Genetic Diversity and Tissue ... - Journal of Virology

JOURNAL OF VIROLOGY, Feb. 1998, p. 1640–1646 0022-538X/98/$04.0010 Copyright © 1998, American Society for Microbiology

Vol. 72, No. 2

Genetic Diversity and Tissue Compartmentalization of the Hepatitis C Virus Genome in Blood Mononuclear Cells, Liver, and Serum from Chronic Hepatitis C Patients SONIA NAVAS, JULIO MARTIŃ, JUAN ANTONIO QUIROGA, ˜ O* INMACULADA CASTILLO, AND VICENTE CARREN Department of Hepatology, Fundacio ń Jimeńez Dıáz, and Fundacio ń Estudio Hepatitis Virales, Madrid, Spain Received 21 August 1997/Accepted 30 October 1997

The degree of genetic variability in the hypervariable region 1 of hepatitis C virus (HCV) was analyzed by cloning and sequencing HCV genomes obtained in paired samples of serum, liver tissue, and peripheral blood mononuclear cells (PBMC) from four chronic hepatitis C patients. Genetic variability in serum was higher than in liver tissue or PBMC at the level of complexity (the number of different sequences obtained from each type of tissue) as well as at the level of genetic distance between all pairs of sequences within each tissue (compared by the Student t test; P < 0.001 for two patients and P < 0.01 for another). The spectrum of viral genomes differed among the three types of tissue, as shown by segregation of sequences according to their tissue of origin in phylogenetic analysis and by statistical analysis of mean genetic distances observed between sequences obtained from different tissues (P < 0.001), but sequences from liver tissue and PBMC were more closely related to each other than to those from serum. RIBA III (Ortho). To this end, four patients were selected according to the following criteria: (i) no previous antiviral or immunomodulatory treatment and (ii) presence of HCV RNA of genotype 1b in paired serum, liver, and PBMC samples taken at the time of liver biopsy. This genotype was selected due to its high prevalence in our population (41). Table 1 shows the clinical features of the patients. RNA was extracted from 200 ml of serum or tissue (frozen PBMC at 280°C or liver biopsy products stored in liquid nitrogen) by a modification of the guanidinium-phenol-chloroform method as previously described (11, 38). Total RNA from PBMC and liver tissue was measured, and 1.5 mg of tissuederived RNA or the whole-serum-derived RNA was used for cDNA synthesis and nested PCR with strand-specific reverse transcription (RT) and amplification (RT-PCR) with primers from the 59 noncoding region of the HCV genome, as previously described (38). HCV genotyping was performed with a restriction fragment length polymorphism analysis of the 59 noncoding amplified products, as previously described (39). Genomic HCV RNA strands were amplified in the E2 region of the HCV genome by RT-PCR with specific primers for HCV type 1b (outer sense [nt 1019 to 1038], 59-TCCCCCAA GCCGTCTTGGACC-39; outer antisense [nt 1273 to 1292], 59-TCATTGCAGTTCAGGGCAGT-39); inner sense [nt 1054 to 1074], 59-CACTGGGGAGTCCTGGCGGGC-39; inner antisense [nt 1258 to 1278], 59-GGCAGTCCTGTTGATGTGC CA-39). cDNA synthesis was performed with Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, Wis.) in a 20-ml volume containing 50 pmol of the antisense primer, followed by heat (94°C, 30 min) and RNase (100 mg/ml) treatment. PCR amplification was performed for 30 cycles (94°C for 60 s, 55°C for 90 s, and 72°C for 120 s), and 5 ml of the first PCR product was amplified for 30 additional cycles as described above. To avoid false-positive results, the contamination prevention measures of Kwok and Higuchi (33) were followed, and negative controls were included in all experiments. In order to eliminate contamination of PBMC by HCV RNA present in plasma, the cells were washed twice

Since 1989, when the hepatitis C virus (HCV) was isolated and recognized as the agent responsible for most cases of non-A, non-B hepatitis (12, 13), several studies have demonstrated a high nucleotide sequence variability in its RNA genome (1, 6–9, 14, 47, 48). Comparison of HCV isolates has shown the existence of a hypervariable region (called HVR1) of 81 nucleotides (nt), located in the 59-terminus region of the envelope glycoprotein 2 (E2) gene, that may account for more than 60% of the amino acid substitutions of the complete E2 protein (24, 54). Some authors have reported that changes in the serum spectra of HVR1 sequences can be observed during the course of chronic HCV infection (18, 20, 32). Recently, Farci et al. (21) have demonstrated that an anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees. Thus, as HVR1 appears to contain a linear B-cell epitope (18, 29, 31, 52), it has been suggested that HVR1 might be implicated in one of the mechanisms whereby HCV may evade the host immune response (23, 46, 51, 52, 55). In addition to the HCV hepatotropism, we, as well as others, have previously reported the presence of both genomic- and antigenomic-stranded HCV RNA in peripheral blood mononuclear cells (PBMC) from patients with chronic hepatitis C (2, 4, 36, 38, 57), although whether HCV replicates in extrahepatic tissues is still a controversial subject (34). Interestingly, different human T-cell lines have been shown to be susceptible to in vitro infection with HCV (37, 49). The aim of this study was to investigate the degree of genetic variability in the HVR1 region of the HCV genome, in paired PBMC liver, and in serum samples (taken at the time of liver biopsy) from patients with histologically proven chronic hepatitis C were included. All of the patients presented anti-HCV antibodies as detected by enzyme-linked immunosorbent assay III (Ortho Diagnostic Systems, Raritan, N.J.) and confirmed by * Corresponding author. Mailing address: Department of Hepatology, Fundacio ń Jimeńez Dıáz, Avda. Reyes Cato ´licos, 2, 28040 Madrid, Spain. Phone: 34-1-543.19.64. Fax: 34-1-544.92.28. E-mail: [email protected]. 1640

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TABLE 1. Clinical, histological, and virological features of patients Patient no.

Age (yr)

Sexa

Acute hepatitis

Known duration of disease (mo)

Histological diagnosisb (Knodell index)

Liver enzymes (ALT/AST)c (IU/I)

Cryoglobulins

Leukocyte count (103/ml)

Neutrophils/ lymphocytes/ monocytes (%)

Serum HCV RNA (copies/ml)d

1 2 3 4

40 30 40 25

M F F M

No Yes Yes No

108 98 116 85

CAH (12) CPH (4) CAH (6) CAH (9)

97/40 187/92 93/60 95/41

No No No No

6.4 7.8 7.6 8.1

53/38/8 48/47/5 52/40/7 51/41/7

5.5 3 103 6.0 3 103 9.1 3 103 1.4 3 104

a

M, male; F, female. CAH, chronic active hepatitis; CPH, chronic persistent hepatitis. ALT, alanine transaminase; AST, aspartate transaminase. d Tested by AMPLICOR HCV Monitor Assay (Roche Diagnostics System, Inc., Basel, Switzerland). b c

with phosphate-buffered saline after centrifugation on FicollHypaque gradient and the last wash of PBMC was stored for further analysis by RT-PCR as a negative control. Furthermore, PBMC were treated with trypsin (0.05%) and EDTA (0.02%) and washed again, in order to eliminate a possible attachment of the virus to the PBMC membrane. The RT-PCR analysis of the last washes always produced negative results. The amplified E2 products (228 bp) were cloned in Escherichia coli (TA cloning kit; Invitrogen, San Diego, Calif.) and sequenced by the dideoxynucleotide chain termination method with phage T7 DNA polymerase (Sequenase; United States Biochemicals, Cleveland, Ohio). Genetic diversity within each tissue type. In order to study a representative sample of sequences from each type of tissue, we sequenced a total of 139 HVR1 clones (range, 9 to 16 clones per tissue type). Heterogeneity was studied qualitatively and quantitatively. First, we defined the complexity coefficient (CC) index as the number of different sequences obtained from each type of tissue divided by the number of clones analyzed (Table 2). Thus, for one individual (patient 3) the complexity of sequences obtained was very high in the three tissues, on both nucleotide and amino acid levels. In two cases (patients 1 and 4) the CC index from serum was very high in both nucleotide and amino acid sequences, while CC indexes from liver tissue were equal to or lower than those from serum, and CC indexes from PBMC were always lower than those

from serum or liver tissue. Finally, in the last case (patient 2) the complexities of the sequences obtained from serum and liver tissue were very similar and, remarkably, the most abundant nucleotide sequence in serum (representing 33% of the mutant spectrum) was also predominant in liver tissue (54%). In summary, the results obtained demonstrated the existence of complex distributions of nonidentical but closely related genomes, usually called quasispecies (5, 17, 19), on two levels: individual patient and tissue. In addition, the spectrum of viral genomes found in serum seemed to be more complex than those found in liver tissue or PBMC. The next step was to calculate the genetic distances between all possible pairs of sequences within each type of tissue of each individual by the Kimura two-parameter modification method (30); the distribution is shown in Fig. 1. According to this distribution, it seemed that a greater diversity could be observed in the HVR1 sequences amplified from serum than in those obtained from liver tissue or PBMC. In order to investigate this heterogeneity quantitatively, mean genetic distances within each type of tissue (Table 3) were compared by the Student t test. The mean genetic distance between nucleotide sequences from serum was significantly higher than those of sequences from liver tissue or PBMC in patient 1 (serum versus liver or versus PBMC, P , 0.001), patient 3 (serum versus liver or versus PBMC, P , 0.001), and patient 4 (serum versus liver or versus PBMC, P , 0.01); and the mean genetic

TABLE 2. Qualitative analysis of genetic variability in HVR1 of the HCV genome at nucleotide and amino acid levels Patient no.

Tissue

No. of clones analyzed

1

PBMC Liver Serum

2

Nucleotides

Amino acids

Sequences (CC)a

Appearances (no. of clones)b

Sequences (CC)

Appearances (no. of clones)

Nonconserved/ conserved

16 11 15

9 (0.56) 8 (0.73) 14 (0.93)

1 (8), 8 (1) 1 (3), 1 (2), 6 (1) 1 (2), 13 (1)

8 (0.50) 8 (0.73) 11 (0.73)

1 (8), 1 (2), 6 (1) 1 (3), 1 (2), 6 (1) 1 (3), 2 (2), 8 (1)

13/14 21/6 3/24

PBMC Liver Serum

NAc 13 12

6 (0.46) 6 (0.50)

1 (7), 1 (2), 4 (1) 2 (4), 4 (1)

5 (0.38) 4 (0.33)

1 (8), 1 (2), 3 (1) 1 (9), 3 (1)

23/4 22/5

3

PBMC Liver Serum

11 15 12

11 (1.00) 15 (1.00) 11 (0.92)

11 (1) 15 (1) 1 (2), 10 (1)

10 (0.91) 14 (0.93) 11 (0.92)

1 (2), 9 (1) 1 (2), 13 (1) 1 (2), 10 (1)

10/17 10/17 7/20

4

PBMC Liver Serum

12 13 9

5 (0.42) 10 (0.77) 8 (0.89)

1 (8), 4 (1) 1 (4), 9 (1) 1 (2), 7 (1)

5 (0.42) 9 (0.70) 7 (0.78)

1 (8), 4 (1) 1 (5), 8 (1) 1 (3), 6 (1)

14/13 9/18 8/19

a b c

CC, complexity coefficient, defined as the number of sequences obtained divided by the number of clones analyzed. No. of clones refers to the number of times each sequence was observed in the clones analyzed. NA, not available.

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FIG. 1. Distribution of genetic distances between all possible pairs of sequences within each type of tissue studied (PBMC, liver tissue, and serum) on nucleotide (A) and amino acid (B) levels. Genetic distances (in increments of 0.05) are recorded on the x axes, while the percentages of times scored are recorded on the y axes.

distance between amino acid sequences from serum was also significantly higher than those between sequences from liver tissue or PBMC in patient 1 (serum versus liver or versus PBMC, P , 0.001) and patient 3 (serum versus liver tissue or versus PBMC, P , 0.001). Therefore, diversity observed in HVR1 sequences obtained from serum is greater than that observed in sequences from liver tissue or PBMC. Phylogenetic analysis of HVR1 sequences. In order to evaluate the relationship among the sequences obtained within each tissue, distances between all possible pairs of sequences were calculated by the Kimura two-parameter modification method (30) and unrooted phylogenetic trees were constructed by the neighbor-joining method (42) from the Windows Easy Tree software package (version 1.3) (http://www.tdi.es/ programas/WET-i.html). Phylogenetic analysis showed that sequences obtained from each individual segregated according to their tissue of origin. Figure 2 shows phylogenetic trees corresponding to nucleotide and amino acid HVR1 sequences of patient 4. In order to investigate whether the distribution of sequences according to their tissue of origin was confirmed by

statistical analysis, mean genetic distances observed between sequences obtained from different types of tissue (Table 3) were compared by the Student t test. Thus, on the nucleotide level, sequences from PBMC were more closely related to those from liver tissue than to those from serum in patients 1, 3, and 4 (P , 0.001); sequences from liver tissue were more closely related to those from PBMC than to those from serum in patients 1, 3, and 4 (P , 0.001); and sequences from serum were more closely related to those from liver tissue than to those from PBMC in patient 1 (P , 0.001). Finally, on the amino acid level, sequences from PBMC were more closely related to those from liver tissue than to those from serum in patients 1, 3, and 4 (P , 0.001); sequences from liver tissue were more closely related to those from PBMC than to those from serum in patients 1, 3, and 4 (P , 0.001); and sequences from serum were more closely related to those from liver tissue than to those from PBMC in patients 1 (P , 0.05) and 4 (P , 0.02). Overall, statistical analysis of distances between types of tissue within each individual confirmed that HVR1 sequences from PBMC

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TABLE 3. Mean genetic distancesa of all pairs of sequences, within and between tissues, at nucleotide and amino acid levels Patient no.

1

2

3

4

a b

Tissue

Nucleotides PBMC

Liver

PBMC Liver Serum

0.157 6 0.101 0.178 6 0.033 0.341 6 0.222

PBMC Liver Serum

NAb

PBMC Liver Serum

0.179 6 0.062 0.219 6 0.104 0.271 6 0.104

0.197 6 0.121 0.269 6 0.132

PBMC Liver Serum

0.098 6 0.137 0.146 6 0.112 0.888 6 0.292

0.096 6 0.133 0.859 6 0.291

Amino acids Serum

PBMC

Liver

Serum

0.033 6 0.020 0.251 6 0.250

0.360 6 0.337

0.190 6 0.132 0.329 6 0.059 0.558 6 0.373

0.078 6 0.036 0.462 6 0.475

0.574 6 0.571

0.015 6 0.013 0.021 6 0.012

0.015 6 0.012

0.040 6 0.027 0.072 6 0.031

0.026 6 0.033

0.290 6 0.111

0.351 6 0.144 0.385 6 0.155 0.465 6 0.163

0.326 6 0.162 0.442 6 0.174

0.468 6 0.162

0.293 6 0.391

0.183 6 0.254 0.323 6 0.218 0.857 6 0.219

0.193 6 0.286 0.758 6 0.225

0.310 6 0.359

Mean genetic distances expressed as mean 6 standard deviation. NA, not available.

were more closely related to those from liver tissue than to those obtained from serum in every case. In the light of these results, it is likely that the HCV circulating population (as detected in serum) does not directly reflect either the liver tissue or the PBMC viral quasispecies. Hypothetically, the circulating HCV might be constituted from a heterogeneous population: (i) HCV virions forming an immunocomplex (25) (and thus theoretically neutralized) and (ii) a pool created by trafficking between blood and liver tissue. Also, considering the high diversity of sequences from serum, the possible contribution of released HCV populations which are infecting other tissues or organs cannot be excluded (40, 45). Another possibility is the presence of circulating virions with defective genomes. However, we have found only one HVR1 clone with a stop codon (in the PBMC sample from one patient), so our study has not indicated the possible implication of a high production of defective viral genomes in the establishment of a persistent viral infection, as has been demonstrated for other viruses (3, 27). Overall, the HVR1 tissuespecific compartmentalization is similar to previous observations in the V3 loop of the human immunodeficiency virus types 1 and 2 gp120 glycoprotein (15, 28, 44, 53). Recently, a few studies have attempted to analyze HVR1 in plasma and liver tissue (10, 43), serum and PBMC (22), serum and ascites (56), and serum, liver tissue, and PBMC from persistently HCV-infected patients (35). The results of these studies demonstrated similar HVR1 patterns among types of tissue (43), differences found in only a portion of the patients studied (10, 22, 56), and different patterns without tissue-specific trends (35). It should be observed that most of these studies were performed using the single-strand conformation polymorphism (SSCP) technique alone or in combination with sequencing of a few particular SSCP bands of individual patients. On the other hand, Cabot and colleagues (10) sequenced liver and plasma samples from four patients, and they found differences in HCV quasispecies in two of them, with a more complex population of sequences in the liver than in serum; although no genetic distance evaluation or phylogenetic analysis was performed, they concluded that circulating virus was a subset of replicating virus in the liver. On the contrary, our results clearly demonstrated different spectra of viral genomes (i.e., higher complexity in serum than in liver tissue or PBMC) and different levels of diversity (i.e., a closer relationship between

HCV quasispecies in liver tissue and PBMC) among the three types of tissue studied, emphasizing the unknown mechanism(s) of HCV evolution and pathogenesis. Studies of predicted amino acid sequences. The analysis of the HVR1 sequences at the level of amino acids showed that the number of conserved and nonconserved residues was different for each type of tissue and patient studied, although the number of variable residues tended to be higher in serum than in liver or PBMC samples (Table 2). Among the 139 HVR1 sequences studied, only one residue (no. 7, G) was absolutely conserved in all tissues and patients. We have also analyzed the number of positively charged residues in the HVR1 sequences. The average net charge (defined as the average of the number of charged residues in all the sequences derived from each type of tissue and patient) tended to be lower in PBMC (range, 1.4 to 4.6) or liver tissue (range, 2.0 to 4.6) than in serum (range, 1.8 to 5.5). In human immunodeficiency virus type 1, determinants of tropism have been mapped in the V3 loop of the gp120 glycoprotein (28). A lower net charge of the V3 loop has been associated with the non-syncytium-inducing or macrophage-tropic phenotype that is found in brain tissue (48). Furthermore, tissue-tropic variants have been found in the envelope surface glycoprotein of the lymphocytic choriomeningitis virus (16). In this study, we have found the lowest average net charge in HVR1 sequences from PBMC, but the possible implications of this fact remain to be further evaluated, as determinants of tropism in HCV have not yet been discovered. Shimizu et al. (50) have found that a particular HCV strain may exhibit a tropism for lymphocytes in vivo, as detected by sequencing in HVR1 of HCV genomes recovered from experimentally infected chimpanzees. We have not found a particular nucleotide sequence or amino acid “motif” that could suggest the existence of tissue-specific HVR1 determinants in our patients. Recently, mapping of the HVR1 region has demonstrated that this domain contains a linear B-cell epitope (18, 29, 31, 46). In our samples, the study of the hydrophilicity patterns in the consensus sequences from each tissue, by the method described by Hopps and Woods (26), showed that significant changes in the maximum antigenicity position occurred in two patients: in patient 1, this position was altered in PBMC compared to those in liver tissue and serum, and in patient 4, the position was altered in liver tissue compared to those in PBMC

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FIG. 2. Unrooted phylogenetic trees constructed by the neighbor-joining method from the analysis of the HVR1 sequences obtained from paired samples of PBMC, liver tissue, and serum of patient 4, on nucleotide (left) and amino acid (right) levels.

and serum sequences. In these individuals, the different quasispecies distributions among the three types of tissue studied might generate the coexistence of two different B-cell epitopes for the same envelope region, and this might increase the possibility of escape from the host’s humoral immune response. This result could suggest a possible relationship between the HVR1 region and a mechanism of viral persistence. Conclusions. Our results demonstrated, by means of sequencing, phylogenetic analysis, and comparison of the genetic distances of a large number of HVR1 sequences, a tissue compartmentalization of HVR1 sequences from PBMC, liver tissue, and serum samples taken at the same time from chronic hepatitis C patients. To our knowledge, this is the first time that different HCV quasispecies have been demonstrated to segregate in different types of tissue of individuals. Remarkably, our findings indicate the need for caution in considering as artifactual the detection of HCV RNA in lymphoid tissue, such as PBMC, and point out the need to further characterize the biological significance of HCV lymphotropism. Finally, our

observations have critical implications for the development of an effective vaccine, as the variability in the natures of HCV RNA quasispecies among individuals and types of tissue should be evaluated. Nucleotide sequence accession numbers. The GenBank accession numbers for the sequences presented in this article are AF018288 through AF018426. This work was supported by Fundacio ń Estudio Hepatitis Virales (Madrid, Spain). S. Navas and J. Martıń were supported by Fundacio ń Estudio Hepatitis Virales. We are indebted to Joaquıń Dopazo (Glaxo-Wellcome, Madrid, Spain) for his valuable advice, review of the manuscript, and phylogenetic analysis. REFERENCES 1. Adams, N. J., R. W. Chamberlain, L. A. Taylor, F. Davidson, C. K. Lin, R. M. Elliott, and P. Simmonds. 1997. Complete coding sequence of hepatitis C virus genotype 6a. Biochem. Biophys. Res. Commun. 234:393–396. 2. Artini, M., G. Natoli, M. L. Avanttaggiati, C. Balsano, P. Chririllo, A. Constanzo, M. S. Bonavita, and M. Levrero. 1993. Detection of replicative

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