ILAR Journal Online, Volume 42(2) 2001: Animal Models of Hepatitis

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ILAR Journal V42(2) 2001
Animal Models of Hepatitis

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Clones

Molecular Clones of Hepatitis C Virus: Applications to Animal Models
Michael Gale, Jr., and Michael R. Beard

Michael Gale, Jr., Ph.D., is Assistant Professor in the Department of Microbiology, University of Texas Southwestern Medical Center, Dallas; and Michael R. Beard, Ph.D., is Assistant Professor in the Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Texas.

Abstract

Hepatitis C virus (HCV) is a global public health problem, with approximately 3% of the world population now infected. The clinical course of HCV often involves chronic infection, which can lead to liver dysfunction and hepatocellular carcinoma. Because HCV cannot be efficiently propagated in cell culture, researchers have relied heavily on animal models to study the physical characteristics of HCV and the course of events associated with HCV infection. The chimpanzee is the only nonhuman primate actually proven to be susceptible to HCV infection and has commonly been used to study viral hepatitis induced by HCV. Molecular cloning of the HCV genome has now allowed HCV transmission studies in chimpanzees to progress from the early work of characterizing infectious serum to a current focus of characterizing infectious HCV molecular clones. Moreover, the cloned HCV genome has paved the way for the development of alternative animal models for HCV, most notably transgenic mouse models for the study of HCV pathogenesis. The authors review these animal model applications of the HCV molecular clones, including construction and transmission of mutant viral genomes. The expression of specific viral protein products in these animal models will provide important insight into the structure-function relation that specific HCV genome sequences impart on virus replication and pathogenesis.

Key Words: animal model; chimpanzee; hepatitis; hepatitis C virus; infectious clone; liver; transgenic mouse; virus

Introduction

Hepatitis C virus (HCV¹) is the etiological agent responsible for the majority of non-A, non-B hepatitis (NANBH1) cases worldwide. Since the recognition of a viral NANBH agent (Alter et al. 1975; Prince et al. 1974), and the subsequent molecular cloning of HCV genome (Choo et al. 1989), animal models for the study of the virus have played an important role in our understanding of the disease processes associated with HCV infection. Early studies, in which serum from documented NANBH patients was passed first through filters to eliminate bacteria and then by inoculation of the filtrate into the chimpanzee (Pan troglodyte), established the validity of the chimpanzee model for NANBH and current HCV research. These studies demonstrated that chimpanzees could serve as a susceptible host to the NANBH viral agent and that the infected animals developed a disease course with many similar characteristic of humans infected with NANBH agent.

Over the past 20+ years, the chimpanzee model has served as the backbone for advancements in the HCV research field. The model has proved invaluable for the early characterization of the NANBH agent and for the current understanding of HCV infection, including the natural history of infection, routes of transmission, immunity, and pathogenesis. More recently, this animal model has been used to characterize the virological properties of HCV infectious molecular clones (reviewed by Major and Feinstone [2000]). Despite this remarkable progress, the chimpanzee model has some important disadvantages. Perhaps most importantly, chimpanzees are rare, are expensive and difficult to handle, and must be housed and cared for in appropriate nonhuman primate research facilities. Such research facilities must possess proper surgical support and specialized veterinary care. Moreover, the chimpanzee has been listed as an endangered species since 1988, and appropriate safeguards must be considered whenever selecting such a model for research purposes (Booth 1988; Ryazanov et al. 1991).

These and other limitations to the chimpanzee model have stimulated progress toward developing alternative animal models for HCV research. Transgenic technology, coupled with the relative ease and low cost by which mice can be reared and maintained, along with the availability of inbred mouse strains, have made the laboratory mouse an attractive animal model for HCV research. An HCV infection cannot be propagated in mouse tissues, which obviously limits the research application of mouse models. However, expression of the HCV genome or subgenomic fragments of HCV within inbred strains of mice has demonstrated the utility of mouse models for research geared toward understanding mechanisms of HCV pathogenesis. In this article, we present an overview of current animal models for HCV research. The first part of this article is focused on the recent application of the chimpanzee model for the characterization of infectious HCV molecular clones, followed by a brief summary of novel nonhuman primate models for the study of HCV (for general reviews of the chimpanzee in HCV research, the reader is directed to Bradley [2000]; Major and Feinstone [2000], and Prince and Brotman [1994]). The second part of the text is focused on the emerging role of the transgenic mouse model in HCV research and the power of genetics that this system brings to studies of HCV pathogenesis. We conclude with a brief presentation on future directions for these animal models in the HCV research field.

HCV Molecular Biology and Epidemiology

The genome of HCV was first cloned in 1989 from the plasma of a chimpanzee that had been experimentally infected with the NANBH agent (Alter et al. 1975; Prince et al. 1974). Characterization of the isolated clones revealed that the HCV genome was composed of a positive-sense, single-stranded RNA molecule (Choo et al. 1989). Further work has led to the complete characterization of the HCV genomic RNA and classification of six major HCV genotypes into a single genus within the family Flaviviridae (Bukh et al. 1995). The HCV RNA is approximately 9600 nucleotides (nt) in length and encodes a large polyprotein within a single open reading frame (ORF¹) of approximately 3010 amino acids (aa) (Figure 1). Once translated within the infected cell, the HCV polyprotein is cleaved into individual structural and nonstructural proteins through the actions of host cell signal peptidase and viral-encoded protease activities (Reed and Rice 1998). The viral structural proteins derived from cleavage of the HCV polyprotein include the viral core protein and the E1 and E2 envelope glycoproteins. The viral structural proteins are flanked by p7, a small protein of unknown function that precedes the nonstructural protein region of the HCV polyprotein. The viral nonstructural (NS¹) protein products most likely participate in replication of the viral RNA and include NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The HCV ORF is preceded in the viral genome by a 341 nt 5' untranslated region (UTR¹), which is highly conserved among viral isolates and functions as an internal ribosome entry site to direct the translation of the HCV polyprotein (Figure 1). The HCV ORF is followed by a 3' UTR composed of a variable sequence of approximately 40 nt, followed by poly U-U/C tracts. The HCV RNA terminates with a conserved 98 nt sequence that includes a region of secondary structure. HCV genome replication involves the generation of antisense strand RNA intermediates, which serve as the template for synthesis of the genomic RNA. The presence of the antigenomic HCV RNA defines active viral replication within the human or chimpanzee host and is usually identified by RT-PCR of serum-derived RNA (Fong et al. 1991).

More than 200 million people worldwide are now believed to be infected with HCV, including 4 million individuals within the United States (Alter et al. 1997, 1999; Cohen 1999). HCV accounts for the deaths of at least 8000 to 10,000 Americans each year and is now one of the most common indicators for adult liver transplants in developed countries. Before the introduction of anti-HCV screening in mid-1990, HCV accounted for 80 to 90% of new cases of post-transfusion hepatitis in the United States (Cuthbert 1994). Currently, injection drug use is probably the most common risk factor for HCV infection, with approximately 80% of this population seropositive for HCV (McHutchison et al. 1992). A high rate of HCV infection is also seen in individuals with bleeding disorders or chronic renal failure, groups that have had frequent exposure to blood and blood products.

Exposure to HCV may result in one of three general outcomes: (1) acute infection with recovery, (2) development of persistent infection without apparent disease (often referred to as "silent" disease), or (3) persistent infection with chronic hepatitis. The majority of those infected with HCV will eventually fall into the latter group (Alter 1997). Clinical and serological signs of acute HCV infection include a significant increase in the level of liver enzymes (e.g., alanine aminotransferase [ALT¹]) in the serum demarked by the presence of circulating HCV RNA and serum reactivity with the current enzyme immunoassays for anti-HCV antibodies (Esteban et al. 1998). A normalization of serum ALT levels and the absence of circulating HCV RNA characterize resolution of acute infection. In contrast, the development of persistent HCV infection can be accompanied by the constitutive presence of circulating HCV RNA and abnormal serum ALT levels, even in the presence of an antibody response to HCV (Esteban et al. 1998). The lack of a protective antibody response is attributed, in part, to the remarkable level of genetic variation of HCV, which replicates as a heterogeneous pool of quasispecies (Martell et al. 1992; Simmonds 1998). In particular, the hypervariable region 1 (HVR¹¹) of the E2 envelope glycoprotein is thought to represent an important neutralizing antibody epitope (Bassett et al. 1999). Thus, variation in this region may facilitate persistent infection by mediating escape from a neutralizing host humoral immune response.

The treatment options for HCV are limited to interferon-based therapies (Fried and Hoofnagle 1995; Gretch et al. 1996). Unfortunately, standard treatment regimens lead to sustained viral clearance and clinical remission in only 15 to 25 % of treated patients. The introduction of ribavirin as a treatment adjunct to interferon alpha has only a moderately improved therapeutic outcome (McHutchison et al. 1998). For those who fail antiviral therapy, persistent HCV infection often results in progressive liver damage and the development of cirrhosis (Alter 1995). In these patients, liver damage may progress to the development of hepatocellular carcinoma. In general, hepatocellular carcinoma is a late occurrence in HCV infection (Kiyosawa et al. 1990). The relative contribution of viral or host factors in determining disease progression is not well understood, underscoring the importance of animal models for the characterization of HCV-induced disase.

HCV Replicates Inefficiently in Vitro

Our poor understanding of HCV-host interactions comes, in part, from an inability to efficiently propagate the virus in cell culture. Scant reports of sustained viral replication in cell culture systems do exist, but these studies have not been followed up or accurately reproduced (Dash et al. 1997; Ikeda et al. 1998; Yoo et al. 1995). The inability to grow the virus in cell culture imposes severe limitations to the HCV research field by precluding molecular studies aimed at understanding HCV virology. The recent development and characterization of infectious molecular clones of HCV (see below) now provide a starting base for the development of in vitro culture systems for HCV. A recent highlight in this arena has been the successful development of a subgenomic HCV replicon RNA capable of autonomous replication in human liver cell lines (Lohmann et al. 1999). This incomplete HCV genome is essentially a bicistronic construct in which the HCV 5' UTR and internal ribosome entry site direct the synthesis of a neomycin resistance gene for stable selection in cell lines. This construct is followed by the viral nonstructural proteins and 3' UTR placed under the control of a heterologous ribosome entry site from the murine encephalomyocarditis virus. The HCV replicon system represents a major breakthrough in our ability to study HCV replication in vitro. However, the lack of viral structural proteins precludes the study of virion propagation and assembly within this replicon system. In contrast to the subgenomic HCV replicon system, attempts to generate and propagate an infection in cell culture by introducing synthetic full-length genomic RNA from an infectious HCV clone into susceptible cells has met little success, even though a respective clone may confer infection in vivo. The reasons for this discrepancy are not clear, but they highlight the continued importance of animal models for HCV research.

Development of Infectious Molecular Clones of HCV

As a positive-strand RNA virus, the HCV genome serves directly as the mRNA for synthesis of the HCV polyprotein. This relation is advantageous from the molecular virology perspective because transcripts from a synthetic cDNA, derived from the viral RNA, will confer infection when introduced into a permissive cell. An important highlight in last 3 yr of HCV research has been the development and characterization of infectious molecular clones of HCV. In these studies, distinct cDNA clones derived from HCV genomic RNA were produced from infectious serum. The resulting cDNA was cloned into a plasmid downstream from a bacteriophage T7 promoter and just upstream from a transcription runoff site conferred by a unique restriction enzyme. In each case, cleavage of the plasmid DNA by the restriction enzyme at the runoff site produces a linear DNA construct that terminates at the authentic 3' terminus encoded by the HCV RNA. As shown in Figure 1, generation of synthetic transcripts in vitro by T7 RNA polymerase results in a representative HCV genomic RNA. Several studies have now demonstrated the infectious potential of the HCV molecular clones using the chimpanzee as a host. Importantly, the infectious HCV clones now provide the molecular tools to examine the salient features of HCV genomic structure on viral replication and disease.

Infectious Molecular HCV Clones and the Chimpanzee Model for HCV Research.

Construction of Genotype-specific Infectious Clones

HCV has been classified into six different genotypes based on nucleotide sequence homology scores of viral isolates (Bukh et al. 1995; Simmonds 1998). The spectrum of disease associated with each viral genotype varies. However, several studies have identified a significant correlation between infection with HCV genotype 1 and severity of disease, including the development of persistent infection and chronic hepatitis (Amoroso et al. 1998; Bruno et al. 1997). To begin to understand the mechanisms contributing to the apparent genotypic differences in disease severity, it is of interest to examine the disease profile associated with experimental infection of the chimpanzee by HCV genotype-specific clones.

Infectious HCV molecular clones of genotypes 1A, 1B, and 2A have so far been constructed and characterized. Although all of the genotype 1 clones have been derived from the well-characterized Hutchinson H77 isolate of HCV (Feinstone et al. 1981), it is important to note that they are not identical, but differ slightly in aa composition of the encoded polyprotein. Initial attempts to infect chimpanzees with transcripts derived from cloned isolates of H77 HCV proved unsuccessful. Only after extensive nucleotide sequence analysis of several noninfectious genomic clones was it determined that nucleotide substitutions and deletions, which likely occurred during the cloning process, affected the viability of each clone in vivo. Therefore, the construction of the infectious HCV clones followed a tedious process of nucleotide sequence analysis of several HCV isolates to define a genotype "consensus sequence." In each case, nonconsensus sequences of the cloned HCV genome were identified and repaired to the consensus sequence using various recombinant DNA techniques. Chimeric genomic clones of HCV have also been constructed and characterized, including a clone encoding the HCV 1B polyprotein flanked by the 5' and 3' UTR from a cloned HCV 1A H77 genome (Yanagi et al. 1997, 1998). In other studies, this group constructed a series of genotype 1A-2A chimeras in which the majority of the 5' UTR, entire NS region, and 3' UTR were derived from HCV genotype 1A, while the remaining sequences encoding the structural proteins and 5' UTR were derived from HCV genotype 2A (Yanagi et al. 1999a).

In Vivo Characterization of the HCV Infectious Clones

In their seminal studies defining the infectious potential of the cloned HCV 1A genome, Rice and colleagues prepared 10 full-length consensus clones from the H77 HCV isolate, and each differed in the presence of additional 3' nt, having a poly U sequence of either 75 or 133 nt in length (Kolykhalov et al. 1997). Full-length RNA was transcribed and inoculated into two healthy chimpanzees by direct intrahepatic injections. Over the initial 13-wk monitoring period, both animals became viremic and exhibited significant elevations in serum ALT levels, consistent with the development of hepatitis (summarized in Table 1). Both animals developed a specific humoral immune response to HCV within 13 wk after inoculation. Histological examination of liver biopsies prepared from the infected animals revealed classical signs of hepatic disease, including the presence of multinucleated hepatocytes, cytoplasmic inclusions, and developing steatosis. Collectively, these results demonstrated for the first time that (1) HCV is sufficient to cause hepatitis, and (2) the cloned HCV genome can successfully transmit infection in a chimpanzee. Moreover, nucleotide sequence analysis of cloned viral RNA recovered from the infected animals revealed an in vivo preference for viral sequences encoding the longer 133 nt poly U region. This finding suggests that the sequence composition and length of the viral 3' UTR may affect HCV replication in vivo.

Two other HCV genotype 1A infectious clones have now been described, and each represents an isogenic variant of the H77 inoculum. Bukh and colleagues constructed a consensus HCV 1A clone, termed pCV-H77-C (Yanagi et al. 1997). Initial studies demonstrated that synthetic full-length transcripts generated from this clone could cause infection when introduced directly into the liver of a chimpanzee. A second study using the pCV-H77-C clone again demonstrated the infectious nature of the clone-derived HCV RNA (Yanagi et al. 1999a). Here, the investigators directly compared this clone with the infectivity of a molecular clone of an HCV 2A genome. Direct inoculation of full-length HCV 2A transcripts into the liver of a chimpanzee resulted in viremia over an 8-wk period, with normal serum ALT levels (Table 1). Thereafter the infection was apparently resolved. Previous inoculation of the same animal with RNA derived from HCV 1A/2A chimeric clones failed to produce infection. This lack of in vivo viability of the chimeric clones suggests that genotype-specific interactions encoded within the HCV genome are critical for supporting viral replication. The same animal was subsequently superinfected with RNA derived from the pCV-H77-C clone. Superinfection resulted in sustained levels of HCV 1A RNA in the serum and eventual seroconversion, demonstrating that the chimpanzee was susceptible to HCV infection. However, no indication of hepatitis induced by the HCV 1A clone was demonstrated. Taken together, these studies demonstrate (1) a remarkable contrast in the ability of the two HCV genotypes to establish persistent infection, and (2) evidence that the outcome of monoclonal HCV infection can include an asymptomatic "silent" disease. The latter observation is notable in that infection of chimpanzees with NANBH serum stocks has also demonstrated induction of biochemically and histologically silent disease even in the presence of persistent viremia (Bradley 2000; Prince and Brotman 1994). Importantly, this characteristic mirrors the clinical picture in a significant proportion of the HCV-infected human population (Esteban et al. 1998).

With the exception of the genotype 2A clone, all of the studies described above have utilized the HCV 1A, H77 inoculum, as the source of viral sequences for construction of infectious molecular clones of HCV. In a departure from these studies, two representative genotype 1B clones have now been described (Beard et al. 1999; Yanagi et al. 1998). Importantly, this genotype comprises a significant number of HCV-infected individuals, including a predominance of 1B infections within the HCV-positive population of Europe and Japan (Alter 1997; Mansell and Locarnini 1995). Moreover, it has been suggested that infection with HCV 1B may confer a more severe liver pathology than the other HCV genotypes (Esteban et al. 1998). These characteristics of HCV 1B infection can now be assessed from the molecular level using the infectious HCV 1B clones. Three chimeric HCV clones, differing by 7 to 9 aa overall, were constructed that encode the HCV 1B polyprotein derived from a Japanese serum isolate (HC-J4/91 [Okamoto et al. 1992] flanked by 5' and 3' UTR sequences derived from the HCV 1A H77 inoculum [Yanagi et al. 1998]). Thus, although replication of each resulting virus is dependent on the encoded HCV 1B polyprotein products, the HCV 1A sequences must confer the signals for protein translation and genome replication. Direct liver inoculation of pooled RNA derived from three clones resulted in accumulation of viral titers and sustained viremia in a chimpanzee over an 18-wk period. Waning viremia returned between weeks 21-24, indicating persistent infection. However, no biochemical signs of hepatitis developed over the study period. Nucleotide analysis of RNA isolated during acute infection revealed that only a single clone (pCV-J4L6S) was viable in vivo, even though all three clones encoded the same 5' and 3' UTR sequences. However, each nonviable clone had at least one mutation in highly conserved positions within the E1 or E2 glycoprotein sequences, which may have affected viability in vivo. In contrast to the nonviable phenotype of the intertypic HCV 1A/2A chimeric clone (Yanagi et al. 1999a), it appears that the sequence conservation within HCV genotypes sufficiently tolerates intratypic chimeric sequences to support HCV replication in vivo. However, it is possible that the presence of chimeric sequences may affect the natural history and even confound the characterization of HCV 1B-associated disease in the chimpanzee.

To avoid the potential limitations associated with chimeric clones, Beard and colleagues (1999) constructed a full-length HCV 1B clone with authentic 5' and 3' UTR sequences. This clone, pHCV-N, was derived from viral sequences originally cloned from a Japanese patient infected with the N strain of HCV 1B. Comparison of the pHCV-N and pCV-J4L6S sequences revealed extensive polymorphism between the polyprotein encoded by each clone, with minor nucleotide sequence divergence found within the 5' and 3' UTR sequences. Overall, the polyprotein encoded by pHCV-N possessed aa differences at 280 sites, including a unique insert of 4 aa within the NS5A coding region. However, in contrast to the minimal 7 to 9 aa polymorphism that attenuated the viability of pCV-J4L6S-related clones (Yanagi et al. 1998), this extensive polymorphism did not affect the viability of the encoded virus; transcripts from pHCV-N conferred infection when inoculated into the liver of a seronegative chimpanzee. The inoculated animal became viremic and virus persisted in the serum throughout a 20-wk study period (Table 1). By week 13, there was a very strong seroconversion, and viremia became intermittent. No biochemical or histological evidence of liver disease was observed. Together these results demonstrate that RNA synthesized from an authentic HCV 1B molecular clone can mediate persistent infection in a chimpanzee. Because clone viability was maintained in the presence of extensive sequence divergence within the encoded polyprotein, the HCV-N genome most likely represents a natural HCV 1B variant, rather than an extensive series of mutations introduced during the cloning process.

Transmissible Virions Can Be Propagated from an Infectious HCV Molecular Clone

The studies described above demonstrate that RNA from a cloned HCV genome is capable of producing viable and replicating virus when inoculated into the liver of a chimpanzee. In addition, overall at least two studies have verified that cloned HCV RNA is sufficient to cause hepatitis when introduced into the chimpanzee (Table 1). It is likely that the silent infections observed in other studies using molecular HCV clones will become symptomatic in future long-term follow-up studies. A key question for future HCV transmission studies is whether the molecular clones can generate transmissible infectious virions. To answer this question, Lau and coworkers constructed a consensus HCV 1A clone (pET/T7/HCV-H) derived from previously cloned sequences of the H77 isolate (Hong et al. 1999). RNA transcribed from this clone was infectious when introduced directly into the liver of a chimpanzee. The animal became persistently infected with HCV, as fluctuating viremia was observed over an 11-mo study period during which time the animal seroconverted to anti-HCV positive. A bimodal rise in serum ALT levels correlated with acute and chronic infection. To verify that the RNA from clone pET/T7/HCV-H could generate transmissible virions, the serum from this first chimpanzee was injected intravenously into a second chimpanzee. The second animal became infected within 1 wk of receiving the serum and remained viremic for 11 wk of the 14-wk study period. An increase in the titer of circulating viral RNA accompanied a significant rise in serum ALT levels. Seroconversion to anti-HCV positive at week 9 was followed by a drop in viral RNA titers, but viremia soon returned, indicating that this animal was persistently infected with the clone-derived, passaged HCV. Examination of liver biopsies taken from both animals in this study revealed histological evidence of mild hepatitis (Hong et al. 1999).

The results described above demonstrate that (1) RNA derived from HCV 1A clone, pET/T7/HCV-H, is infectious and can induce disease in a chimpanzee; and (2) the resulting virions produced from this clone are likewise infectious and transmissible. It will now be of interest to conduct transmission studies to follow the genetic and pathologic evolution of the infectious HCV clones in the chimpanzee, and viral genotype differences, if any, in disease severity. The latter is of particular interest because studies of HCV-infected humans have correlated HCV genotype with disease severity (Amoroso et al. 1998; Bruno et al. 1997). Such genotypic differences can be elucidated only through multiple long-term transmission studies of chimpanzees infected with genotype-specific HCV molecular clones. These studies may provide clues into the molecular mechanisms that differentiate an acute, self-limiting infection from the development of persistent infection and chronic disease.

Follow-up Studies and Structure-Function Analyses

The successful construction and characterization of viable infectious HCV molecular clones can now be followed by long-term observation of clone-infected chimpanzees. Moreover, new studies to examine the structure-function relation of the HCV genome can now be initiated. Major and coworkers have reported follow-up observations of the first two chimpanzees to be infected with a molecular clone of HCV (Major and Feinstone 2000; Major et al. 1999). Although no additional signs of disease were observed beyond those previously reported (Kolykhalov et al. 1997), the animals remained persistently infected throughout the 60-wk study period, even in the presence of a constitutive antibody response. Analysis of the specific antibody response to the envelope glycoproteins revealed that both animals developed only very low amounts of E1/E2-specific antibody soon after infection.

Thus, it is likely that an insufficient neutralizing antibody response resulted in failure to clear the virus during acute infection. Sequence analysis of the HVR1 region of the E2 protein revealed that this element remained remarkably stable, with only one aa substitution present within the dominant HVR1 sequence of one animal. Sequencing of the entire HCV genome from each animal at week 60 revealed the presence of several aa substitutions within the dominant sequence and demonstrated a nucleotide substitution rate of 1.48-1.57 × 10-3 substitutions/site/year (Major et al. 1999). Nucleotide substitutions clustered to within the E1, E2, NS2, NS3, and NS5 regions of the genome. In the absence of significant HVR1 evolution, it is possible that one or more of these mutations could represent an immuno-escape variant. Collectively, these observations suggest that HCV genome evolution in regions outside of the HVR1 may facilitate persistent infection. Additional studies to address the functional role of independent mutations will help define the mechanisms of HCV persistence.

Structure-function analyses of the infectious HCV molecular clones are now being conducted. In an effort to understand the role of the viral 3' UTR sequences in HCV replication, Bukh and colleagues conducted studies to map the critical regions of the 3' UTR that were required for infection (Yanagi et al. 1999b). A series of deletions were made within the 3' UTR of the HCV 1A genome encoded in clone pCV-H77-C (Yanagi et al. 1997), including deletions within the 3' terminal 98 nt conserved region, the poly U-U/C region, and the variable region (see Figure 1). Serial inoculation of a chimpanzee with RNA from this series of viral mutants demonstrated that the poly U-U/C and 98 nt conserved regions of the viral 3' UTR were essential for HCV viability in vivo, whereas the variable region was not. These results may reflect the putative roles of poly U-U/C and 98 nt conserved regions in binding to cellular factors to facilitate RNA stability and translation and functioning as a replication signal for the virus-encoded polymerase, respectively (Reed and Rice 1998).

Additional functional analyses of the HCV genome have demonstrated essential roles for the enzymatic activities encoded by HCV. Rice and coworkers constructed mutations that inactivated the activities of the NS2/3 protease, NS3 serine protease/helicase, and the NS5B viral polymerase in the p90 clone of HCV 1A (Kolykhalov et al. 2000). Two other clones were constructed with a deletion of the entire 98 nt conserved region or deletion of the invariable region after the poly U-U/C sequence. RNA derived from these mutant clones was inoculated concurrently into a chimpanzee to assess clone viability in vivo. None of the clones replicated to detectable levels, and the animal remained seronegative over the 8-mo study period. Inoculation with RNA from the wild type clone resulted in HCV infection. Taken together, these studies demonstrate that the enzymatic activities encoded by HCV are essential for viral replication in vivo. Moreover, this work confirms an essential role for the conserved 98 nucleotide and invariant regions of the viral 3' UTR sequence for HCV replication. This study affirms that the enzymatic activities and 3' UTR of HCV represent potentially attractive targets for the therapeutic intervention of HCV replication in vivo.

Alternative Nonhuman Primate Models for HCV Research

The chimpanzee model for HCV infection has moved from the early role of characterizing NANBH disease into a new role of characterizing the structure-function relation of infectious HCV molecular clones. This new role will certainly create a bottleneck for the HCV research community as more and more chimpanzees will be required to conduct the many studies needed to fully understand the role of HCV sequences in replication and disease. Are there other animal models that may sufficiently fit the need for future HCV transmission studies? One group has assessed the replication of HCV in tree shrews (Tupaia belangeri chinensis) (Xie et al. 1998). Wild-caught tree shrews were inoculated intravenously with infectious serum containing HCV 1A, or a pool of HCV 1A, HCV 1B, and HCV 3. Animals receiving the pooled virus source were either inoculated directly, or first exposed to whole-body X-ray irradiation. Approximately one of three animals inoculated with HCV 1A became viremic and seroconverted to anti-HCV positive. Of those animals that received the pooled virus source, only 20% of the directly inoculated animals became viremic, and two of four that first received X-ray exposure became viremic. Viremic animals exhibited transient elevations in serum ALT levels, consistent with HCV-induced hepatitis. These studies should be regarded with caution, as the pathophysiology of liver disease and overall responses to stress have not been characterized in T. belangeri chinensis. Further characterization of this species, including the response of these animals to captivity, is required to ascertain their utility as an alternative nonhuman primate model for studies of HCV infection. Besides the chimpanzee, other nonhuman primate species do not appear to be susceptible to HCV infection (Abe et al. 1993; Garson et al. 1997). Thus, the tree shrew may eventually represent an attractive alternative to the chimpanzee for in vivo studies of HCV.

Transgenic Mouse Models of HCV Pathogenesis

The chimpanzee model has certainly proved invaluable for understanding HCV-related disease and characterization of infectious molecular clones of HCV. However, the HCV-infected chimpanzee rarely develops chronic liver disease to the extent seen in HCV-infected humans, making the chimpanzee an inadequate model for studying the mechanisms of HCV pathogenesis. The molecular mechanisms responsible for HCV-related liver pathology are poorly understood, but pathogenesis is generally believed to be a consequence of a potent cell-mediated immune response to the HCV-infected hepatocyte. A number of HCV proteins have now been implicated in abrogating cellular processes in vitro that may be contributing factors in the development of liver disease and hepatocellular carcinoma (HCC¹) in vivo. To examine the effect of HCV protein expression on hepatocyte physiology within the in vivo milieu of the liver, a number of groups have now established transgenic mice that express HCV proteins from tissue-specific promoters (e.g., Feitelson and Larkin 2001). Such mice have already proved to be useful models for characterizing pathogenesis due to HCV protein expression. The major findings from these transgenic animal studies are summarized below.

HCV Transgene Expression

The majority of HCV transgenic mice developed to date have focused on expression of the viral structural proteins, core/E1/E2, and examination of the effect that expression of these proteins has on liver pathology. These proteins have been expressed either collectively (Kato et al. 1996; Kawamura et al. 1997; Koike et al. 1995, 1997; Wakita et al. 1998) or individually (Moriya et al. 1997a,b, 1998; Pasquinelli et al. 1997) under the transcriptional control of either liver-specific or generic promoter elements (Table 2). In most of these studies, HCV protein expression in the liver is relatively abundant, in possible contrast to some of the findings from studies of HCV-infected individuals. In these transgenic animals, HCV proteins are readily detected by immunoblot and visualized by immunohistochemistry, whereas HCV transgene mRNA can be detected by Northern hybridization. This is not always the case in liver biopsies from most patients with chronic hepatitis C in which HCV proteins can be difficult to identify, and HCV RNA can usually only be detected by RT-PCR. Thus, the overexpression of HCV proteins in transgenic mice and any pathological phenotype should perhaps be interpreted with caution. What is evident from these transgenic mouse studies is that the HCV core, E1 and E2 proteins, are located within the cytoplasm, consistent with findings from in vitro cell culture models. However, some reports show the presence of HCV core protein in the nucleus by immunohistochemistry, subcellular fractionation, and immunoelectron microscopy (Kawamura et al. 1997; Moriya et al. 1997a, 1998), supporting the idea that the core may have transcriptional regulatory properties. It was also demonstrated using immunohistochemistry that E1 was present in the nucleus of some hepatocytes (Koike et al. 1995). The significance of these observations is unknown and may be a consequence of high expression levels in these mice. Moreover, expression of the envelope proteins, E1 and E2, individually or in combination with the core protein results in expression of E1 and E2, which are correctly processed and glycosylated (Koike et al. 1995, 1997; Pasquinelli et al. 1997). In addition, immunoprecipitation experiments revealed that the HCV E1 and E2 proteins associate with each other in vivo when expressed in transgenic mice (Koike et al. 1995).

Consistent with expression of HCV proteins from birth, conventional transgenic mice are immunotolerant to these proteins. This immunotolerance makes it difficult to study HCV-related immune-mediated mechanisms of liver injury in transgenic animals. To overcome this problem, transgenic mice have been produced that conditionally express core/E1/E2 using the Cre/loxP system, producing a mouse that is immunocompetent for the transgene product (Wakita et al. 1998). A full description of the Cre/loxP system is beyond the scope of this review; however, it has been used successfully to alter gene expression in the liver of transgenic mice (Wang et al. 1996). Briefly, core/E1/E2 expression is induced when the chicken b -actin promoter with the cytomegalovirus immediate to early enhancer (CAG1) promoter is placed in-frame with the core/E1/E2 ORF by the removal of an intervening sequence through the action by Cre recombinase, delivered to the liver by recombinant adenovirus. Cre recombinase catalyses DNA cleavage between two loxP sequences resulting in positioning of the CAG promoter in the correct context with the core/E1/E2 ORF. Once induced, these mice expressed core/E1/E2 in the majority of hepatocytes with no evidence of lymphocytic infiltrate or liver pathology, although they were followed for only 1 wk (Wakita et al. 1998). Clearly, these mice must be followed for longer periods before firm conclusions can be made regarding the nature of immune-mediated liver injury induced by HCV protein expression.

Development of Steatosis

We have developed transgenic mice that constitutively express low levels of the complete HCV polyprotein in a liver-specific fashion, under the transcriptional control of the albumin promoter (Lerat et al. 2000). In addition, we have produced mice in the same genetic background that express the HCV structural proteins only. Both lines of mice develop primarily microvesicular or mixed micro- and macrovesicular liver steatosis with a pericentral predominance in animals greater than 3 mo of age that progresses with time and is predominantly male-gender specific (Figure 2) (S. Lemon, University of Texas Medical Branch at Galveston, personal communication, 2000). These results are similar to those of Moriya and colleagues (1998) who showed transgenic mice expressing the HCV core protein alone develop steatosis. However, we note that our transgenic mice, which, in addition to core, express all the HCV proteins to a low level commonly seen in HCV-infected human patients. Nonetheless, these data indicate that the core protein is most likely responsible for the steatosis, although other HCV proteins may contribute to this phenotype in transgenic animals. The exact mechanism by which core induces steatosis is not clear. It has been suggested, based on use of in vitro cell culture expression systems, that core physically associates with lipid-laden vesicles and that this alters normal lipid metabolism and leads to steatosis (Barba et al. 1997). It has also been suggested that core protein may associate with the mitochondria, and induce steatosis, through impairment of mitochondrial fatty acid oxidation (Moriya et al. 1998). Alternatively, transgenic expression of the HCV core protein may lead to the accumulation of free radicals, oxidative stress and lipid peroxidation.

Importantly, steatosis is a common finding in human chronic HCV infection, although it is largely unknown whether the occurrence is coincidental or directly related to HCV protein expression. These data provide strong evidence that expression of HCV proteins, particularly the viral core protein, plays a direct role in the development of steatosis. Steatosis described in transgenic mice appears similar in breadth and microvesicular distribution to the human form associated with chronic HCV infection (see Figure 2). Thus, these transgenic mice provide an excellent model to study the molecular mechanisms of steatosis associated with HCV infection in the human population.

It is interesting to note that other groups of investigators that describe transgenic mice expressing the core, E1 or E2 proteins, have found no evidence of steatosis, even when these animals have been followed for up to 2 yr (Honda et al 1999; Kawamura et al. 1997; Koike et al. 1995, 1997; Pasquinelli et al. 1997). The reasons for these differences are unknown; however, they cannot be explained by insufficient levels of protein expression or differences in HCV genotype. Importantly, the differences may be related to the different genetic backgrounds on which the various transgenic models were produced. In this respect, mice that develop steatosis were produced on a C57BL/6 background otherwise exhibit a low rate of spontaneous steatosis. These findings may suggest that HCV proteins can function as cofactors in the development of steatosis and that host genetics may play a role in susceptibility to steatosis associated with HCV infection and viral protein expression.

Liver pathology has been described in only one other transgenic mouse study. Liver-specific transgenic expression of the HCV structural proteins resulted in infiltration of lymphocytes, hepatocyte necrosis, degeneration and hepatocellular-altered foci in mice after 6 mo of age (Honda et al. 1999). The hepatocytes in these animals showed a higher sensitivity level to liver injury mediated by apoptosis, an observation also seen within in vitro-cultured human hepatoma cells expressing HCV core. Thus, expression of the HCV structural proteins may augment the apoptosis signal transduction pathway in hepatocytes. However, these studies should be interpreted with caution, because relatively large amounts of HCV core protein were expressed in the transgenic animals and results were obtained from only one mouse line. The question remains whether insertational mutagenesis of the transgene construct could possibly play a role in the pathophysiology of this transgenic mouse model.

Hepatocellular Carcinoma

There is now considerable evidence from in vitro studies implicating HCV core protein as a modulator of gene expression from a variety of cellular promoters (reviewed by McLauchlan [2000]). Furthermore, core expression can transform rat embryo fibroblasts in combination with the cellular oncogenes c-myc and H-ras (Ray et al. 1996). This potential suggests that the HCV core could be a co-factor in regulating cellular proliferation and the development of HCC during HCV infection. In support of this hypothesis, there have been two reports in which HCC has developed in HCV transgenic mice (Lerat et al. 2000; Moriya et al. 1998). In their studies, Moriya and colleagues described the development of HCC in transgenic C57BL/6 mice expressing the core protein only. HCC occurred in approximately 30% of mice at 16 to 19 mo of age and was observed predominantly in males, an observation consistent with the epidemiological data that men chronically infected with HCV are more likely to develop HCC (Alter 1997). We also observed HCC in C57BL/6 transgenic mice expressing the HCV structural proteins and the complete HCV ORF (S. Lemon, personal communication, 2000). These animals showed similar pathology to that observed by Moriya et al. (1998), with the exception that HCC developed earlier and at approximately 13 mo of age (see Figure 2). Importantly, studies conducted by both groups demonstrated that HCC failed to develop in nontransgenic littermates. This failure is very consistent with the extremely low spontaneous incidence of hepatic tumors in the C57BL/6 mouse strain. In both studies, tumor histology was variable, with some lesions revealing highly differentiated hepatic cords with proliferating hepatocytes, while other lesions contained proliferating hepatocytes distorted by oversized lipid vesicles (Moriya et al. 1998). The focal lesions that were also observed occasionally could represent premalignant centers. In contrast, mice expressing the envelope proteins at high levels did not develop neoplastic lesions (Koike et al. 1995, 1997). Taken together, these studies strongly suggest that the intrahepatic expression of the core protein, and possibly in combination with other HCV proteins, significantly enhances the risk of cancer, even in the absence of an immunologically mediated inflammatory response to the infection. As seen in the development of steatosis, not all mice expressing the core protein develop HCC, a finding that again most likely relates to different genetic backgrounds on which the transgenic models were produced. What is clear is that steatosis was present in transgenic mice from both groups that develop HCC. The consistent correlation between steatosis and HCC in transgenic mouse models suggests that steatosis may be an early event in the development of HCC during persistent HCV infection.

Extrahepatic Pathology in HCV Transgenic Mice

HCV structural protein expression has been demonstrated in tissues other than the liver, including lung, intestine, kidney, testes, heart, thymus, spleen, and the salivary glands (Honda et al. 1999; Koike et al. 1995). Although no pathology was noted in the majority of these extrahepatic sites, transgenic expression of the E1 and E2 envelope proteins, under the transcriptional control of the hepatitis B virus regulatory elements, resulted in exocrinopathy in the salivary and lachrymal glands resembling Sjögren's syndrome (Koike et al. 1997), which has been associated with human HCV infection. Interestingly, this pathology correlated with E1/E2 expression levels. However, pathology was not evident within the livers of these transgenic mice. The transgenic mouse model should prove useful for characterizing extrahepatic sites of pathogenesis associated with persistent HCV infection.

Conclusion

As the cornerstone of HCV research, the chimpanzee has provided the model system for the initial characterization of the NANBH agent and, more recently, characterization of the various infectious HCV molecular clones. The successful development of infectious HCV molecular clones provides a means for scientists to study the structure-function relation of the HCV genome in great detail and in the context of a relevant animal model for HCV infection. For example, it will now be possible to follow the evolution of a monoclonal inoculum of HCV into distinct viral quasispecies and to study quasispecies "fitness" in response to selective pressures applied by the host immune system, novel vaccines, and antiviral therapies. It is important to continue long-term follow-up studies examining the in vivo behavior of wild type and mutant HCV genome constructs. Such studies will make it possible to define the role of specific aa residues and sequence motifs in HCV replication, persistence, and pathogenesis.

Development of infectious molecular clones of HCV has spawned a new era in HCV research. The development of new and alternative animal models for HCV research should become a priority because future studies will most certainly place huge demands on the limited number of chimpanzees available for study. This demand will become even more important as the HCV field pushes ahead into developing and testing vaccines and anti-HCV drug therapies. Further development and characterization of HCV transgenic mouse models and the tree shrew model of HCV infection are warranted, including assessing the susceptibility of the latter to infection by RNA derived from HCV molecular clones.

Transgenic mice expressing HCV proteins are only beginning to provide insights into the pathobiology of HCV infection. From the studies presented, it is clear that expression of HCV proteins per se is not directly cytopathic to hepatocytes, even when expression levels are greater than those observed in HCV-infected individuals. This evidence supports that notion that the host immune response to HCV may play a significant role in HCV pathogenesis. The use of conditional transgenic expression systems, such as the Cre/LoxP system, offers exciting prospects to study the immune-mediated mechanisms of HCV-related liver injury in transgenic mice. However, the recent development of mice immunocompetent for the HCV proteins using this system awaits full characterization. It is clear that in some transgenic mouse strains, expression of the HCV genome or the core protein alone results in the development of steatosis and HCC. How does the core protein induce these pathologies, and how may the other HCV proteins contribute to this phenotype? It is still unclear whether this phenotype is a result of HCV protein expression directly or whether it occurs in combination with environmental or host-derived factors leading to chronic liver cell injury. Importantly, steatosis and HCC are relevant clinical manifestations seen in HCV-infected individuals. These transgenic mice should therefore play a pivotal role in elucidating the molecular mechanisms of HCV-related liver pathology.

Acknowledgments

M.G. is supported by National Institutes of Health grant R01AI48235, State of Texas Applied Research Program grant 010019-0138, and by University of Texas Southwestern Medical Center Endowed Scholars program. M.B. is supported by National Institutes of Health grant U1940035 awarded to Stanlely M. Lemon. We also thank Steven Weinman, Michiari Okuda, Kim Loesch, Shu-Yuan Xiao, Hervé Lerat and Stanley M. Lemon, who have contributed to the characterization of transgenic mice described in this paper.

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¹Abbreviations used in this article: aa, amino acid; ALT, alanine aminotransferase; CAG, chicken b -actin promoter with the cytomegalovirus immediate-to-early enhancer; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HVR1, hypervariable region 1; ORF, open reading frame; NANBH, non-A, non-B hepatitis; NS, nonstructural; RT-PCR, reverse transcriptase-polymerase chain reaction; UTR, untranslated region.

Figure 1 Structural features of the hepatitis C virus (HCV) genome and the infectious molecular HCV clones inserted into a plasmid vector. The vector encodes a bacteriophage T7 promoter element just upstream of the HCV cDNA. The HCV cDNA encodes the entire HCV RNA genome. Digestion of the plasmid at a unique restriction site immediately following the 3' end of the HCV genome enables transcription of the entire full-length HCV RNA using T7 RNA polymerase. The structural elements within the HCV genome are indicated, including the internal ribosome entry site (IRES), the protein coding, the poly U/UC region of the 3' UTR (U/UC), and the conserved 98 nt region (98 nt). The positions of each of the individual HCV polyprotein cleavage products are indicated.

Figure 2 Liver histology in hepatitis C virus (HCV) transgenic mice expressing the complete HCV open reading frame. A, liver section from a transgenic male at 3 mo of age showing normal hepatic architecture. B, section prepared from a transgenic male at 10 mo of age, showing microvesicular steatosis. C, section of a tumor nodule prepared from a transgenic male 13 mo of age, showing compression of adjacent normal liver tissue. D, section prepared from a well-differentiated hepatocellular carcinoma from a transgenic male animal 13 mo of age. All sections were fixed and stained with hematoxylin and eosin.

Table 1 In vivo characteristics of infectious hepatitis C virus (HCV) molecular clones

^aDefined as an abnormal increase in serum alanine aminotransferase levels and/or abnormal liver histology.
^bND: not determined, UTR, untranslated region.
^cThis clone consisted of the HCV 1B polyprotein coding region flanked by HCV 1A 5' and 3' UTR sequences.

Table 2 Characteristics of hepatitis C virus transgenic mice

^aCAG, chicken b-actin promoter with the cytomegalovirus immediate-to-early enhancer; HCC, hepatocellular carcinoma; MUP, mouse urinary promoter.