Journal of Medical Virology 71:195–204 (2003)

Characterization and Evolution of NS5AQuasispecies of Hepatitis C Virus Genotype 1b inPatients With Different Stages of Liver Disease

Sandra Franco, Mireia Gimenez-Barcons, Francesc Puig-Basagoiti, Ivana Furcic,Jose-Marıa Sanchez-Tapias, Juan Rodes, and Juan-Carlos Saiz*

Liver Unit, Institut de Malalties Digestives, Departament de Medicina (IMD),Institut d’ Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Hospital Clınic,University of Barcelona, Barcelona, Spain

Thequasispeciesnatureof hepatitis Cvirus (HCV)is thought to play a central role in modulatingviral functions. Recent work has linked NS5Aprotein with viral replication, resistance to inter-feron (IFN), and control of cellular growth, pro-bably through the interaction of its protein kinaseR (double strandedRNA-activatedprotein kinase,PKR) binding domain (PKR-bd) with cellular PKR,but knowledge of how PKR-bd viral populationevolves during disease progression is limited.Since we have previously described an associa-tion between amino acid composition of the PKR-bd and the presence of HCC, in this report wefurther investigated thedynamic behavior of viralpopulation parameters by sequencing an aver-age of 20 clones per sample in 27 samples from19 untreated patients with different degrees ofliver disease, 8 ofwhomwere followedover time.Viral population parameters varied widely frompatient to patient, but no differences wereobserved in the complexity, diversity, types ofnucleotide changes, or evolutionary pattern ofthe quasispecies according to the stage of liverdisease. In five samples, we detected ‘‘quasispe-cies-tails’’; that is, clones whose minimum ge-netic distance to the remaining clones of theirown quasispecies were higher than the max-imum genetic distance found between any othertwo clones of the same sample. In summary,independent of the degree of liver disease, orthe mutations detected within the consensussequence of the PKR-bd, the NS5A of HCV pre-sents a flexible and variable quasispecies struc-ture that remains largely stable during thenaturalcourse of an HCV infection, highlighting thecentral role of NS5A protein in viral life cycle.J. Med. Virol. 71:195–204, 2003.� 2003 Wiley-Liss, Inc.

KEY WORDS: hepatitis; hepatocarcinoma;PKR-bd; viral dynamics

INTRODUCTION

Hepatitis C virus (HCV), the unique Hepacivirus ofthe Flaviviridae family [Choo et al., 1989], is anenveloped, positive single-stranded RNA virus. Acuteinfection with HCV very often progresses to chronicinfection, which may eventually lead to cirrhosis andhepatocellular carcinoma (HCC). Although the factorsthat contribute to the development of HCC are largelyunknown, it seems very likely that the pathogenesis ofHCV infection is directly related to a strong interplaybetween the host defense mechanisms and the ability ofthe virus to evade them.

One of the most important features of HCV is that itsgenome exhibits significant genetic variability asso-ciated primarily with the error-prone nature of its RNA-dependent RNA polymerase. As a consequence, HCVcirculates in an infected individual as a swarm of gene-tically different but related variants [Martell et al.,1992], called quasispecies. This appears to favor rapidadaptability of the virus in the event of environmentalchanges, and it is believed to serve as one of the survivalstrategies to circumvent selective forces. To date, thevast majority of studies aiming at defining the role ofthe quasispecies structure of HCV in the pathogenesisof chronic infection have focused on the hypervariableregion 1 (HVR-1) of the viral E2 gene. However, theassociation between HVR-1 evolution and diseaseprogression remains controversial, and little is known

Grant sponsor: Ministerio de Ciencia y Tecnologıa; Grantnumber: SAF01-1799; Grant sponsor: Fundacio Clinic (FC); Grantsponsor: Instituto de Salud Carlos III (ICIII); Grant sponsor:Asociacion Espanola de Cooperacion Internacional (AECI).

*Correspondence to: Dr. Juan-Carlos Saiz, Departamento deBiotecnologıa, Instituto Nacional de Investigaciones Agrarias,Ctra. Coruna Km. 7.5, 28040 Madrid, Spain.E-mail: jcsaiz@inia.es

Accepted 15 April 2003

DOI 10.1002/jmv.10470

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2003 WILEY-LISS, INC.

about the relevance of the dynamics of HCV quasispe-cies in other viral genomic regions [Forns et al., 1999].

Recently, there has been considerable focus on thenonstructural 5Aprotein (NS5A) ofHCV. In the absenceof an appropriate animal model or culture system, thefunctions of this protein in the actual setting of HCVinfection have not yet been established; nevertheless, ithas been suggested that the NS5A protein plays apivotal role in viral replication [Lohmann et al., 1999;Blight et al., 2000] and resistance to IFN therapythrough the interferon (IFN) sensitivity determiningregion (ISDR) [Enomoto etal., 1996].Although the latterissue is stillmuchdebated [Pawlotsky, 1999], analysis ofmost of the published information appears to confirmthe relationship between theamino acid (aa) sequence ofthe ISDR (encompassing aa 2209–2248) and the typeof response to IFN [Gimenez-Barcons et al., 2001;Witherell and Beineke, 2001]. Furthermore, in vitrostudies showed that the NS5A region responsible forresistance to IFN therapy was able to interact withcellular protein kinase R (PKR) through the so-calledPKR-bindingdomain (PKR-bd) [Gale et al., 1997],whichinclude the ISDR plus an additional 26 aa stretchlocated at the C-terminal portion (aa 2209–2274). Thisinteraction leads to dramatic effects onviral translation,cell growth and apoptosis [Gale et al., 1998, 1999];therefore, it may contribute to HCC development.

In this regard,we have previously observed [Gimenez-Barcons et al., 2001] that the consensus aa sequence ofthe PKR-bd of HCV strains from patients with HCCshowed a much higher aa heterogeneity than that frompatients with liver cirrhosis (LC), and that such differ-ences were not observed in other regions of the viralgenome, including core, HVR-1, PePHD, NS3, or the 50

and 30 flanking regions to the PKR-bd.As there are strong arguments linking NS5A protein

to resistance to IFN, replication, and pathogenesis ofHCV, we addressed whether the quasispecies structureand evolution of HCV in this region may affect thecomplex interaction between the virus and the host and,consequently, contribute to liver disease progression.

PATIENTS AND METHODS

Patients

The study included 19 patients with chronic hepatitisCwith different degree of liver disease severity: group Aincluded3patientswith chronic hepatitis (CH); groupB,6 patients with LC; and group C, 10 patients with LCand superimposed HCC. All patients were infected by1b-HCV, and none had received antiviral therapy.Samples were randomly retrieved from a larger cohortof patients, whose clinical, biochemical and epide-miological features have been previously described[Gimenez-Barcons et al., 2001]. Samples were denotedby consecutive numbers, preceded by a capital letterdepending on the liver histology of the patient (A, B,or C) and followed by a lowercase letter that indicatesthe first (a) or second (b) sample analyzed for eachpatient.

Viral evolution was analyzed in a subgroup of eightpatients from whom sequential serum samples wereavailable: during chronic hepatitis in three cases, inthe presence of LC in one case, before and after HCC inone case, and once the tumor was diagnosed in threecases.

HCV RNA Extraction, Amplification,Genotyping, and Quantification

HCV-RNA extraction and specific amplification byreverse transcription-polymerase chain reaction (RT-PCR) of the 50 noncoding (50NC) and nonstructural 5A(NS5A) regions of HCV have been previously described[Saiz et al., 1997; Gimenez-Barcons et al., 2001]. HCVgenotypewasdeterminedbyrestriction fragment lengthpolymorphism (RFLP) of the amplified 50NC region asdescribed elsewhere [Lopez-Labrador et al., 1997].HCV-RNA concentration in serum was measured bythe COBAS Amplicor HCV Monitor test v.2.0 (RocheDiagnostic Systems).

NS5A Cloning and Sequence Analysis

PCR-amplified products of 571 base pairs (bp),encompassing the PKR-bd (198 nt) of the NS5A, werepurified, directly ligated to the TA cloning plasmidpGEM T-Easy (Promega, Madison, WI), and cloned aspreviously described [Sanchez-Fueyo et al., 2001]. Anaverage of 20 (14–25) randomly selected clones fromeach time point of every patient were analyzed.

Clones were sequenced using previously describedinner primers [Saiz et al., 1998; Gimenez-Barconset al., 2001] with the DyeDeoxy Terminator CycleSequencing Kit (Perkin-Elmer Applied Biosystems,Warrington, UK) in a 310 DNA sequencer (AppliedBiosystems,Westerstad,Germany). Electropherogramswere analyzed using the Sequence Navigator software(Perkin-Elmer). HCV sequences have been submit-ted to GenBank under accession numbers AF464197-AF464734.

Quasispecies Analyses

The sequence of all clones from each time point ofevery patient was aligned, and their respective within-consensus (Intra-Cons) sequences were computed.Genetic complexity of the PKR-bd of all samples wasaddressed by calculating the Shannon entropy[Wolinsky et al., 1996; Pawlotsky et al., 1998], definedas the different sequences or clusters of sequences thatcan appear at a given time point

S ¼ �X

piln pið Þ

where pi is the frequency of each sequence in the viralpopulation. The normalized entropy, Sn, was calculatedat both nucleotide and amino acid levels as

Sn ¼ S=lnN

where N is the total number of sequences analyzed.

196 Franco et al.

Nucleotide and aa derived sequences were alignedwith the CLUSTAL W 1.6 program [Thompson et al.,1994], and genetic distance between pairs of sequencesand phylogenetic trees was analyzed with the PHYLIPpackage [Felsenstein, 1993]. For each time point ofevery patient, Ks and Ka values for all the sequencesanalyzed (where Ks is the frequency of synonymoussubstitutions per synonymous site and Ka is the fre-quency of nonsynonymous substitutions per nonsynon-ymous site) were calculated by pairwise comparisonof every single sequence obtained using the MEGAprogram [Kumar et al., 1994].

When sequential serum samples were available, theinter-sample genetic distance, Ka and Ks were calcu-lated by pairwise comparison of sequences from bothtime points [Pawlotsky et al., 1998], as well as by com-parison of each single clone with the derived consensussequence of the first time point analyzed [Wolinskyet al., 1996].

Statistical comparisons between groups were madeby the Fisher’s exact test for categorical variables andby the Mann-Whitney test for quantitative variables.A P-value of <0.05 was considered significant.

RESULTS

Characterization of the PKR-bd Quasispecies

A 571-bp PCR fragment of the NS5A gene of HCV-1b,encompassing thePKR-bd,was amplified in each case.Atotal of 537 cloneswere analyzed in the PKR-bd (198 nt),with a mean of 20 clones (range, 14–24) per sample.

Alignment of nucleotide (nt) and deduced aa se-quences of all clones disclosed amino acid changes inalmost all residues of the PKR-bd, and especially atpositions 2216, 2218, 2251, 2252, 2260, 2265, and 2268,which were more prone to accept aa substitutions.Insertions or deletions were not observed in the total of537 clones analyzed, except in sample C19b, where aninsertion and a deletion were observed at positions 2237and 2261, respectively (Fig. 1).

Further analysis of the amino acid composition ofthe quasispecies showed that in all cases, except insamples A1a and C18b, the intra-sample aa derivedconsensus sequence (Intra-Cons) and the more repre-sented sequences of each sample were identical (Fig. 1).Major variants of each quasispecies represented 30–91% of all detected subpopulations.

Table I summarizes the viral population parametersin the 19 patients studied. Overall, analysis of the gene-tic complexity of the samples showed that the mean ntand aa normalized Shannon entropies were relativelyhigh and varied widely from one patient to the next(0.76� 0.16; range, 0.39–0.95; and 0.48�0.19, range,0.11–0.74, respectively). Similarly, the degree of qua-sispecies diversity, calculated as the average geneticdistance (gd) within the quasispecies, was 0.018�0.0082, and also greatly varied from patient to patient.All gd within each individual quasispecies were in therange of 0–0.086, except for sample C19, in which theyvaried from 0 to 0.327. This fact was due to the presence

of a mixed infection with two different HCV strains inthis patient (see below and Figs. 2 and 3). In addition,the proportion of synonymous substitutions per syno-nymous site, Ks, were higher than that of nonsynon-ymous substitutions per nonsynonymous site, Ka (0.046and0.012, respectively). Phylogenetic analysis discloseda variety of quasispecies patterns, from highly homo-geneous to quite heterogeneous populations (Fig. 3).

Remarkably, a more detailed analysis of the quasis-pecies structure showed the presence of what we havetermed ‘‘quasispecies-tails’’ (qs-tails). These ‘‘qs-tails’’can be defined as clones that belong to a given quasi-species distribution and whose minimum gd to the re-maining clones of their own quasispecies is greater thanthemaximumgd observed between any other two clonesof the same sample. The likelihood of a laboratory cross-contamination of samples was ruled out by phylogeneticanalysis of all sequenced clones, aswell as thepossibilityof a mixed infection with two different strains, as ‘‘qs-tails’’ were more related genetically to the remainingclones of their respective sample than to any clone froman unrelated sample (Fig. 2). In any case, no differenceswere observed in any of the viral variables analyzedbetween quasispecies in which ‘‘tails’’ were or were notdetected.

PKR-bd Quasispecies and Liver Histology

The 19 patients studied were grouped according totheir clinical status as follows: group A, patients withCHand no histological signs of LC and/orHCC; groupB,patientswithLCwithout superimposedHCC;andgroupC, patients with LC andHCC. As shown in Table I, viralload was similar in the three groups of patients (5.51,5.55, and 5.29 log10 IU/ml for group A, B, and C, re-spectively). Likewise, no statistically significant differ-ences were observed in their mean nt or aa normalizedShannon entropies (0.73, 0.77, and 0.77, and 0.44, 0.55,and 0.45, respectively), nor in their average gd (0.0174,0.0168, and 0.0205, respectively). Phylogenetic analysisdid not showany specific quasispecies pattern regardingthe liver histology of the patient (Fig. 2).

Similarly, no specific nt or aa changes associated withthe degree of liver disease and no amino acid motifsuniquely shared by sequences from a group of patients(A, B, or C) could be identified within the quasispecies(Fig. 1). The mean number of synonymous and non-synonymous substitutions within the region of interestalso varied widely from patient to patient but wascomparable among the three groups, A, B, and C(0.049, 0.034, and 0.054, and 0.008, 0.012, and 0.013,respectively).

PKR-bd Quasispecies Evolution

After characterization of the quasispecies structureof the PKR-bd in basal samples, we explored the gene-tic evolution of this region from a subgroup of 8 patientsin sequential serumsamples taken10–72monthsapart.Atotal of330cloneswereanalyzed (20clonespersample;range, 17–24). As shown in Table II, the dynamics of

NS5A-HCV Quasispecies in Chronic Hepatitis C 197

viral load fluctuated over the period of time studied inall patients: viremia slightly decreased in three of thepatients and increased in five. The rate of fixation ofmutationswas similar in all samples (Table II), with the

exception of samples from patient C19, inwhomamixedinfection was observed (see below).

Nucleotide changes in the intra-sample derived con-sensus sequences between time points (a) and (b) were

Fig. 1. Alignment of the deduced amino acid intra-sample consensussequences of the protein kinase R binding domain (PKR-bd) (aa 2207–2278), including the interferon (IFN) sensitivity determining region(ISDR) (aa 2209–2248), of the NS5A protein of every analyzed sample.The deduced inter-sample consensus (Inter-Cons) sequence of allanalyzed clones and the aa sequence of hepatitis C virus HCV-J

prototype [Kato et al., 1990] have been included for comparison. Aminoacid residues are indicated by standard single letter code, and dashesindicate residues identical to those in their respective Inter-Conssequence. In sequences from sample C19b, a single amino acid deletionis denoted by d. Number of individual sequences detected and relativefrequencies of the major variant of each sample are shown.

198 Franco et al.

observed in all patients, but except in samples fromC18and C19, the consensus amino acid sequences remainedunchanged (see intra-cons; Fig. 1). The average inter-sample genetic distances (gd) (when comparing end offollow-up versus first sample) were not significantlydifferent to the average genetic distances of clones frombasal time, except in one case (C18). Pairwise compar-ison of clones from both time points disclosed higherKs values in all cases, suggesting that evolutionarychanges in this region were mainly due to randomgenetic drift during the period studied. Similar resultswere obtained by comparison of each single clone withthe derived consensus sequence of the first time pointanalyzed (data not shown).

Phylogenetic analysis of viral diversification overtime in individual cases is presented in Figure 3. Ingeneral, substantial intermingling of viral sequencesfrom both time points analyzed was found, a findingconsistent with the lack of significant genetic evolutionobserved during the follow-up period. Clustering ofsequenceswas detected in only two cases, patients 9 and18.A clear shift to anew, but closely relatedquasispeciespopulation was observed in patient 9 (Fig. 3), albeit nochanges in the consensus derived aa sequence weredetected (Fig. 1). In fact, the inter-sample gd was onlyslightly higher than the intra-sample gd of the first timepoint (0.0213 and 0.0251, respectively; Table II).

In contrast, in patients 18 and 19, the consensusamino acid sequences between both time points differ-ed in 5 and 15 amino acid residues, respectively, andnucleotide complexity, assessed by the normalizedentropy (Sn), was lower in the follow-up samples forboth patients. In patient 18, the more represented

population detected at basal time (C18a)was completelyreplaced and evolved after 48 months into a mixture oftwo major subpopulations (C18b) that derived fromvariants already present at basal time (Fig. 3). Con-sistent with this analysis, the inter-sample gd wasalmost double than the intra-sample gd (0.0229 and0.0432, respectively) In patient 19, a mixed infectionwith two major genetically distant viral strains wasobserved (Figs. 2 and 3), being the more representedsequence of the first sample completely replaced by aminor variant that was already present at basal time.Important differences in quasispecies complexity anddiversity between the two samples were observed(Table II), but when minor variant strains of eachquasispecies were excluded from the analysis, the com-plexity and diversity of each variant were similar tothat of the remaining samples analyzed. Phylogeneticanalysis confirmed these data (Fig. 3).

DISCUSSION

The quasispecies structure of HCV [Martell et al.,1992] may have implications in viral persistence andin the long-term outcome of HCV infection since, selec-tion of pre-existing, or emerging subpopulations of thequasispecies may confer important biological propertiesto thevirus [DomingoandHolland, 1997;Domingo et al.,1998]. To date, most of the studies on the possible im-plications of the structure and evolution of HCV quasi-species during chronic infection have focused on theHVR-1 of the E2 envelope protein with no conclusiveresults, and little is known about other genomic regionsof HCV [Forns et al., 1999].

TABLE I. Characteristics of the PKR-bd Quasispecies of the Samples Analyzed

Patient Liver histologyMutations onthe PKR-bda

Viral loadb

(log10 IU/ml)No. ofclonesc

Snd Type of aa changed

nt aa gdd Ks Ka

A1 CH 3 5.60 22 0.81 0.74 0.0247 0.0599 0.0138A2 CH 4 4.95 21 0.71 0.48 0.0117 0.0243 0.0078A3 CH 3 6.00 23 0.68 0.11 0.0158 0.0631 0.0011B4 LC 0 6.35 19 0.43 0.20 0.0051 0.0147 0.0021B5 LC 2 5.27 14 0.91 0.69 0.0251 0.0468 0.0182B6 LC 2 5.72 18 0.88 0.64 0.0136 0.0257 0.0099B7 LC 2 6.15 18 0.94 0.52 0.0293 0.0729 0.0155B8 LC 4 4.11 15 0.71 0.71 0.0065 0.0115 0.0086B9 LC 2 5.73 18 0.73 0.56 0.0213 0.0313 0.0037C10 HCC 2 5.38 21 0.39 0.28 0.0104 0.0141 0.0092C11 HCC 1 5.65 21 0.79 0.50 0.0215 0.0494 0.0128C12 HCC 2 5.62 17 0.86 0.48 0.0281 0.0660 0.0164C13 HCC 2 5.16 21 0.79 0.25 0.0187 0.0570 0.0069C14 HCC 5 5.19 19 0.70 0.38 0.0088 0.0187 0.0056C15 HCC 5 5.20 18 0.56 0.25 0.0145 0.0475 0.0043C16 HCC 11 4.34 24 0.92 0.57 0.0337 0.0916 0.0165C17 HCC 3 4.84 20 0.77 0.61 0.0187 0.0479 0.0093C18 HCC 2 5.62 23 0.95 0.56 0.0229 0.0714 0.0078C19 HCC 0 5.86 19 0.95 0.64 0.1211 0.1727 0.1048

aa, amino acid; nt, nucleotide; gd, genetic distance; CH, chronic hepatitis; LC, liver cirrhosis, HCC, hepatocellular carcinoma.aNumber of aamutations detected in the intra-sample consensus PKR-bd sequence (Intra-Cons) when comparedwith the inter-sample consensussequence (Inter-Cons) of all clones analyzed.bViral load was determined by COBAS Amplicor HCV Monitor test v.2.0 (Roche Diagnostic Systems).cNumber of different clones analyzed.dNormalized nt and aa entropy (Sn), within and between genetic distance (gd) and the proportion of synonymous substitutions per synonymoussite (Ks) and nonsynonymous substitutions per nonsynonymous site (Ka) was calculated as described in Patients and Methods.

NS5A-HCV Quasispecies in Chronic Hepatitis C 199

Fig. 2. Phylogenetic tree (a) and intra-sample genetic distancevalues of the quasispecies-tails (b). a: For simplicity, apart from thetails, only the intra-sample consensus (Intra-Cons) sequence of eachpatient is included in the tree. The deduced inter-sample consensus(Inter-Cons) sequence of all analyzed clones is used as outgroup.Numbers in italics represent bootstrap proportions in support of theadjacent node based on 100 resampling iterations. Only bootstrapvalueshigher than70%are shown.Scale bars represent 10%nucleotidesequence divergence. Circles indicate clones defined as ‘‘tails’’ (seeResults and Discussion). Squares represent the deduced Intra-Cons

sequences of the two different isolates co-infecting patient C19. b: Dataobtained frompatientswithdisparate liver disease severity: (A) chronichepatitis; (B) liver cirrhosis; and (C) hepatocellular carcinoma. Thewhite bars correspond to the mean genetic distances (gd) of all clonesof the quasispecies, excluding the ‘‘tails.’’ Filled and hatched barscorrespond to the mean gd of the clones considered as ‘‘tails’’ to theremaining clones of their respective sample. Mean gd were calculatedas described in material and methods. The high genetic distance ofsampleC19 is due to thepresence of amixed infectionwith twodifferentHCV-strains.

Fig. 3. Phylogenetic trees of the protein kinase R binding domain(PKR-bd) of the NS5A protein of hepatitis C virus (HCV) quasispeciessequences from the 8 patients sequentially analyzed. The scale barsrepresent percentage of nucleotide sequence divergence. Circles,triangles, and squares represent samples obtained while the liverhistology of the patients was at the chronic hepatitis (CH), liver

cirrhosis (LC), and hepatocellular carcinoma (HCC) stages, respec-tively. White and black symbols represent clones from the first (a)and second (b) time points analyzed, respectively. The deduced inter-sample consensus (Inter-Cons) sequence of all analyzed clones isincluded in the trees as outgroup.

200 Franco et al.

Fig.3.

NS5A-HCV Quasispecies in Chronic Hepatitis C 201

TABLE

II.PKR-bdQuasisp

eciesEvolution

*

Patien

tHistologya

Tim

ebetwee

nsa

mples

Mutation

swithin

the

PKR-bdb

Viralloadc

(log

10IU

/ml)

No.

ofclon

esd

Normalized

Shannon

entrop

y(e)

Mea

ngen

etic

distance

eTypeof

nt

changes

e

Rate

offixation

ofmutation

sf

ab

ab

ab

nt

aa

Intra

Inter

Ks

Ka

(�10�3)

(mo)

ab

ab

1A

toA

11

33

5.60

6.09

22

17

0.81

0.83

0.74

0.39

0.0247

0.0243

0.0649

0.0117

1.91

2A

toA

10

44

4.95

5.01

21

21

0.71

0.81

0.48

0.51

0.0117

0.0151

0.0340

0.0091

<2.10

3A

toA

12

33

6.00

5.88

23

22

0.68

0.79

0.11

0.11

0.0158

0.0156

0.0607

0.0017

1.75

7B

toB

54

22

6.15

5.39

18

20

0.94

0.97

0.52

0.82

0.0293

0.0348

0.1004

0.0141

0.77

9B

toC

72

22

5.73

6.12

18

18

0.73

0.57

0.56

0.33

0.0213

0.0251

0.0701

0.0109

1.46

17

Cto

C12

33

4.84

5.43

20

21

0.77

0.84

0.61

0.61

0.0187

0.0195

0.0488

0.0102

<1.75

18

Cto

C48

28

5.62

4.73

23

23

0.95

0.82

0.56

0.62

0.0229

0.0432

0.0873

0.0293

4.37

19g

Cto

C11

015

5.86

5.97

19

24

0.95

0.36

0.64

0.28

0.1129

0.1181

0.2280

0.1759

63.09

aa,aminoacid;nt,nucleotide.

*Twotime-pointsa

mpleswerecomparedin

each

patien

t(a

andb).

aA,ch

ronic

hep

atitis;B,liver

cirrhosis;C,hep

atocellularcarcinom

a.

bNumber

ofaamutation

sdetectedin

theintra-sample

consensu

sPKR-bdsequen

ce(Intra-C

ons)

when

comparedwiththeinter-sa

mple

consensu

ssequen

ce(Inter-Con

s)of

allclon

esanalyzed.

c Viralloadwasdetermined

byCOBASAmplicorHCVMon

itor

test

v.2.0

(RocheDiagnosticSys.).

dNumber

ofdifferentclon

esanalyzed.

eMea

nntandaaShannon

entrop

y,within

andbetweengen

etic

distancesandKsandKavalues

werecalculatedasdescribed

inPatien

tsandMethod

s.f Rate

offixation

ofmutation

swascalculatedasnumber

ofnucleotidesu

bstitution

sbetweentheIn

tra-C

onssequen

ceof

each

patien

tper

thetotalnumber

ofnucleotides

sequen

cedper

yea

r.gHighgdandmutation

ratesfoundweredueto

thepresence

ofamixed

infection,w

hen

only

clon

esof

thesa

mequasisp

eciesweretaken

into

accou

nt,therate

wereof

1.91and7.65�10�3forea

chof

thestrains.

202 Franco et al.

Although the actual functions of NS5A protein ofHCV in vivo are not well known [Pawlotsky, 1999],this protein became the focus of much attentionbecause of its possible role in IFN resistance [Enomotoet al., 1996] and in control of cellular growth andtumorogenesis [Gale et al., 1998, 1999]. In this regard,we have previously observed a possible associationbetween amino acid heterogeneity of PKR-bd in theNS5A protein and presence of HCC, suggesting apossible link between NS5A protein and HCV patho-genesis [Gimenez-Barcons et al., 2001]. Since verylittle is known about NS5A population turnover andevolution in the natural course of HCV infection,and because many important functions have been sofar attributed to NS5A protein, we have characterizedthe quasispecies composition and dynamics of theNS5A central region protein during the natural courseof the disease. The study was carried out in 27 samplesfrom 19 untreated patients with different degreesof disease severity, some of which were followed overtime.

No specific residues or motifs associated with theoutcome of the liver disease were found. Some positionsseemed to accept amino acid changes more frequentlythan others, probably reflecting the tolerance of theseresidues to accept changes.

Cross-sectional analysis of the PKR-bd quasispeciesshowed that the complexity and the diversity of thepopulations in the different samples widely varied frompatient to patient and that they were unrelated to thestage of liver disease. Quasispecies were composedof either highly homogeneous populations or a cloudof relatively divergent molecules. By phylogeneticanalyses, clustering of sequences in relation with theseverity of liver damage was not observed.

Interestingly, a detailed analysis of the quasispeciesstructure revealed the presence of what we have termed‘‘quasispecies-tails’’ in 5 of the 27 viral populations.These ‘‘tails’’ are clones whose genetic distances to anyclone of their own quasispecies are higher than themaximumgenetic distance found betweenany other twoclones of the same specimen. ‘‘Tails’’ were found insamples from twopatientswithCHand threewithHCC.At this moment, the biological meaning, if any, of such‘‘tails’’ is unknown. It could be argued that they cor-respond to subpopulations that are replicating in placesother than the liver; however, a significant extra-hepatic contribution of HCV variants to the circulatingpool is still a controversial issue [Afonso et al., 1999;Cabot et al., 2001]. A possible hypermutational event, asdescribed in other RNA viral models [Cattaneo et al.,1988], was not observed in the tails. Finally, they couldbe interpreted as a molecular record of a past quasis-pecies composition, as if amolecularmemorywas acting[Ruiz-Jarabo et al., 2000]. This ‘‘memory’’ has beendescribed as minor variants of the quasispecies, whichwill represent past evolutionary events and might pro-vide additional mechanisms to respond to externalstimuli that were previously experienced by the samepopulation.

Evolutionary analyses showed that NS5A protein is aquite stable element of the viral genome since, inmost ofthe cases, variation of the quasispecies structure did notyield changes in the consensus amino acid sequence.Despite this relative stability, a detailed analysis of thequasispecies distribution provided evidence for severaldifferent patterns of PKR-bd evolution (Fig. 3). In somecases, there were no extensive sequence changes overthe period studied (patients 3 and 17), while othersexhibited sporadic variation with fluctuations in themajor andminor variants, both present at the same timebut with changes in their relative proportions (patients1 and 7). Amore profound variationwas also possible, inwhich either completely different PKR-bd sequencesbecome dominant, or distinct PKR-bd lineages coexistwithin an infected patient with different proportionsof both populations over time (patients 18 and 19,respectively; see below and Figs. 1 and 3). These datasupport the notion that, during the natural course of theliver disease, the quasispecies distribution of the PKR-bd of the NS5A fluctuates over time in its genomiccomplexity and diversity and that both rapid sequenceturnover or relatively evolutionary stasis are observedin the infecting populations. However, no correlationwas found between the qualitative changes of thequasispecies distribution, either at the nucleotide oramino acid level, and liver disease severity.

The rate of fixation of mutations observed in all oursamples (0.77–7.65�10�3 s/nt/y) was similar to thatpreviously described for this region [Polyak et al., 1998;Maekawa et al., 2000; Soguero et al., 2000; Gimenez-Barcons et al., 2001]. A mixed infection with two dif-ferent HCV strains was detected in one patient (C19).Minor variants were found in all samples analyzed butinterestingly, except in one case (patient C18), imposi-tion of such variants was not observed in the evolu-tionary analysis. Thiswould be in keepingwith previousstudies on evolution of NS5A during antiviral therapyshowing that selection of variants is an unusual event[Pawlotsky et al., 1998; Polyak et al., 1998; Saiz et al.,1998]. Likewise, Ks values were always higher thanKa;thus, no strong selective pressure appears to be actingover this region in the circumstances analyzed in thepresent study. Therefore, since no known neutralizingor cytotoxic epitopeshavebeendescribed in this genomicregion, quasispecies fluctuationmight be a consequenceof selective forces acting at other viral regions, such asthe HVR-1, that would select for some specific strains ina given environment. Whatever the specific nature ofthe forces that drive these changes, they have beenfound to occur irrespective of the clinical status of thepatients, suggesting that these changes are the result ofa continuous reorganization of the quasispecies struc-ture, rather than of profound changes in the environ-mental conditions, which is consistent with previousreports analyzing other genomic regions of HCV [Cabotet al., 2001].

In summary, our results indicate that the PKR-bdof the NS5A of HCV is a quite stable element of theviral genome. This fact would be in agreement with the

NS5A-HCV Quasispecies in Chronic Hepatitis C 203

putative important biological properties so far attri-buted to this protein. Although a larger number ofsamples should be analyzed, the complexity, diversity,and evolutionary rate of the genomic region analyzedappear to be independent of the clinical status of thepatients.

ACKNOWLEDGMENTS

This work was supported in part by Ministerio deCiencia y Tecnologıa (SAF01-1799); and by grants fromFundacio Clinic (FC) (to S.F.), from Instituto de SaludCarlos III (ICIII) (to M.G.B. and F.P.B.), and fromAsociacion Espanola de Cooperacion Internacional(AECI) (to I.F.). We thank the sequencing core ofUniversity of Barcelona (UB) for technical assistancewith the sequences of this work.

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