100 indi-idual clones) the enzymatic activities of three HCV NS3/4Arotease quasispecies (Franco et al., 2007a). This report showedhat 67% of the analyzed enzymes displayed detectable proteasectivity. Moreover, within this 67%, a huge range of catalyticfficiencies was observed, demonstrating that HCV replicationenerates variant NS3/4A proteases with quantitatively differ-nt functional properties. An intriguing question is why suchwide range of catalytic efficiencies is found among masterS3/4A proteases isolated from different HCV-infected individ-als. Three possibilities might explain these findings: (i) there iswide range of optimum proteolytic activity, and only activitiesutside this range confer a selective advantage or disadvantage;ii) NS3 catalytic efficiency is not a selectable trait, and geneticrift or selection pressures in other viral genomic regions drivehe evolution of the NS3 protease genotype and phenotype oriii) the most advantageous viral catalytic efficiency is selectedy the interaction of the virus with its host. The present data sup-ort the latter possibility because they indicate that the activityf the HCV NS3 protease can be precisely regulated by theS4A cofactor. A few amino acid changes within the NS4A orS3 protein can drastically affect the proteolytic activity of theS3 protease, suggesting that finely tuning the activity of theS3 protease may be advantageous for the virus in its inter-

ction with the host. Although the exact biological function ofhe NS4A cofactor remains to be elucidated, it has been sug-ested that the existence of the NS4A cofactor may reflect aeed for regulating protease activity during the viral life cycleWright-Minogue et al., 2000). Recently, infectious HCV cell

ulture systems have been established that will likely improveur knowledge of virus-host interactions and the virus life cycleLindenbach et al., 2005; Wakita et al., 2005; Zhong et al.,005).

2 esear

osuHMSNHa(trr(ofiidwtdrbiao

A

cF

R

A

A

B

B

B

B

C

C

C

C

D

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F

F

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68 S. Franco et al. / Virus R

Several NS3/4A PIs have been developed to block this stepf the viral life cycle, and in some cases they have been shown toignificantly decrease the plasma viral load of infected individ-als (Bogen et al., 2006; Goudreau and Llinas-Brunet, 2005;uang et al., 2006; Lamarre et al., 2003; Lin et al., 2006;alcolm et al., 2006; Perni et al., 2006; Reesink et al., 2006;

arrazin et al., 2007b; Thomson and Perni, 2006). Nevertheless,S3 protease variants resistant to PIs have been isolated fromCV replicon cells (Lin et al., 2004; Lu et al., 2004; Tong et

l., 2006; Zhou et al., 2007), as well as from treated individualsSarrazin et al., 2007a; Wohnsland et al., 2007). Because all ofhe above-mentioned PIs are substrate-based inhibitors, cross-esistance to diverse PIs can be achieved by the mutation of a fewesidues, or in some cases by a single amino acid substitutionLin et al., 2005, 2004; Yi et al., 2006). Thus, the combinationf multiple PIs could have limited value in clinical practice. Thending that some NS4A cofactors can be lethal for the activ-

ty of some NS3 protease domains suggests the possibility ofesigning small peptides or peptidomimetic PIs that competeith the binding of the natural NS4A cofactor to the NS3 pro-

ease. Because modulation of NS3 protease activity seems to beependent on the interaction of only a few NS4A amino acidesidues, the design of small peptidomimetic PIs may be feasi-le. Current therapies for the treatment of HIV-1 infection havendicated that combining several antiviral compounds directedgainst different viral targets will likely provide better treatmentptions for HCV-infected individuals.

cknowledgments

This work was supported by the Spanish Minsiterio de Edu-acion y Ciencia (MEC) project BFU2006-01066/BMC andondo de Investigacion Sanitaria (FIS) project PI050022.

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