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Honda, M., S. Kaneko, A. Sakai, M. Unoura, S. Murakami, and K. Koba- yashi. 1994. Degree of diversity of hepatitis C virus quasispecies and pro- gression of ...
JOURNAL OF VIROLOGY, Feb. 1997, p. 1732–1734 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 71, No. 2

Structure of Replicating Hepatitis C Virus (HCV) Quasispecies in the Liver May Not Be Reflected by Analysis of Circulating HCV Virions ` , VICTOR VARGAS, BEATRIZ CABOT, JUAN I. ESTEBAN,* MARI´A MARTELL, JOAN GENESCA ´ MEZ RAFAEL ESTEBAN, JAIME GUARDIA, AND JORDI GO Liver Unit, Department of Medicine, Hospital General Universitari Vall d’Hebron, Universitat Auto ´ma de Barcelona, Barcelona, Spain Received 14 June 1996/Accepted 22 October 1996

We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum samples from four patients with chronic hepatitis C. Sequences from three different biopsy specimens from a liver explant from one patient were compared with each other and with the circulating sequences. Our results demonstrate that the circulating quasispecies does not necessarily reflect the viral population replicating in the liver and that this is not due to a macroscopic anatomic compartmentalization of HCV replication. This finding has important implications for the pathogenesis and natural history of chronic HCV infection. the E2-NS2 region (outer set, MJJ3 and MJJ4; inner set, MJJ5 [59-CTAGAATTCAAAAATATTGTAACCACCA-39 at positions 2547 to 2530] and MJJ6 [59-ACAGGATCCAGTCCTT CCTTGTGTTCTTCT-39 at positions 2299 to 2317]). The E2 hypervariable region (HVR) was amplified by seminested PCR with specific primers E2 (59-TCCCGGATCCGGGATATGA TGATGAATTGGT-39; positions 959 to 979) and E31b (59-A GCGAATTCATGTGTGTAGAACAGCGCGGCA-39; positions 1334 to 1314) for the first round of PCR and with E2 and an inner primer, E71a (59-CGAGAATTCATCATTGCAGT TCAGGGCCGT-39; positions 1292 to 1273), for a second round. Amplified products were purified and cloned in Escherichia coli JM109. Individual clones were sequenced by the dideoxy chain termination method with either T7 DNA polymerase (Pharmacia) or AmpliTaq FS (Perkin-Elmer). For the E2 HVR, the consensus nucleotide sequence was determined directly from the PCR product. Table 1 summarizes the results obtained for the four patients. The characteristic quasispecies structure found in HCV with a master sequence and a variable spectrum of mutants was observed in all samples. Patients A and C had a more complex population of sequences in the liver with respect to serum and an inverted proportion of silent mutations; the ratios of number of polymorphic sites to number of nucleotides sequenced (Pn ratios) were two- and threefold higher in the liver than in the serum, respectively. For the other two patients (B and D), the viral quasispecies were very similar in the complexity and proportion of synonymous mutations in liver and serum specimens. In all cases, the liver population of sequences was complex and appeared strongly selected for synonymous replacements (the proportion of silent mutation being higher than 60%), while the circulating viral population structure was not uniform among the patients. In patients B and D, the populations of viral sequences in the liver and serum were very similar, and at least 10% of the liver mutant sequences were also present in serum, suggesting that circulating viruses had been released from their corresponding hepatic samples. In contrast, in patient A and C, the circulating quasispecies were more homogeneous and had only 37.5 and 33% silent mutations, respectively, and no more than 1.5% of the mutants present in the liver were also present in the serum. We subsequently compared HCV sequences from three dif-

Hepatitis C virus (HCV) is one of the most important causes of chronic liver disease worldwide (7). The quasispecies nature of the single-strand RNA genome of HCV is thought to play a central role in maintaining and modulating viral replication (5, 14, 15). Although there is compelling evidence that HCV replicates only in hepatocytes (13) and the dynamics of the release of HCV from hepatic cells to blood is largely unknown, most studies of population structure parameters have focused on serum-circulating particles. In this study, we performed a detailed analysis of the HCV population structure of the wellconserved 59-untranslated region (59-UTR) and both genomic and antigenomic strands of a variable fragment encompassing the envelope 2-nonstructural region 2 junction (E2-NS2) in paired liver and serum samples from four patients with chronic hepatitis C of different severities. Liver RNA was isolated by a modification of the method described by Chomczynski and Sacchi (2). Viral circulating RNA was isolated with DS6MP solution (6 M guanidine thiocyanate, 0.25% Sarkosyl, 12.5 mM sodium citrate [pH 7], 0.1 M sodium acetate [pH 4], 0.5% 2-b mercaptoethanol, and phenol) and two chloroform extractions. Isolated HCV RNA was reverse transcribed into cDNA by using primers specific for genotype 1b to copy the plus strand (NR5, 59-TCTCGTAGA CCGTGCACCATGAGCA-39 at positions 7 to 218 for 59UTR; E11b, 59-ATAGAATTCACAGCTGGCCATGCGCTC TGGGCA-39 at positions 1376 to 1354 for the E2 region; and MJJ3, 59-CTCGAGCGTTGAGGGGGG-39 at positions 2602 to 2585 for the E2-NS2 region) or minus strand (MJJ4, 59-TG TGCCTGCTTGTGGATG-39 at positions 2194 to 2211 for E2-NS2). Minus-strand cDNA was treated at 378C for 30 min with RNase A (100 mg/ml) and heated at 908C for 10 min to minimize the amplification of the undesired RNA viral strand. Nested PCR was performed with specific oligonucleotides to amplify the 59-UTR region (outer set, NF5 [59-GTGAGGAA CTACTGTCTTCACGCAGAAAGCG-39 at positions 2295 to 2265] and NR5; inner set, KF2 [59-TTCACGCAGAAAG CGTCTAG-39 at positions 2279 to 2260] and NR4 [59-CCT TGTGGTACTGCCTGAT-39 at positions 243 to 263]) and * Corresponding author. Mailing address: Servei de M. InternaHepatologia, Hospital General Universitari Vall d’Hebron, P Vall d’Hebron 119, 08035 Barcelona, Spain. 1732

VOL. 71, 1997

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TABLE 1. Sequence complexity of E2-NS2 genomic regions in different patientsa Patient

Sample

No. of clones sequenced

No. of identical nucleic acid sequences (%)

No. of mutations

No. of synonymous mutations (%)

Pn

A

Liver Serum Liver

64 22 10

53 8 18

32 (60) 3 (37.5) 15 (83)

1/256 1/583c 1/112

Serum Liver Serum Liver Serum

9 10 10 9 9

15 (23) 15 (68)b 2 (20) 2 (20) 2 (22) 0 4 (40) 3 (33) 3 (33)

8 19 6 8 6

7 (87.5) 15 (79) 2 (33) 6 (75) 5 (83)

1/226 1/93 1/301c 1/219 1/292

B C D

a Patients A, B, and D had severe chronic active hepatitis and elevated levels of serum aminotransferases (patients A and B had liver cirrhosis), and patient C had chronic persistent hepatitis and persistently normal levels of aminotransferases. The population complexity was determined by the relative frequency of identical sequences (master sequence) with respect to the total number of clones and the degree of polymorphism represented by the ratio between mutated sites and the total number of nucleotides analyzed. The phenotypic polymorphism is represented by the percentage of synonymous or silent mutations with respect to the total number of nucleotide mutations. b P , 0.001 for liver versus serum proportion of master sequence (chi-square test with Yates correction). c P , 0.05 for liver versus serum Pn ratio (chi-square test with Yates correction). The rest of the comparisons are not significant.

ferent liver specimens obtained from the liver explant of patient A. The same consensus sequence, which coincided with the master sequence, was found in each sample analyzed (serum and liver samples L1, L2, and L3) for the two genomic regions (59-UTR and E2-NS2). However, a distinct unique mutant spectrum accompanied each master sequence. In the 59-UTR region, the proportion of master sequence in the three liver samples was variable, while the frequency of any other mutant never surpassed 5%. In serum, the master sequence was predominant and the Pn ratio was lower than those in the liver samples. In the E2-NS2 region, the master sequence was predominant in the serum, while liver samples had more heterogeneous quasispecies structures in both genomic and antigenomic strands. As shown in Table 2, the sequences from the three specimens were very similar with regard to the complexity and proportion of synonymous mutations in both genomic and antigenomic strands. In addition, 12 of 53 mutated positions were shared by the three different liver specimens and were uniformly distributed among them: 4 between L1 and L2, 3 between L2 and L3, 2 between L1 and L3, and 3 among all. Direct sequencing of the E2 HVR 1 (HVR-1) at the 59 terminus of the envelope 2-coding gene in all four patients revealed no differences between the consensus sequences of serum and liver sample pairs. These results show that there is no general correlation between liver and serum quasispecies and raise the question of

the potential origin of the circulating viruses in some of these patients (patients A and C). Although extrahepatic replication of HCV might account for the observed differences, since there is compelling evidence that HCV replicates only in hepatocytes, we assumed that circulating virus was indeed a subset of replicating virus in the liver. Data from patient A reveal that HCV population structure is highly homogeneous throughout the liver; when the 59-UTR and the E2-NS2 regions were examined, the major sequence was maintained in similar proportions in the three hepatic specimens and the mutants could not be divided into subgroups. Moreover, the overall parallelism between genomic and replicating-strand (negative-strand) populations in the latter in all three liver specimens suggests that, in contrast to the situation in human immunodeficiency virus infection (1, 3), there is no such macroscopic anatomic compartmentalization of HCV replication in the liver but rather that the whole liver quasispecies intercommunicates as a unique evolutionary unit, at least after such a long-standing infection. Thus, an alternative explanation should account for the observed differences between serum and liver quasispecies in patients A and C. One possibility might be the positive selection of a minor mutant by the humoral immune response. Direct and indirect evidence strongly suggests that HCV-infected patients generate virus-neutralizing antibodies (8, 11) which seem to target the E2 HVR-1 (18) and may limit, to some extent, the spread of the infection. However, we could

TABLE 2. Sequence complexity in the liver and serum quasispecies of patient Aa Genomic region (strand polarity)

59-UTR E2-NS2 (1)

E2-NS2 (2) 59-UTR E2-NS2 a

No. of clones sequenced

No. of identical nucleic acid sequences (%)

No. of mutations

L1 L2 L3 L1 L2

12 11 10 25 19

7 7 5 23 20

L3 L1 L2 L3 Serum Serum

20 24 28 22 9 22

4 (33) 6 (54.5) 8 (80) 7 (28) 2 (10.5) 2 (10.5) 6 (30) 5 (21) 7 (25) 6 (27) 7 (78) 15 (68)

Samplea

25 19 23 23 3 8

No. of synonymous mutations (%)

18 (78) 11 (55) 16 (64) 10 (53) 14 (61) 16 (70) 3 (37.5)

Pn

1/338 1/310 1/394 1/230 1/201 1/170 1/246 1/258 1/203 1/591 1/583

L1, L2, and L3 are three small pieces of 0.1 to 0.15 g of hepatic tissue obtained from three different parts of the right hepatic lobule from the liver explant of a patient (patient A) undergoing liver transplantation for end-stage HCV-related cirrhosis; a serum sample was obtained at the same time.

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not confirm this hypothesis, since the same consensus sequence was found in the HVR-1 of E2 in both serum and liver specimens in all patients, indicating that an immune-selected mutant, if any, would map outside this region (16). Recently, application of mathematical models of human immunodeficiency virus turnover (9, 17) to study the dynamics of HCV replication in patients receiving interferon has suggested that the vast majority of circulating viruses are produced through a dynamic process of continuous rounds of de novo virus infection of susceptible cells and rapid cell turnover and not from cells chronically producing virions (19). Assuming that HCV replicates only in hepatocytes, this suggests the existence of distinct functional compartments with different replication kinetics. In this regard, the high percentage of synonymous mutations and the high complexity of the replicating quasispecies in the liver might be indicative of a more ancient origin and a higher degree of adaptation of the hepatic quasispecies to liver cells. In contrast, the structure of the circulating population in patients A and C with a narrow mutant spectrum and a random distribution of silent mutations indicates a more recent origin of most circulating virus (4). This implies the existence of hepatocytes with different kinetics of viral replication. Some might be chronically infected cells replicating well-adapted quasispecies at low levels and capable of avoiding clearance by the immune system. Others might be recently infected hepatocytes, which would replicate quasispecies expanded from a mutant or mutants having some kind of replicative advantage and inducing liver cell damage compensated by rapid cell turnover. Whether the circulating virus is preferentially released by one type of cell or another could depend on several factors and might fluctuate over time (10, 12). Analysis of sequential liver-serum sample pairs from the same patients would help to clarify this issue. Our findings may have important practical implications. The relative size of the chronically infected pool would increase over time and explain the resistance of long-standing infections to interferon treatment (6, 10). In addition, the stochastic nature of mutant selection from the chronic pool might explain the wide spectrum of disease outcomes among HCV-infected individuals (7). Finally, because of the lack of consistent correlation between the complexity and nature of the circulating and the predominant intrahepatic replicating quasispecies, one should be cautious when trying to correlate viral population parameters, from circulating virions with response to antiviral treatment or disease outcome. Nucleotide sequence accession numbers. The GenBank accession numbers for the sequences presented in this article are Z75850 through Z75869, Z76782 through Z76899, Z76913 through Z76922, Z76935 through Z76943, Z76958 through Z76988, Z77005 through Z77013, Z77028 through Z77046, Z77061 through Z77102, and Y07774. We are grateful to Esteban Domingo (Centro de Biologia Molecular, CSIC, Madrid, Spain) and Josep Casacuberta (Centro de Investigacio ´n y Desarrollo, CSIC, Barcelona, Spain) for valuable reviews of the manuscript. We also thank Josep Quer for helpful technical advice and Teresa Otero for excellent technical assistance. This investigation was supported in part by grants BIO 94-0297 and 94/1682 from the Comisio ´n Interministerial de Ciencia y Tecnologı´a and the Fondo de Investigaciones Sanitarias.

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