THE MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

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TREATMENT OF CHRONIC HEPATITIS C

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THE MOLECULAR BIOLOGY OF HEPATITIS C VIRUS Genotypes and Quasispecies Xavier Forns, MD, PhD, and Jens Bukh, MD

Hepatitis C virus (HCV) is a member of the Flaviviridae family.124 The Flaviviridae family of viruses are associated with human and animal diseases and encompass at least three different genera: pestiviruses, such as bovine viral diarrhea virus and classic swine fever virus that cause disease in cattle and pigs, respectively; flaviviruses, the most important cause of arthropod-transmitted viral diseases, for example, dengue fever and yellow fever; and hepacivirus, whose sole member is HCV. The GB viruses, which are also members of the Flaviviridae, are most closely 145 89, related to HCV and infect humans and rnonke~s.'~, One of the signature characteristics of HCV is its genetic heterogeneity.16 Thus, HCV isolates from around the world have been divided into six major genotypes and more than 100 subtypes.16,45 Moreover, genetic heterogeneity of HCV within infected individuals is manifested as a mixture of closely related but distinct genomes called a quasispecies.l6?loo More than 2% of the world population is chronically infected with HCV.66,120, 170 In developed countries, HCV is the main cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; HCV-related endstage liver disease is now the leading reason for liver transplantati~n.'~~ Hepatitis C virus infection is also associated with a number of extrahepatic diseases, such as cryoglobulinemia and gl~merulonephritis.~~ The most important feature of HCV infection is its high rate of chronicity. More than 80% of HCV-infected individuals develop a chronic

From the Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

CLINICS IN LIVER DISEASE VOLUME 3 * NUMBER 4 NOVEMBER 1999

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infection.66Unfortunately, treatment of chronic hepatitis C with interferon (IFN), the principal recognized therapeutic agent, succeeds only in 20% of the cases.'09 The response rate is even lower in patients infected with genotype 1 HCV. Recently, a combination treatment of IFN and ribavirin has achieved higher response rates both in patients infected with genotype 1 and in patients infected with other genotypes.101,118 Despite the heartening progress achieved in treating chronic hepatitis C with combination therapy, the overall response rate in these patients is still below 50%. A better understanding of the molecular biology of HCV has highlighted targets for new therapeutic strategies. The genetic heterogeneity of HCV is a major confounding factor; thus, the design of effective drugs might be facilitated by targeting conserved regions of HCV. Drugs directed against conserved sequences of the 5' untranslated region (UTR)8,53, lz3,I3O and the serine-protease domain within NS328,84 have already been tested in vitro. The lack of a reliable cell culture system or small animal model for HCV (the chimpanzee is the only recognized animal model) presents significant obstacles for the study of these new therapeutic agents. The recent availability of infectious HCV cDNA clonesso,172, 174 may facilitate the establishment of useful in vitro replication systems. GENOMIC ORGANIZATION OF HEPATITIS C VIRUS

Hepatitis C virus is an RNA virus with a positive-sense singlestranded genome of approximately 9600 nucleotides encoding a single polyprotein of about 3000 amino acids (ranging from 3008 to 3037).96, 123,124 The open reading frame (OW) is flanked at each terminus by UTRs. Translation of the HCV OW produces a polyprotein that is cleaved into at least 10 proteins by a combination of host and viral proteases (Table 1).

Table 1. HCV STRUCTURAL AND NONSTRUCTURAL PROTEINS AFTER CLEAVAGE OF THE POLYPROTEIN BY HOST SIGNALASES AND VIRAL PROTEASES Protein

Molecular Mass (kda)

Functions

C El E2 P7 NS2 NS3

21 31-35 (21 if not N-glycosylated) 68-72 (36 if not N-glycosylated) 7 23 70

NS4A NS4B NS5A

8 27 58

NS5B

68

Nucleocapsid protein Virion envelope protein Virion envelope protein Unknown NS2-3 protease component NS2-3 protease component, serineprotease, NTPase and helicase Cofactor for NS3 serine-protease Unknown Unknown, possibly involved in interferon resistance RNA-dependent RNA polymerase

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The Untranslated Regions

The 5’ UTR is a highly conserved region of approximately 340 nu~leotides.’~,Portions of the HCV 5’ UTR sequence have high homology with the 5 ’ UTR sequences of pestiviruses and GB virus B (GBVB).”, 17, 23, 145 In pestiviruses, translation of the polyprotein is controlled through an internal ribosome entry site (IRES) within the 5’ UTR.lZ4An IRES is a relatively large and highly structured RNA sequence that directs ribosomes to the initiating methionine codon and thus bypasses the requirement for a mG cap at the 5’ terminus. Computer and enzyme protection analyses suggested that the 5’ UTR of HCV and the GB viruses contain IRES-like secondary structures.”, 63, 87, lZ3,143, 163-165 For HCV, these include four stem-loop structures ( H 1 4 ) and a pseudoknot at the 87, 1M In vitro analyses have suggested that base of the H3 ~tem-loop.6~, H2, H3, and H4 stem-loops and the initial portion of the OW are necessary for IRES activity.63,lZ3Generally, it is believed that the structure of double-stranded segments within the 5‘ UTR stem-loops (and not their actual sequences) are relevant for IRES function; in contrast, the actual sequence within the single-stranded RNA of these loops seems to be essential for IRES functi0n.8~ Besides the importance of the 5’ UTR in initiating translation, sequences within the 5’ UTR may be used as replication signals, either by direct interaction with the 3’ end of the viral genome or by indirect RNAprotein interaction^.^^, 87 Analogy with other members of the Flaviviridae family suggests that HCV replication is initiated at the 3’ end of the genome by a membrane-associated complex that would include the HCV RNA polymerase and cellular proteins.123,lZ4The 3’ UTR of HCV contains, in order, a short region that varies in sequence and length, a polypyrimidine stretch of variable length, and, finally, a highly conserved sequence of 98 nucleotides that constitutes the 3’ terminus of the HCV genome.81,153*154,171 In other positive-strand RNA viruses, conserved elements within the 3’ UTR are relevant for RNA synthesis and genome packaging.81Furthermore, mutagenesis analysis of conserved sequences within the 3‘ UTR of the related flaviviruses demonstrates that such 99 Secondary structure sequences are important for viral repli~ation.~~, analyses show that the conserved sequence of the 3’ UTR of HCV can potentially form three stable stem-loop structures, including a highly stable stem-loop of 46 nucleotides at the extreme 3’ end of HCV.9, 153,154 Deletion of these structures wjthin the 3’ UTR of an infectious cDNA clone of HCV makes the corresponding transcripts unable to infect a chimpanzee, thus indicating they are required for viral replication.’” The presence of a polypyrimidine tract within the 3’ UTR is unique to HCV and GBV-B ’&, 154; although its function is not known, this region seems to be essential for the infectivity of HCV because its elimination is lethal.’” In contrast, deletion of the proximal 60% of the variable region of the HCV 3’ UTR (which is part of two putative stem-loop structures) from an infectious cDNA clone did not diminish infectivity of the encoded sequence in ~ i v 0 . Similarly, l~~ the variable

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sequences found between the ORF and the conserved sequences of the lo2 3’ UTR of flaviviruses are not critical for viral replicati~n.~~, The high degree of genetic conservation of elements within the 5’ and 3‘ UTRs among different HCV genotypes makes these regions preferred targets for antiviral therapy. In fact, antisense oligonucleotides complementary to conserved 5’ UTR sequences and ribozymes designed to cleave conserved elements within the 5’ UTR have already been tested in ~ i t r o ,90,~ lz3, ~ , 130 and they significantly inhibited expression of reporter genes. The Hepatitis C Virus Virion and the Structural Proteins

Hepatitis C virus is a small enveloped virus. The viral nucleocapsid consists of core protein (C) and the viral genomic RNA. It is presumably enveloped by a lipid bilayer containing the two viral glycoproteins, E l and E2, to produce the infectious virions. However, the detailed structure of the assembled virion is not known. Filtration studies have shown that HCV is 30 to 60 nm in diameter.55Because HCV is inactivated by chloroform, it is presumed to have an envelope.44The extremely inefficient replication of HCV in cell culture and the relatively low titers of HCV in serum of infected individuals or animals has made it difficult to visualize the viral particles by electron microscopy (EM). However, virus-like particles have been observed as aggregates in cytoplasmic vesicles by EM both in Daudi cells infected with HCV and in high137 density fractions of plasma from patients with high HCV RNA More recently, HCV-like particles were obtained in insect cells infected with a recombinant baculovirus expressing the HCV structural proteins4: the viral proteins assembled into 40 to 60 nm particles that accumulated in large cytoplasmic vesicles. During sucrose gradient centrifugation, HCV from sera and plasma was fractionated into low-density (1.06-1.13) and high-density (1.171.25) components containing HCV-RNA.61The high-density fractions probably are virus complexed with immunoglobulins; low-density fractions are believed to contain virus associated with low-density lipoproteins (LDL). The HCV genome contains a single ORF, and all viral proteins are therefore initially contained within a polyprotein precursor. Cellular signal peptidases in the lumen of the endoplasmic reticulum (ER) cleave the precursor to release the three structural proteins.57* lZ3,lZ4 The core protein of HCV is a highly conserved protein of 191 amino acidslg with a molecular mass of 21 kd57,yl; its basic nature and the presence of RNAbinding motifs in its N-terminus suggest that the core associates with the HCV genome.lz4 In fact, binding of the core to the 5’ UTR of the HCV genome might be important for encapsidation and particle assembly.6yThe C terminus of the core protein contains a hydrophobic region, which acts as a signal sequence for transport of E l into the ER

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and for membrane-dependent processing of core. In immunofluorescence studies, the core protein is localized to the cytoplasm, with an ERassociation pattern.54,131, 135 Nuclear localization of smaller forms of the 151; translocation of core into the nucleus protein has also been In of cells has been suggested as a mechanism of cell tran~formation.'~~ fact, HCV core protein has been proposed as an important factor in the development of hepatocellular carcinoma in patients with chronic hepatitis C. In vitro studies have shown that HCV core protein can interact with cellular proto-oncogenes; in particular HCV core cooperated in Ras-mediated transformation of rat embryo fibroblasts.1z2In a recent study,lo7transgenic mice expressing the HCV core protein developed steatohepatitis and hepatocellular carcinoma. In another study, however, cell growth did not change when the HCV core protein was expressed in a human cell line.lo5It is possible that the different cell substrates and variable levels of expression may explain the differences observed in these studies. The E l and E2 envelope proteins of HCV are N-glycosylated proteins with apparent molecular masses of 31 kd and 68-70 kd, respectively.96,lW, lZ4 The function of the small hydrophobic protein p7 is not known; cleavage at the E2-p7 site is incomplete and two forms of E2 are lo4,136 The E l and E2 proteins both contain Cproduced: E2 and E2-~7.8~* terminal hydrophobic domains, which function as membrane anchors. The E l and E2 glycoproteins interact and are thought to function as lZ1 Previous studies using the vaccinia/ T7 and Sindbis heter~dimers.~~, virus expression systems showed that E l and E2 complexes form slowly, and a significant proportion are m i ~ f o l d e d .Only ~ ~ a fraction of the glycoproteins mature to form heterodimers, which are stabilized by noncovalent interactions with the intervention of ER chaperons.31A signal for retention of E2 in the ER has been localized within the 29carboxy-terminal amino acids of the E2 proteinz4;if this signal is replaced by a transmembrane domain of a protein anchored in the cell membrane, E2 is directed to the cell surface.z4,47 It is not known whether the retention signal present in E2 is sufficient to retain E l and E2 complexes or whether a separate signal is necessary for El. Retention of HCV glycoprotein complexes in the ER suggests that HCV budding might occur in this cellular compartment, especially because flaviviruses acquire their envelopes at internal membranes.1z4 The E l and E2 proteins of different HCV isolates exhibit a high degree of genetic heterogeneity.16The N terminus of the E2 protein is so variable that the region encompassing amino acid positions 384-410 has 74, 166 In genotype l b isobeen called hypervariable region 1 (HVR1).58, lates, a second hypervariable region within the envelope E2 protein was There are data indicating that identified downstream from HVRl .58* HVRl is on the surface of the envelope protein and that it represents a neutralization domain.4z,139, 155, 168 The mechanism by which HCV enters cells to initiate infection is not known. The lack of a reliable HCV culture system makes it difficult to analyze the different steps in viral infection. It seems reasonable,

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however, to believe that E l and E2 are involved in receptor binding and subsequent fusion. By preparing a cDNA expression library from a cell line that exhibited high E2-binding capacity, Pileri et a1115have recently identified CD81 as a potential HCV receptor. This molecule is a 25-kd protein that is a member of the tetraspanin superfamily; these proteins are expressed on the cell surface and span the membrane four times, generating two extracellular loops. The major extracellular loop is heterogeneous in sequence among different species; however, the sequence is conserved between humans and chimpanzees, the only animal species known to support HCV replication. In addition, CD81 is expressed in the membrane of hepatocytes and lymphocytes, cells that support HCV rep1icati0n.l~~ The Nonstructural Proteins

The 3’ part of the OlW encodes seven nonstructural (NS) proteins The NS2 gene encodes a hydrophobic protein that, together (Table 1).96*124 with the N-terminal third of the NS3 protein, forms a zinc-dependent protease. Although initially proposed as a metalloprotease, based on structural and functional studies51,59, lz3 the NS2/3 protease is now thought to be a cysteine protease. This NS2/3 protease mediates the 59 Several studies have shown cleavage at the NS2/3 junction, in that mutations inactivating the NS3 serine-protease domain have no effect on the cleavage at the NS2/3 junction, suggesting that this second protease function of NS3 is not essential for the processing of NS2/3.51,59 It was demonstrated that cleavage of NS2/3 is partially dependent on the presence of microsomal membranes, indicating that a cellular cofac132 tor is required for efficient pro~essing.~~, The NS3 gene encodes a 70-kd protein with multiple functions. The aminoterminal part of NS3 has serine-protease activity and has been shown to cleave NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions of the polyprotein.2,27, 32, 59, 60, 161 The protease forms a stable complex with the NS4A protein. In fact, NS4A functions as an essential cofactor in the processing of NS3/4A and NS4B/5A sites and enhances cleavage at the other sites.*= Recently, two research groups described the threedimensional crystal structure of the NS3 protease domain and of the protease domain complexed with a synthetic NS4A cofactor ~ e p t i d e .94~ ~ , These studies indicate that the enzyme contains a zinc atom with a structural function and assumes a chymotrypsin-like folding pattern. Determining the crystal structure may make it possible to design specific inhibitors of the enzyme for therapeutic use as inhibitors of viral replicat i ~ n84,~149; ~ ,however, the substrate-binding groove of HCV NS3 is too shallow and not sufficiently unique to make the development of specific inhibitors simple. Potential target sites other than the cleavage site are the NS4A binding site or the structural zinc site. Functional and structural analyses of the carboxy-terminus of the NS3 protein of HCV have shown that it contains an NTPase and an

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RNA-helicase.5” 77, 82, lsO,Is2, 175 Helicases are nucleotide triphosphate-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication; hydrolysis by the NTPase provides the necessary energy to unwind the nucleic acids. Purified NS3 expressed in Escherchia coli exhibited NTPase and RNA helicase activity, unwinding RNA:RNA, RNA:DNA and DNA:DNA duplexes in a 3‘ to 5’ d i r e c t i ~ n .In ~ a~ recent , ~ ~ ~ study, it was found that poly (U) inhibited the helicase activity of NS3 but stimulated its protease activity.’06Morgenstern et aPo6have proposed that the binding of NS3 to the poly (U) region of the 3’ UTR serves to localize NS5A/NS5B processing to the 3‘ UTR and thus facilitate assembly of the RNA replication complex. Conserved amino acids surround the enzyme pocket in its three-dimensional structure of the helicase, and molecules binding to this conserved region could potentially inhibit the helicase activity.82, 176 The function of NS4B and NS5A are still unknown. It has been shown, however, that NS5A is ph~sphorylated~~; protein phosphorylation regulates protein-protein interactions as well as protein-nucleic acid interactions. A region containing amino acids 2209 to 2248 of the NS5A protein has been implicated in the modulation of the host IFN-mediated antiviral response.36Mutations in this region, called the IFN-sensitive determining region (ISDR) appeared to correlate with the sensitivity of HCV genotype l b viruses to IFN treatment. The 68 kd NS5B protein is believed to function as an RNA-dependent RNA polymerase. Purified NS5B expressed in E coli or insect cells infected with recombinant baculoviruses has RNA polymerase activity.’, 6, 92, In The HCV NS5B protein is able to bind and copy different RNA templates, suggesting that specific sequences might not be required for RNA synthesis.6 Some RNA-dependent RNA polymerases require a priming mechanism for initiation of RNA synthesis; this primer can be an exogenous RNA molecule or the 3’ end of the RNA folded onto itself. In HCV, it is proposed that RNA-dependent RNA synthesis takes place by a copy-back mechanism, in which the 3’ nucleotide of the template is used to prime synthesis of the complementary The RNA polymerase might also provide a target for inhibition of viral replication; in fact, compounds binding to the RNA-dependent RNA polymerase of dengue or kunjin viruses inhibited their activity in cell cu1tu1-e.~ 1753

INFECTIOUS cDNA CLONES OF HEPATITIS C VIRUS

Recently, two laboratories demonstrated that RNA transcripts of full-length HCV cDNA clones were infectious in vivo when inoculated into the liver of chimpanzees.80,172 To obtain infectious cDNA, clones were engineered that contained the consensus sequence of the viral strain (H77, genotype la) used as the source of HCV. These studies confirmed that the sequence, including the 5’ and 3’ termini, was complete and represented the infectious sequence. The need to engineer clones containing a consensus or near-consensus sequence became ap-

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parent when attempts to obtain infectious cDNA clones directly amplified from the original source of HCV were repeatedly unsuccessful.s0, Although spurious mutations introduced during the amplification or cloning procedures cannot be excluded as a cause of nonviability, these data suggest that a proportion of HCV-circulating genomes are probably not infectious. This conclusion is supported by the fact that the infectivity titer is lower than the polymerase chain reaction (PCR) titer determined in chimpanzees of the human HCV strain used in these experiment^.'^^ The course of HCV infection in chimpanzees after intrahepatic transfection of RNA transcripts from the HCV cDNA clones did not differ significantly from the course observed in animals infected intravenously with the original virus. Chimpanzees transfected with these clones developed acute hepatitis, thereby proving that HCV, as expected, is a cause of liver disease.2O. 97 As noted previously, studies of the mutagenesis of infectious cDNA clones showed that all regions of the 3' UTR except the variable region are critical for infectivity.'" Another mutagenesis study produced a chimeric virus.174In this case, the chimeric genome encoded a polyprotein of genotype l b but replicated by means of the 5' and 3' termini of genotype la, demonstrating that chimeras between the UTRs of different subtypes can be viable. Availability of infectious cDNA clones should also facilitate the study of the structural and nonstructural proteins of HCV. Long-term follow-up of chimpanzees transfected with infectious HCV cDNA clones will be important for studying the evolution of HCV and the development of quasispecies populations. The existence of infectious plasma pools containing monoclonal HCV populations are important for vaccine development, because they will make possible, for the first time, dissection of the immune response following true homologous challenge of chimpanzees immunized with monoclonal recombinant HCV proteins. Finally, infectious cDNA clones may be useful in the development of in vitro propagation systems. GENETIC HETEROGENEITY OF HEPATITIS C VIRUS: GENOTYPES AND QUASISPECIES Like other RNA viruses, HCV has a relatively high mutation rate, which results from an error-prone RNA-dependent RNA polymerase that lacks proofreading activity.lZ4Sequence analysis of HCV during long-term follow-up in infected hosts suggested that the mutation rate nucleotide substitutions/genome of HCV is about 1.44 to 1.92 x site/ year."', 113 The highest mutation rates were observed in the envelope regions, and in particular in the HVRl. In general, the genetic heterogeneity of HCV has been described under two different headings: genotypes and quasispecies.I6 The term genotype refers to the genetic heterogeneity among HCV isolates world-

THE MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

wide and reflects the accumulation of mutations evolution of these viruses. The term quasispecies heterogeneity within an infected individual, as infection with a heterogeneous virus population mutations during the course of the infection.

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during the long-term refers to the genetic a result of de novo and accumulation of

Classification of Hepatitis C Virus Isolates into Different Genotypes Genetic analyses demonstrated that HCV exists as multiple genotypes, worldwide. These genotypes reflect differences as high as 35%.l14 The most diverse isolates belong to different major genotypes and have been assigned arabic numerals (e.g., 1, 2, 3); more closely related isolates within these major genotypes belong to subtypes and are identified with lower case letters (e.g., a, b, c). Sequence analysis of the E l gene (576 nucleotides) suggested that HCV could be classified into six major genotypes with 12 subtypes.18This classification was confirmed by analyzing the C gene (573 nucleotides) of the same isolate^.'^ Analysis of partial NS5B genome sequences (222 nucleotides) identified the same Analyses of full-length ORF six major genotypes with 11 sequences of representative isolates have confirmed this original 21 Sequence analysis of a huge number of isolates from clas~ification.'~, around the world has led to a great increase in the number of subtypes; more than 100 subtypes have been recognized within HCV genotypes 1, 2, 3, 4 and 6.15,95 Classification of some isolates from Southeast Asia has 142 Based on analysis of subgenomic regions, the been isolates were initially classified as new major genotypes 7 through 11,157, 159, I6O but further analysis of nearly full-length sequences suggested that these viruses could instead be classified within the six major genotypes originally described: genotype 10 as a divergent subtype of genotype 3 and genotypes 7, 8, 9 and 11 as divergent subtypes of genotype 6.14, IZ5, 158 Based on these observations, it was recently proposed that HCV be classified into six clades (Table 2).

Table 2. PROPOSED CLASSIFICATION OF HEPATITUS C VIRUS ISOLATES BASED ON THEIR PHYLOGENETIC ANALYSIS Clade

1 2 3

4 5 6

Genotype 1 2 3, 10 4

5 6, 7, 8, 9, 11

Subtypes

Isolates

Many Many Many Many 5a Many

Many Many Many Many Many Many

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Although the different genotypes can be found worldwide, there are clear differences in their distribution (Fig. 1).16,45* 95 Genotypes la, lb, 2a, 2b, 2c, and 3a account for more than 90% of the HCV infections in North and South America, Europe, Russia, China, Japan, Australia, and New Zealand. Genotypes l a and l b each account for more than 40% of the HCV isolates in the United States. Genotype l b is especially prevalent in Southern and Eastern Europe, and in China and Japan, where it accounts for the majority of infections. Genotype 3a is more common among younger populations. Other subtypes of genotype 3 are highly prevalent in Nepal, Bangladesh, India, and Pakistan. Most infections in Egypt are gentotype 4a, and this and other subtypes of genotype 4 are found in Central Africa. Genotype 5a accounts for about 50% of infections in South Africa. Genotypes 4 and 5 are found only sporadically outside Africa. Genotype 6 isolates (including former genotypes 7 through 9 and 11) are primarily found in Southeast Asia. It should be noted that the genotype distribution can vary significantly among different population groups in the same geographical area. An example is the high prevalence of genotype 3a in the younger population of Western countries, particularly among intravenous drug users.15 Effect of Hepatitis C Virus Genotypes on Long-term Outcome and Therapy

The effect of HCV genotypes on the long-term outcome of HCV infection is still controversial.134The results of some studies suggest that the genotype of HCV may be one of the factors influencing the severity of the associated liver disease. Infection with genotype l b has been associated with more advanced liver disease and the development of liver cirrhosis and hepatocellular carcinoma.1z, 119, 140 This association, however, might not reflect a direct causative relationship because genotype l b may have existed for longer periods in some areas, and the longer duration of the disease might explain the association with disease progre~sion.~, 93, 117 The inherent difficulties in performing long-term follow-up studies, coupled with the limited epidemiologic information, make it very difficult to define the role of genetic variability in the course of HCV infection. The HCV genotype has been associated with response to IFN treatment in many studies. Patients infected with genotype 1, and in particular with lb, respond poorly to IFN treatment compared with patients infected with genotypes 2 or 3.26Patients infected with genotype 4 also have a low response rate.34As reviewed by Davis et in 15 studies that reported long-term results after short courses of IFN treatment, sustained response was achieved in only 18.1% of 536 patients with genotype 1 compared with 54.9% of 288 patients with other genotypes. Hepatitis C virus genotype is also strongly associated with the response to the more effective combination therapy of IFN and ribavirin, both in patients treated for chronic hepatitis C for the first time and in patients treated after relapse following treatment with IFN alone.25,lol It seems

v

W

0

Figure 1. Genotype distribution worldwide. Hepatitis C virus strains initially classified 2s genotypes 7 to 9, and 11 have been included within genotype 6, and strains initially classified as genotype 10 have been included in genotype 3. Only the two more common subtypes l a and 1b are depicted. (Data from references 16, 45, and 95.)

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clear that patients infected with genotype 1benefit from a long treatment regimen with combination therapy,'"', 118 suggesting that genotyping should be considered in the management of treatment. Besides the stated differences among the different HCV genotypes, isolates within a genotype may also exhibit a different response to interferon therapy. For example, Enomoto et a136found that genotype l b isolates identical to the HCV-J prototype within the ISDR of the NS5A protein were resistant to IFN. In contrast, patients infected with genotype l b strains that had amino acid substitutions within this region responded better to IFN therapy.36,lZ9It should be emphasized, however, that the correlation between the ISDR sequence and the response to IFN could not be confirmed in other studies.56,147, 178 The mechanism by which IFN sensitivity might be regulated by the NS5A region is unknown, but it was recently shown that NS5A interacts with the IFN-induced cellular protein kinase PKR.49 A recent study has also shown an interaction The interaction of NS5A or E2 with PKR between HCV E2 and PKR.155a could be a mechanism used by the virus to escape antiviral activity. Hepatitis C Virus Genotyping

The relevance of HCV genotype as a predictive factor for response to therapy makes HCV genotyping useful. Most HCV genotyping methods rely on PCR and include a first step of universal amplification followed by type-specific amplification, specific hybridization, specific enzymatic digestion or sequence analysis.&Methods that rely on the 5' UTR are advantageous because reverse transcription PCR (RT-PCR) of this region is routinely used for diagnosis of HCV infection; however, such methods cannot distinguish all of the different genotypes. Methods based on other genomic regions have the potential for distinguishing among additional genotypes. In addition to determining the infecting genotype, it is possible to detect genotype-specific humoral immune responses. Such indirect methods are more reproducible and less expensive 95 Although sequence than PCR-based assays, but they are less ~ensitive.4~, analysis of appropriate genomic regions (such as the E l and NS5B genes) is the only definitive method for determining the HCV genotype, the most frequently used genotyping methods (such as the line probe assay and restriction fragment length polymorphism) are actually capable of identifying the infecting genotype in approximately 90% of cases in the United States, Europe, and Japan, where genotypes 1, 2, and 3 predominate. Such HCV genotyping methods should, however, be interpreted more cautiously in geographic areas where the accuracy of these methods may be confounded by the presence of different genotypes. The Study of Hepatitis C Virus Quasispecies in an Infected Individual

The quasispecies composition of HCV in an infected individual is the result of mutations that accumulate over time during infection or

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mutations that are present from the onset of the infection resulting from simultaneous transmission of multiple viral species. A new dominant HCV sequence can result from the accumulation of mutations over time or from the selection of a preexisting minor viral species.29,33, 62 In an acute HCV infection, in which multiple HCV species contained in the inoculum are transmitted simultaneously, all species may not be equally capable of infecting and replicating in a host, so selection can occur during the acute phase of an infection.4O. 148, 169 Sequence analysis of the HVRl region has been used most frequently to characterize the distribution of HCV quasispecies in infected individuals.16Farci et a142performed an extensive analysis of the viral genome population in H77, the acute-phase plasma from patient H, who had post-transfusion hepatitis C. A total of 104 molecular clones derived from amplified products spanning part of the E l and E2 regions were sequenced. The analysis demonstrated that at least 19 different viral strains were simultaneously present; the most prevalent strain represented 69% of the clones. Although some strains may appear identical after analysis of subgenomic regions, it is well known that genomic heterogeneity in RNA viruses extends throughout the entire genome. In fact, sequence analysis of 18 full-length HCV ORFs derived from plasma H77 demonstrated that no two sequences were identical, and that heterogeneity was present throughout the ORE’” The same observation was made by sequence analysis of the ORF of nine clones obtained from an acute-phase plasma pool from an experimentally infected ~himpanzee.’~~ Therefore, when studying the quasispecies distribution of HCV by sequence analysis of a short subgenomic region, it is important to recognize that heterogeneity outside the target region might exist among genomes with identical sequences in the region analyzed. It also should be noted that minor false quasispecies can result from polymerase mistakes introduced during the PCR amplifi~ation’~~ or from the selection of certain clones during the amplification and cloning procedure^.^^ The complexity of the quasispecies population might influence both the outcome of the HCV-associated liver disease and the response to interferon therapy?” 41, 43 By sequence analysis of the C-El region, Honda et a P showed that the magnitude of the intrapatient quasispecies variation increases with the progression of liver disease. Koizumi et a179also showed that a high degree of diversity is related to the progression of liver disease, independent of the duration of the infection. Farci et a141 reported that the quasispecies complexity at the beginning of an HCV infection can be useful in predicting the long-term outcome of the associated liver disease. There is still controversy~however, over the role of quasispecies complexity in the outcome of HCV infection, primarily because of the differences in the studied cohorts and the use of different strategies to evaluate the quasispecies comp0sition.4~ Several studies found an association between the HCV quasispecies complexity and the response to IFN; the less divergent the quasispecies, the higher the likelihood of response.5o, 116 In most such studies, the target sequence used to assess the genetic heterogeneity was the HVRl.

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Enomoto et a135demonstrated that IFN treatment can induce changes in the composition of the HCV quasispecies, with selection of specific variants that would be more resistant to the treatment. Recently, Polyak et aP6 analyzed the evolution of HCV quasispecies in HVRl and ISDR in patients during IFN therapy and compared it with the evolution in natural infection. Although only a limited number of patients were studied, it was shown that individuals receiving IFN therapy presented a higher mutation rate in both regions analyzed (and especially in the HVRl), suggesting the existence of a selective pressure. More studies are needed, however, to define which regions are targets for selective pressure during treatment, because mutations that appear in one genomic region may only be a marker for a mutation that results from pressure on a nonanalyzed region.

Hepatitis C Viral Genetic Heterogeneity and Viral Persistence The mechanism by which HCV establishes a chronic infection in most infected individuals is still unknown. The HCV genetic variability, in particular the quasispecies nature of HCV, has been suggested as a potential mechanism for viral persistence, mainly by allowing the virus to escape the host immune response. Genomic changes in the virus seem to emerge in a pattern consistent with selective escape from a previous immune response. Weiner et all6*found that the appearance of antibodies against the HVRl of the predominant sequence in one patient was followed by the emergence of a new variant, against which no antibodies were detectable. The same phenomenon has been documented by other investigator^.^^, 13* It has been suggested that apart from the humoral immune response, the cellular immune response can result in immune pressure and possible immunologic escape. Weiner et a P 7 observed that cytotoxic T lymphocytes (CTL) directed against a conserved epitope within the NS3 region selected for another variant. In a recent study, CTL activity directed against an epitope located within the HVRl region was significantly stronger in patients who resolved HCV infection than in those who developed a chronic infection.162During the acute phase of the infection, the two patients who developed a chronic infection displayed mutations in this epitope; patients who resolved the HCV infection did not. The relevance of immunologic escape as a mechanism for viral persistence of HCV is, however, a controversial issue. The recent development of infectious cDNA clones from HCVso,172, 174 has permitted more detailed analysis of the natural history of HCV infection in chimpanzees. Interestingly, when RNA transcribed from infectious cDNA clones was inoculated intrahepatically into chimpanzees, chronic infection developed in most of the animals. The RNA inoculated into the animals was generated from a single HCV sequence, and the animals, therefore, became infected with a monoclonal viral population. However, three of

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the four animals infected with monoclonal viruses developed a chronic 97 Long-term follow-up of one of these chimpanzees showed infection.20, that there were no amino acid changes within the envelope proteins during the first year.2oAs anti-El and anti-E2 were detected soon after the onset of the infection, escape from the humoral immune response against the envelope proteins could not be shown to be a cause of viral persistence, at least when HCV infection is caused by a monoclonal viral population. It is possible, however, that persistence resulted from the development of mutants that escaped from the cellular immune response, because some amino acid changes did occur in the NS regions during follow-up. It is clear that additional studies are needed to elucidate the mechanism by which HCV establishes a persistent infection in a high proportion of infected individuals. Genetic Variability and Vaccine Development Natural infection with HCV seems not to elicit protective immunity. In a study by Farci et al,38chimpanzees that had resolved their acute experimental HCV infection could again develop hepatitis when challenged with the homologous strain. Studies in thalassemic children show that HCV can reinfect an individual after resolution of a previous HCV infection.86However, antibodies capable of neutralizing HCV seem to exist: chronic-phase plasma from patient H neutralized HCV from the patient's acute-phase plasma.39,138 Antibodies directed against the HVRl of the E2 protein also had neutralizing activity.", 139 The authors used a hyperimmune serum raised against the consensus sequence of the HVRl of HCV strain H77. After in vitro neutralization of the acute phase .inoculum from patient H, infectivity was tested in two chimpanzees." One animal was protected from HCV infection, and the other developed a persistent infection. Sequence analysis of the HVRl of several HCV clones recovered from the serum of the infected animal identified minor species that had been present in the inoculum, but the predominant sequence in the inoculum was not detected. These findings suggest that minor species present in the inoculum that were not neutralized by the hyperimmune serum raised against the HVRl of the predominant species had caused the infection in this animal. Further support for the existence of neutralizing antibodies comes from a study in which infusion of immune globulin containing HCV antibodies markedly prolonged the incubation period of acute hepatitis C in chimpanzees, although it did not prevent HCV infection.83 In the only study in which in vivo neutralization of HCV has been demonstrated, an experimental vaccine produced from expressed envelope glycoprotein protected chimpanzees against a low-dose challenge with an homologous strain (HCV-1).22Challenge with a closely .~~ related heterologous strain (H77), however, resulted in i n f e ~ t i o nSerum samples from the immunized chimpanzees were recently tested for neutralization of binding (NOB) antibodies, which block the binding of a recombinant E2 protein to a human cell line.126Chimpanzees that were

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protected against homologous challenge showed a higher titer of NOB antibodies than chimpanzees that were not protected against challenge.68 The success of a vaccine probably depends on the production of antibodies with adequate neutralizing activity. The existence of different genotypes with a low degree of homology within the envelope proteins16, 18, 114 and the presence of multiple viral species within an infected individual make it unlikely that a highly conserved neutralization epitope will be found. The emergence of minor variants that escape antibodies elicited by a potential vaccine might be a major cause of neutralization failure,4’ 168 and therefore, it is likely that a polyvalent vaccine will be needed to generate broadly reactive neutralizing antibodies. The lack of a reliable cell-culture system for HCV is also a major obstacle for the study of virus neutralization. Several groups have analyzed the humoral and cellular immune responses to immunization with HCV antigens in different animals, such as mice, rabbits, and rhesus macaques. These studies have provided a better understanding of the immune response mounted against structural and nonstructural proteins of HCV, as well as the relative efficiency of different methods of immunization. One such method, vaccination with DNA, has been shown to elicit protective immune responses against several pathogens, including human immunodeficiency virus and flaviviruses.’O,133 Vaccination with DNA can induce humoral and cellular immune responses and, in comparison with protein vaccines, the direct expression of foreign proteins in the host facilitates folding and presentation of the antigens that might better approach those occurring in the natural infe~ti0n.l~~ In addition, DNA vaccines represent a versatile system for studying the immunogenicity of multiple constructs containing different coding sequences, while avoiding the necessity of protein purification. Several studies have already shown that humoral or cellular immune responses to HCV can be obtained by DNA vaccination 48 Regarding protein vaccines, hepatitis of mice70,98, lo8,lZ8and monkeys.47, C virus-like particles synthesized in insect cells (using a recombinant baculovirus containing HCV cDNA) elicited humoral immune response against the core and the E2 proteins in Balb/c mice and New Zealand white rabbits after these were immunized with the purified antigen.5 The lack of a cell-culture system is still a major impediment for understanding the molecular biology of HCV. The existence of an efficient cell-culture system would markedly facilitate both studies of protein functions and the testing of new therapeutic agents. A cell-culture assay for HCV would also allow the study of virus neutralization, which would be highly relevant for determining whether broadly reactive antibodies can be elicited by immunization. An important step toward the development of an efficient HCV replication system was recently published; replication of subgenomic HCV RNA was demonstrated in a hepatoma cell line.92a ACKNOWLEDGMENTS The authors acknowledge Dr. Suzanne U. Emerson and Dr. Robert H. Purcell for careful review of the manuscript.

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References 1. Al RH, Xie Y, Wang Y, et al: Expression of recombinant hepatitis C virus NS5B. Nucleic Acids Symp Ser 36197, 1997 2. Bartenschlager R, Ahlborn-Laake L, Mous J, et al: Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage of the NS3/ 4 and NS4/5 junctions. J Virol 673835, 1993 3. Bartholomeusz A, Tomlinson E, Wright PJ, et al: Use of a flavivirus RNA-dependent RNA polymerase assay to investigate the antiviral activity of selected compounds. Antiviral Res 24341, 1994 4. Baumert TF, Ito S, Wong DT, et al: Hepatitis C virus structural proteins assemble into virus-like particles in insect cells. J Virol 723827, 1998 5. Baumert TF, Vergalla J, Satoi J, et al: Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate. Proceedings of the International Meeting on Hepatitis C Virus and Related Viruses, Venezia, Italy 1998, p 245 6. Behrens SE, Tomei L, DeFrancesco R Identification and properties of the RNAdependent RNA polymerase of hepatitis C virus. EMBO J 15:12, 1996 7. Benvegd L, Pontisso P, Cavalletto D, et al: Lack of correlation between hepatitis C virus genotypes and clinical course of hepatitis C virus-related cirrhosis. Hepatology 25:211, 1997 8. Blight KJ, Kolykhalov AA, Reed KE, et al: Molecular virology of hepatitis C virus: An update with respect to potential antiviral targets. In Schinazi RF,Sommadossi JP, Thomas HC (eds): Therapies for Viral Hepatitis. London, International Medical Press, 1998, p 207 9. Blight KJ, Rice C M Secondary structure determination of the conserved 98-base sequence at the 3’ terminus of hepatitis C virus genome RNA. J Virol 71:7345, 1997 10. Boyer JD, Ugen KE, Wang 8, et al: Protection of chimpanzees from high-dose heterologous HN-1 challenge by DNA vaccination. Nat Med 3:526, 1997 11. Brown EA, Zhang H, Ping L-H, et al: Secondary structure of the 5’ nontranslated regions of hepatitis C virus and pestivirus genomic RNAs. Nucleic Acids Res 20:5041, 1992 12. Bruno S, Silini E, Crosignani A, et al: Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: A prospective study. Hepatology, 253754, 1997 13. Bukh J, Apgar CL: Five new or recently discovered (GBV-A) virus species are indigenous to New World monkeys and may constitute a separate genus of the Flaviviridae. Virology 229:429, 1997 14. Bukh J, Apgar CL, Engle R, et al: Experimental infection of chimpanzees with hepatitis C virus of genotype 5a: Genetic analysis of the virus and generation of a standardized challenge pool. J Infect Dis 178:1193, 1998 15. Bukh J, Emerson SU, Purcell RH: Genetic heterogeneity of hepatitis C virus and related viruses. In Rizzetto M, Purcell RH, Gerin JL, et a1 (eds): Viral Hepatitis and Liver Disease, Turin, Minerva Medica, 1997, p 167 16. Bukh J, Miller RH, Purcell RH: Genetic heterogeneity of hepatitis C virus: Quasispecies and genotypes. Semin Liver Dis 15:41, 1995 17. Bukh J, Purcell RH, Miller RH: Sequence analysis of the 5’ noncoding region of hepatitis C virus. Proc Natl Acad Sci U S A 89:4942, 1992 18. Bukh J, Purcell RH, Miller RH: At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative El gene of isolates collected worldwide. Proc Natl Acad Sci USA 90:8234, 1993 19. Bukh J, Purcell RH, Miller RH: Sequence analysis of the core gene of 14 hepatitis C virus genotypes. Proc Natl Acad Sci U S A 91:8239, 1994 20. Bukh J, Yanagi M, Emerson SU, et al: Course of infection and evolution of monoclonal hepatitis C virus (HCV) strain H77 in chimpanzees transfected with RNA transcripts from an infectious cDNA clone [abstract]. Hepatology 28:628, 1998 21. Chamberlain RW, Adams NJ, Taylor LA, et al: The complete coding sequence of hepatitis C virus genotype 5a, the predominant genotype in South Africa. Biochem Biophys Res Commun 236:44,1997

1

710

FORNS&BUKH

22. Choo Q-L, Kuo G, Ralston R, et al: Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 91:1294, 1994 23. Choo Q, Richman KH, Han JH, et al: Genetic organization and diversity of hepatitis C virus. Proc Natl Acad Sci USA 88:2451, 1991 24. Cocquerel L, Meunier J-C, Pillez A, et al: A retention signal necessary and sufficient for endoplasmic reticulum localization maps to the transmembrane domain of hepatitis C virus glycoprotein E2. J Virol 72:2183, 1998 25. Davis GL, Esteban-Mur R, Rustgi V, et al: Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. N Engl J Med 339:1493, 1998 26. Davis GL, Lau JYN: Factors predictive of a beneficial response to therapy of hepatitis C. Hepatology 26 (suppl 1):122S, 1997 27. De Francesco R, Pessi A, Steinkuhler C: The hepatitis C virus NS3 proteinase: Structure and function of a zinc-containing serine proteinase. In Schinazi RF,Sommadossi J-P, Thomas HC (eds): Therapies for Viral Hepatitis. London, International Medical Press, 1998, p 225 28. Dimasi N, Martin F, Volpari C, et al: Characterization of engineered hepatitis C virus NS3 protease inhibitors affinity selected from human pancreatic secretory trypsin inhibitor and minibody repertoires. J Virol71:7461, 1997 29. Doming0 E, Holland J, Biebricher C, et al: Quasispecies: The concept and the world. In Gibbs A, Calisher C, Garcia-Arena1 F (eds): Molecular Evolution of Viruses. Cambridge, Cambridge University Press, 1994, p 181 30. Dubuisson J, Hsu HH, Cheung RC, et al: Formation and intracellular localization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinina and sindbis viruses. J Virol 68:6147, 1994 31. Dubuisson J, Rice CM: Hepatitis C virus glycoprotein folding: Disulfide bond formation and association with calnexin. J Virol 1996 70778, 1996 32. Eckart MR, Selby M, Masiarz F, et al: The hepatitis C virus encodes a serine protease involved in processing of the putative nonstructural proteins from the viral polyprotein precursor. Biochem Biophys Res Commun 192:399, 1993 33. Eigen M On the nature of virus quasispecies. Trends Microbiol 4:216, 1996 34. El Zayadi A, Simmonds P, Dabbous H, et al: Response to interferon-alpha of Egyptian patients infected with hepatitis C virus genotype 4. J Viral Hepat 3:261, 1996 35. Enomoto N, Kurosaki M, Tanaka Y, et al: Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformational polymorphism analysis. J Gen Virol 75:1361, 1994 36. Enomoto N, Sakuma I, Asahina Y, et al: Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus lb. J Clin Invest 96:224, 1995 37. Enomoto N, Sat0 C: Clinical relevance of hepatitis C virus quasispecies. J Viral Hepat 2267, 1995 38. Farci P, Alter HJ, Govindarajan S, et al: Lack of protective immunity against reinfection with hepatitis C virus. Science 258:135, 1992 39. Farci P, Alter HJ, Wong D, et al: Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization. Proc Natl Acad Sci USA 91:7792, 1994 40. Farci P, Bukh J, Purcell RH: The quasispecies of hepatitis C virus and the host immune response. Springer Semin Immunopathol 19:5, 1997 41. Farci P, Melpolder JC, Shimoda A, et al: Studies of HCV quasispecies in patients with acute resolving hepatitis compared to those who progress to chronic hepatitis [abstract]. Hepatology 242395, 1996 42. Farci P, Shimoda A, Wong D, et al: Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci U S A 93:15394, 1996 43. Farci P, Strazzera R, DeGjonnis D, et al: Evolution of the viral quasispecies tracked by sequence analysis during interferon treatment of chronic hepatitis C [abstract]. Hepatology 28:938, 1998 44. Feinstone SM, Mihalik KB, Kamimura T, et al: Inactivation of hepatitis B virus and non-A, non-B hepatitis by chloroform. Infect Immun 41:816, 1983

THE MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

711

45. Forns X, Bukh J: Methods for determining the hepatitis C virus genotype. Viral Hepatitis Reviews 4:1,1998 46. Foms X, Bukh J, Purcell RH, et a1 How Escherichia coli can bias the results of molecular cloning: Preferential selection of defective genomes of hepatitis C virus during the cloning procedure. Proc Natl Acad Sci USA 9433909, 1997 47. Forns X, Emerson SU, Tobin GJ, et al: DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface. Vaccine 171992, 1999 48. Foumillier A, Vitvitski L, Depla E, et al: Induction of immune responses against HCV E l and E2 proteins in mice and tamarins following DNA vaccination and recombinant canary pox/ protein boosting. Proceedings of the International Meeting on Hepatitis C Virus and Related Viruses, Venezia, Italy 1998, p 54 49. Gale M, Blakely CM, Kwieciszewski 8, et al: Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: Molecular mechanisms of kinase regulation. Mol Cell Biol 185208, 1998 50. Gonzalez-Peralta RP, Qian K, She JY, et al: Clinical implications of viral quasispecies heterogeneity in chronic hepatitis C. J Med Virol49:242, 1996 51. Grakoui A, McCourt DW, Wychowski C, et al: A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A 90:10583, 1993 52. Gwack T, Kim DW, Hang JH, et al: Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochem Biophys Res Commun 225654, 1996 53. Hanecak R, Brown-Driver V, Fox MC, et al: Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes. J Virol 70:5203, 1996 54. Harada S, Watanabe Y, Takeuchi K, et al: Expression of processed core protein of hepatitis C virus in mammalian cells. J Virol 65:3015, 1991 55. He LF, Alling D, Popkin T, et al: Determining the size of non-A, non-B hepatitis virus by filtration. J Infect Dis 156:636, 1987 56. Herion D, Hoofnagle J H The interferon sensitivity determining region: All hepatitis C virus isolates are not the same. Hepatology 25:769, 1997 57. Hijikata M, Kato N, Ootsuyama Y, et al: Gene mapping of the putative structural region of the hepatitis C virus genome by in nitro processing analysis. Proc Natl Acad Sci USA 88:5547, 1991 58. Hijikata M, Kato N, Ootsuyama Y, et al: Hypervariable regions in the putative glycoprotein of hepatitis C virus. Biochem Biophys Res Commun 175:220, 1991 59. Hijikata M, Mizushima H, Akagi T, et al: Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 674665, 1993 60. Hijikata M, Mizushima H, Tanji Y, et al: Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci USA 90:10773, 1993 61. Hijikata M, Shimizu YK, Kato H, et al: Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. J Virol 671953, 1993 62. Holland JJ, de la Torre JC, Steinhouse D A RNA virus populations as quasispecies. Curr Top Microbiol Immunol 1763, 1992 63. Honda M, Brown EA, Lemon SM: Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2:955, 1996 64.Honda M, Kaneko S, Sakai A, et al: Degree of diversity of hepatitis C virus quasispecies and progression of liver disease. Hepatology 20:1144, 1994 65. Hoofnagle JH: Hepatitis C The clinical spectrum of disease. Hepatology 26:15S, 1997 66. Houghton M: Hepatitis C viruses. In Fields BN, Knippe DM, Howley PM (eds): Fields Virology, ed 3. Philadelphia, Lippincott-Raven, 1996, p 1035 67. Houghton M, Choo Q-L, Chien D, et al: Development of an HCV vaccine. In Rizzetto M, Purcell RH, Gerin JL, Verme G (eds): Viral Hepatitis and Liver Disease. Turin, --, Italy, Minerva Medica, 1997, p 656 , 68. Houghton M, Choo Q-L, Coates S, et al: Development of a recombinant HCV subunit

712

FORNS & BUKH

vaccine. Proceedings of the International Meeting on Hepatitis C Virus and Related Viruses, Venezia, Italy, 1998, p 57 69. Hwang S-B, Lo S-Y, Ou J-H et al: Detection of cellular proteins and viral core protein interacting with the 5‘ untranslated region of hepatitis C virus RNA. J Biomed Sci 2:227, 1995 70. Inchauspe G, Major ME, Nakano I, et al: DNA vaccination for the induction of immune responses against hepatitis C virus proteins. Vaccine 15:853, 1997 71. Kaito M, Watanabe S, Tsukiyama-Kohara K, et al: Hepatitis C virus particle detected by immunoelectron microscopic study. J Gen Virol 75:1755, 1994 72. Kakiuchi N, Komoda Y, Komoda K, et al: Non-peptide inhibitors of HCV serine proteinase. FEBS Lett 421:217, 1998 73. Kaneko T, Tanji Y, Satoh S, et al: Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem Biophys Res Commun 205:320, 1994 74. Kato N, Ootsuyama Y, Tanaka T, et al: Marked sequence diversity in the putative envelope proteins of hepatitis C viruses. Virus Res 22107, 1992 75. Kato N, Sekiya H, Ootsuyama Y, et al: Humoral immune response to hypervariable region 1 of the putative envelope glycoprotein (gp70) of hepatitis C virus. J Virol 673923, 1993 76. Khromykh AA, Westaway EG: Subgenomic replicons of the flavivirus Kunjin: Construction and auulications. T Virol 71:1497. 1997 I I . Kim DW, Gwiik Y , Han *JH, et al: C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity. Biochem Biophys Res Commun 215:160, 1995 78. Kim JL, Morgenstem KA, Lin C, et al: Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87343, 1996 79. Koizumi K, Enomoto N, Kurosaki M, et al: Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment. Hepatology 2230, 1995 80. Kolykhalov AA, Agapov EV, Blight KJ, et al: Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277570, 1997 81. Kolykhalov AA, Feinstone SM, Rice CM: Identification of a highly conserved sequence element at the 3’ terminus of hepatitis C virus genome RNA. J Virol 703363, 1996 82. Korolev S, Yao N, Lohman TM, et a1 Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SFl and SF2 superfamilies of helicases. Protein Science 7:1, 1998 83. Krawczynski K, Alter MJ, Tankersley DL, et al: .Effect of immune globulin on the prevention of experimental hepatitis C virus infection. J Infect Dis 173:822, 1996 84. Kumar PK, Machida K, Urvil PT, et al: Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA. Virology 237270, 1997 85. Lai CJ, Bray M, Men R, et al: Evaluation of molecular strategies to develop a live dengue vaccine. Clin Diagn Virol 10:173, 1998 86. Lai ME, Mazzoleni AP, Argiolu F, et al: Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children. Lancet 343:388, 1994 87. Lemon SM, Honda M: Internal ribosome entry sites within the RNA genomes of hepatitis C virus and other flaviviruses. Semin Virol 8:274, 1997 88. Lin C, Lindenbach BD, Pragai BM, et al: Processing in the hepatitis C virus E2-NS2 region: Identification of p7 and two distinct E2-specific products with different C termini. J Virol 68:5063, 1994 89. Linnen J, Wages J, Zhangkeck ZY, et al: Molecular cloning and disease association of hepatitis C virus: A transfusion-transmissible agent. Science 271:505, 1996 90. Littlejohn M, Locamini S, Bartholomeusz A: Targets for inhibition of hepatitis C virus replication. In Schinazi RF, Sommadossi J-I-’, Thomas HC (eds): Therapies for Viral Hepatitis. London, International Medical Press, 1998, p 219 91. Lo S-Y, Selby M, Tong M, et al: Comparative studies of the core gene products of two different hepatitis C virus isolates: Two alternative forms determined by a single amino acid substitution. Virology 199:124, 1994 92. Lohmann V, Korner F, Herian U, et al: Biochemical properties of hepatitis C virus “r)

THE MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

713

NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol 71:8416, 1997 92a. Lohmann V, Komer F, Koch J-0,et al: Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110, 1999 93. Lopez-Labrador FX, Ampurdanes S, Forns X, et al: Hepatitis C virus (HCV) genotypes in Spanish patients with HCV infection: Relationship between HCV genotype lb, cirrhosis and hepatocellular carcinoma. J Hepatol 27959, 1997 94. Love RA, Parge HE, Wickersham JA, et al: The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and structural zinc binding site. Cell 87331, 1996 95. Maertens G, Stuyver L: Genotypes and genetic variation of hepatitis C virus. In Harrison TJ, Zuckerman A (eds): The Molecular Medicine of Viral Hepatitis (Medical Science Series). Chichester, England, John Wiley & Sons, 1997, p 183 96. Major ME, Feinstone SM: The molecular virology of hepatitis C. Hepatology 25:1527, 1997 97. Major ME, Mihalik K, Kolykhalov AA, et al: Long term follow-up of chimpanzees inoculated with the first HCV infectious clone for hepatitis C virus. J Virol 73:3317, 1999 98. Major ME, Vitvitski L, Mink MA, et al: DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid. J Virol 69:5798, 1995 99. Mandl CW, Holzmann H, Meixner T, et al: Spontaneous and engineered deletions in the 3’ noncoding region of tick-borne encephalitis virus: Construction of highly attenuated mutants of a flavivirus. J Virol 72:2132, 1998 100. Martell M, Esteban JI, Quer J, et al: Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: Quasispecies nature of HCV genome distribution. J Virol 663225, 1992 101. McHutchison JG, Stuart CG, Schiff ER, et al: Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 339:1485, 1998 102. Men R, Bray M, Clark D, et al: Dengue type 4 virus mutants containing deletions in the 3’ noncoding region of the RNA genome: Analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70:3930, 1996 103. Mizokami M, Gojobori T, Ohba K-I, et al: Hepatitis C virus types 7, 8 and 9 should be classified as type 6 subtypes. J Hepatol24622, 1996 104. Mizushima H, Hijikata M, Asabe S, et al: Two hepatitis C virus glycoprotein E2 products with different C termini. J Virol 686215, 1994 105. Moradpour D, Englert C, Wakita T, et al: Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 222:51, 1996 106. Morgenstem KA, Landro JA, Hsiao K, et al: Polynucleotide modulation of the protease, nucleoside triphosphatase and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells. J Virol 71:3767, 1997 107. Moriya K, Fujie H, Shintani Y, et al: The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 4:1065, 1998 108. Nakano I, Maertens G, Major ME, et al: Immunization with plasmid DNA encoding hepatitis C virus envelope E2 antigenic domains induces antibodies whose immune reactivity is linked to the injection mode. J Virol 71:7101, 1997 109. National Institutes of Health Consensus Development Conference Panel statement: management of hepatitis C. Hepatology 26(suppl):2S, 1997 110. Nousbaum JB, Pol S, Nalpas 8, et al: Hepatitis C virus type l b (11) infection in France and Italy. Ann Intern Med 122:161, 1995 111. Ogata N, Alter HJ, Miller RH, et al: Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci U S A 88,3392, 1991 112. Okada S-I, Akahane Y, Suzuki H, et al: The degree of variability in the amino terminal region of the E2/NS1 protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients. Hepatology 16:619, 1992 113. Okamoto H, Kojima M, Okada S-I, et al: Genetic drift of hepatitis C virus during an 8.2-year infection in a chimpanzee: Variability and stability. Virology 1902394, 1992

714

FORNS&BUKH

114. Okamoto H, Kurai K, Okada S-I, et al: Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: Comparative study of four distinct genotypes. Virology 188:331, 1992 115. Pileri P, Uematsu Y, Campagnoli S, et al: Binding of hepatitis C virus to CD81. Science 282:938, 1998 116. Polyak SJ, Faulkner G, Carithers RL, et al: Assessment of hepatitis C virus quasispecies heterogeneity by gel shift analysis: Correlation with response to interferon therapy. J Infect Dis 175:1101, 1997 117. Poynard T, Bedossa P, Opolon P, et al: Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 349:825, 1997 118. Poynard T, Marcellin P, Lee SS, et al: Randomised trial of interferon alpha 2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha 2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 3521426, 1998 119. Pozzato G, Kaneko S, Moretti M, et al: Different genotypes of hepatitis C virus are associated with different severity of chronic liver disease. J Med Virol43: 291, 1994 120. Purcell RH: Hepatitis viruses: Changing patterns of human disease. Proc Natl Acad Sci USA 91:2401, 1994 121. Ralston R, Thudium K, Berger K, et al: Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J Virol 676753, 1993 122. Ray RB, Lagging LM, Meyer K, et al: Hepatitis C virus core protein cooperates with ras and transfirms primary rat embryo fibroblasts to tumorigenic phenotype. J Virol 704438, 1996 123. Reed KE, Rice CM: Molecular characterization of hepatitis C virus. Curr Stud Hematol Blood Transfus 62:1, 1998 124. Rice C M Flaviviridue: The viruses and their replication. In Fields BN, Knippe DM, Howley PM (eds): Fields Virology, ed 3. Philadelphia, Lippincott-Raven, 1996, p 931 125. Robertson B, Myers G, Howard C, et al: Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: Proposals for Standardization. Arch Virol 143:2493, 1998 126. Rosa D, Campagnoli S, Moretto C, et al: A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: Cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA 933759, 1996 127. Ruster B, Zeuzem S, Roth W K Hepatitis C virus sequences encoding truncated core proteins detected in a hepatocellular carcinoma. Biochem Biophys Res Commun 219:911, 1996 128. Saito T, Sherman GJ, Kurokohchi K, et al: Plasmid DNA-based immunization for hepatitis C virus structural proteins: Immune responses in mice. Gastroenterology 112:1321, 1997 129. Saiz JC, Lopez-Labrador FX, Ampurdanes S, et al: The prognostic relevance of the nonstructural 5A gene interferon sensitivity determining region is different in infections with genotype l b and 3a isolates of hepatitis C virus. J Infect Dis 177839, 1998 130. Sakamoto N, Wu CH, Wu GY Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes. J Clin Invest 982720, 1996 131. Santolini E, Migliaccio G, La Monica N: Biosynthesis and biochemical properties of the hepatitis C virus core protein. J Virol 68:3631, 1994 132. Santolini E, Pacini L, Fipaldini C, et al: The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J Virol 69:7461, 1995 133. Schmaljohn C, Vanderzanden L, Bray M, et al: Naked DNA vaccines expressing the prM and E genes of Russian spring summer encephalitis virus and central European encephalitis virus protect mice from homologous and heterologous challenge. J Virol 71:9563, 1997 134. Seeff LB: Natural history of hepatitis C. Hepatology 26(suppl):21S, 1997 135. Selby M, Choo Q Berger K, et al: Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J Gen Virol74:1103,1993 136. Selby M, Glazer E, Masiarz F, et al: Complex processing and protein: Protein interactions in the E2:NS2 region of HCV. Virology 204:114, 1994

THE MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

715

137. Shimizu YK, Feinstone SM, Kohara M, et al: Hepatitis C virus: Detection of intracellular virus particles by electron microscopy. Hepatology 23205, 1996 138. Shimizu YK, Hijikata M, Iwamoto A, et al: Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol68:1494, 1994 139. Shimizu YK Igarashi H, Kiyohara T, et al: A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures. Virology 223:409, 1996 140. Silini E, Bottelli R, Asti M, et al: Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: A case-control study. Gastroenterology 111:199, 1996 141. Simmonds P, Holmes EC. Cha T-A. et al: Classification of heuatitis C virus into six major genotypes and a sehes of subtypes by phylogenetic analisis of the NS-5 region. J Gen Virol 742391, 1993 142. Simmonds P, Mellor J, Sakuldamrongpanich T, et al: Evolutionary analysis of variants of hepatitis C virus found in South-East Asia: Comparison with classifications based upon sequence similarity. J Gen Virol 773013, 1996 143. Simons JN, Desai SM, Schultz DE, et al: Translation initiation in GB viruses A and C: Evidence for internal ribosome entry and implications on genome organization. J Virol 70:6126, 1996 144. Simons JN, Leary TP, Dawson GJ, et al: Isolation of novel virus-like sequences associated with human hepatitis. Nat Med 1:564, 1995 145. Simons JN, Pilot-Matias TJ, Leary TF', et al: Identification of 2 flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci U S A 923401, 1995 146. Smith DB, McAllister J, Casino C, et al: Virus 'quasispecies': Making a mountain out of a molehill? J Gen Virol 78:1511, 1997 147. Squadrito G, Leone F, Sartori M, et al: Mutations in the nonstructural 5A region of hepatitis C virus and response of chronic hepatitis C to interferon alfa. Gastroenterology 113:567, 1997 148. Steinhauer DA, Holland JJ: Rapid evolution of RNA viruses. Annu Rev Microbiol 41:409, 1987 149. Sudo K, Matsumoto Y, Matsushima M, et al: Novel hepatitis C virus protease inhibitors; thiazolidine derivatives. Biochem Biophys Res Commun 238:643, 1997 150. Suzich JA, Tamura JK, Palmer-Hill F, et al: Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with related pestivirus and flavivirus enzymes. J Virol 676152, 1993 151. Suzuki R, Matsuura Y, Suzuki T, et al: Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted. J Gen Virol 76:53, 1995 152. Tai C-L, Chi W-K, Chen D-S, et al: The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol 70:8477, 1996 153. Tanaka T, Kato N, Cho MJ, et al: A novel sequence found at the 3' terminus of the hepatitis C virus genome. Biochem Biophys Res Commun 215:744,1995 154. Tanaka T, Kato N, Cho MJ, et al: Structure of the 3' terminus of the hepatitis C virus genome. J Virol 70:3307, 1996 155. Taniguchi S, Okamoto H, Sakamoto M, et al: A structurally flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NSl protein: Implications for an escape from antibody. Virology 195297, 1993 155a. Taylor DR, Shi ST, Romano PR, et al: Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285:107, 1999 156. Tighe H, Corr M, Roman M, et al: Gene vaccination: Plasmid DNA is more than just a blueprint. Immunol Today 19239, 1998 157. Tokita H, Okamoto H, Iizuka H, et al: Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and Zf), tenth (10a) and eleventh (lla) genetic groups. J Gen Virol 77293, 1996 158. Tokita H, Okamoto H, Iizuka H, et al: The entire nucleotide sequences of three hepatitis C virus isolates in genetic groups 7-9 and comparison with those in the other eight genetic groups. J Gen Virol 79:1847, 1998 159. Tokita H, Okamoto H, Luengrojanakul P, et al: Hepatitis C virus variants from Thailand classifiable into five novel genotypes in the sixth (6b), seventh (7c,7d) and ninth (9b,9c) major genetic groups. J Gen Virol 76:2329, 1995

716

FORNS & BUKH

160. Tokita H, Okamoto H, Tsuda F, et al: Hepatitis C virus variants from Vietnam are classifiable into the seventh, eighth and ninth major genetic groups. Proc Natl Acad Sci USA 91:11022, 1994 161. Tomei L, Failla C, Santolini E, et al: NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol 674017, 1993 162. Tsai S-L, Chen YM, Chen MH, et al: Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity. Gastroenterology 115:954, 1998 163. Tsukiyama-Kohara K, Iizuka N, Kohara M, et al: Internal ribosome entry site within hepatitis C virus RNA. J Virol 661476, 1992 164. Wang C, Sarnow P, Siddiqui A: Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 673338, 1993 165. Wang C, Siddiqui A Structure and function of the hepatitis C virus internal ribosome entry site. Cap-independent translation 203:99, 1995 166. Weiner AJ, Brauer MJ, Rosenblatt J, et al: Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NSl proteins and the pestivirus envelope glycoproteins. Virology 180:842, 1991 167. Weiner A, Erickson AL, Kansopon J, et al: Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T-lymphocyte escape variant. Proc Natl Acad Sci USA 922755, 1995 168. Weiner AJ, Geysen HM, Christopherson C, et al: Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: Potential role in chronic HCV infections. Proc Natl Acad Sci U S A 89:3468, 1992 169. Weiner AJ, Thaler MM, Crawford K, et al: A unique, predominant hepatitis C virus variant found in an infant born to a mother with multiple variants. J Virol 674365, 1993 170. World Health Organization. Hepatitis C: Global prevalence. Weekly Epidemiological Record 72341, 1997 171. Yamada N, Tanihara K, Takada A, et al: Genetic organization and diversity of the 3' noncoding region of the hepatitis C virus genome. Virology 223:255, 1996 172. Yanagi M, Purcell RH, Emerson SU, et al: Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci USA 94:8738, 1997 173. Yanagi M, St. Claire M, Emerson SU, et al: In v i m analysis of the 3' untranslated region of hepatitis C virus following in vitro' mutagenesis of an infectious cDNA clone. Proc Natl Acad Sci USA 962291, 1999 174. Yanagi M, St. Claire M, Shapiro M, et al: Transcripts of a chimeric cDNA clone of hepatitis C virus genotype l b are infectious in vim. Virology 244:161, 1998 175. Yao N, Hesson T, Cable M, et al: Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol 4463, 1997 176. Yao N, Weber PC: Helicase, a target for novel inhibitors of hepatitis C virus. In Schinazi RF, Sommadossi J-P, Thomas HC (eds): Therapies for Viral Hepatitis. London, International Medical Press, 1998, p 229 177. Yuan Z-H, Kumar U, Thomas HC, et al: Expression, purification and partial characterization of HCV RNA polymerase. Biochem Biophys Res Commm 232231, 1997 178. Zeuzem S, Lee JH, Roth WK Mutations in the nonstructural 5A gene of European hepatitis C virus isolates and response to interferon alfa. Hepatology 25:740, 1997

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