Hepatitis B viruses and hepatocellular carcinoma.

HEPATITIS B VIRUSES AND HEPATOCELLULAR CARCINOMA Marie Annick Buendia Departement des RBtrovirus, Unit6 de Recombinaison et Expression Gbnbtique, INSERM U163, lnstitut Pasteur, 75724 Paris Cedex 15, France

I. Introduction

11. Epidemiology: Clinical and Immunological Aspects

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IV.

V. VI.

VII. VIII.

A. Prevalence of HBV Infections and Modes of Transmission B. Progression to Chronicity C. Epidemiologic Association of HBV with Hepatocellular Carcinoma Pathogenicity of Hepadnaviruses: Striking Similarities and Obvious Differences A. Mammalian Hepadnaviruses B. Avian Hepadnaviruses Hepadnavirus Genomes A. Genetic Organization of the HBV Genome B. Genome Structure and Replication C. Virion Assembly D. Regulated Expression of Viral Genes Potential Oncogenic Properties of Viral Proteins A. Surface Glycoproteins B. H B A Transcriptional Trans-activator Integrated State of Viral DNA in Chronic Infections and Hepatocellular Carcinoma A. Integrated Sequences: Physical and Functional Aspects B. Cellular Targets for Viral Integration in Human Hepatocellular Carcinoma C. Insertional Activation of inyc Family Genes in Woodchuck Hepatocellular Carcinoma Genetic Alterations in HBV-Related Hepatocellular Carcinoma Conclusions References

I. Introduction

Primary hepatocellular carcinoma (HCC), one of the most common cancers in many parts of the world, is also one of the rare human cancers showing seroepidemiologic association with a viral infection. The role of hepatitis B virus (HBV) as a causal agent of HCC has been clearly established, and the increased risk of developing HCC, estimated to be 100fold for chronic carriers of the virus as compared with noninfected individuals, places HBV in the first rank among known human carcinogens (Szmuness, 1978; Beasley and Hwang, 1991). Besides epidemiologic evidence, the existence of related animal viruses that form 167 ADVANCES IN CANCER RESEARCH. VOL. 59

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with HBV the hepadnavirus group, and induce acute and chronic infections of the liver and eventually HCC (reviewed by Schodel et al., 1989; Robinson, 1990), add substantial weight to theconcept that HBV stands among the few recognized human oncogenic viruses. A potential role of HBV as an insertional mutagen, suggested by the constant finding of integrated viral sequences in the cellular DNA of HBV-associated HCC, has been described in rare cases but appears now highly improbable in a majority of liver tumors (reviewed by Matsubara and Tokino, 1990). The major unsolved question is whether HBV acts through any already known oncogenic mechanism, either directly or indirectly. T h e problems encountered in hepatitis research are better understood by the long difficulty in identifying the viral agent for hepatitis B and in setting u p convenient experimental systems for genetic and biological studies. After the initial discovery of the “Australia” antigen by Blumberg et al. in 1965, and its later identification with an envelope determinant of the infectious agent for hepatitis B, hepatitis research has long been hampered by the strict host specificity of HBV, which infects only humans and chimpanzees, somewhat uneasy experimental models, and by the absence of tissue culture systems capable of carrying out the complete HBV life cycle. Molecular cloning and sequencing of the HBV genome (Galibert et al., 1979), and the concomitant discovery of animal hepadnaviruses (Summers et al., 1978; Mason et al., 1980; Marion et al., 1980), have represented a major breakthrough by allowing intensive studies of the viral genetic organization and replication pathway. During the last decade, the outcome of cell cultures supporting viral replication and the construction of mouse lines carrying viral transgenes have made it possible to better delineate the contribution of individual proteins to the replicative machinery, the regulation of viral gene expression, and the role of their protein products in liver pathogenesis. These advances, and parallel studies of HBV DNA integration patterns into host cell DNA, have led to a different hypothesis on the contribution of HBV to hepatocarcinogenesis. It has been generally admitted for a long time that HBV has no direct oncogenic or even cytopathic effect on the infected hepatocyte; indeed, viral hepatitis appears to be an immunologic disease. Malignant transformation, which occurs after a long period of chronic liver disease and which is frequently associated with cirrhosis, might be triggered in a nonspecific manner by the immune response against infected hepatocytes, which induces a chronic inflammation of the liver and causes cell killing and consequent cell proliferation-known risk factors for cancer (Ames, 1989). In a still indirect, but more specific pathway, persistent production of viral proteins with potential cytotoxic effects might modify endogenous metabolic processes

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and sensitize liver cells to endogenous or exogenous mutagens. Alternatively, the virus might play a direct role as a mutagen, through insertion of its DNA into host cell chromosomes, causing direct activation of protooncogenes or, in a roundabout way, secondary chromosomal aberrations. Integrated HBV sequences might also alter the host cell growth control through unregulated expression of native or modified viral proteins. Until1 recently, little attention has been paid to the underlying cellular pathway leading to malignancy in response to viral induction. To date, although information on risk factors causally linked to HCC has accumulated, the role of viral agents and carcinogenic cofactors is only partially elucidated. No unifying model, accounting for the contribution of viral and cellular factors to liver oncogenesis, has been proposed. In this context, our recent studies of the mechanisms linking woodchuck HCC with chronic infection by woodchuck hepatitis virus (WHV), a virus closely related to HBV, present an amazing contrast. In about 50% of the woodchuck tumors analyzed, we found activation of myc family genes (c-myc and N-myc) by nearby insertion of WHV DNA (Hsu et al., 1988; Fourel et al., 1990; Y. Wei, A. Ponzetto, and M. A. Buendia, unpublished results). This is the first case of a DNA virus producing insertional activation events at such a frequency. Although genetically related viruses showing similar pathobiological properties might be expected to develop common oncogenic strategies, insertional activation of myc protooncogenes by HBV DNA has never been observed in human liver cancer, in which other potentially oncogenic cellular targets for viral integration have been described only rarely (Dejean et al., 1986; Wang et al., 1990). Whether this striking difference is related to intrinsic properties of WHV, or to genetic or epigenetic variability between humans and rodents, remains to be determined. In addition, overexpression of myc genes, either through genetic alterations or by a still unknown trans-acting mechanism, has been observed frequently in liver tumors from woodchucks and ground squirrels infected with hepadnaviruses, identifying myc gene activation as a key step in the oncogenic process induced by hepatitis virus infection in rodents (Fourel et al., 1990; Moroy et al., 1986; Transy et al., 1992). In human HCCs, there is no published evidence for a predominant role of activated myc genes, albeit amplification of c-myc has been occasionally described (Trowbridge et al., 1988), suggesting a different transformation pathway. Rather than presenting an exhaustive survey of the recent advances in HBV-associated liver cancer research, this article will explore and compare different possible mechanisms by which hepadnaviruses may trigger liver cell proliferation and/or transformation, and considers the

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factors that may influence the primacy of some oncogenic pathways over others in tumors induced by different viruses of the same family. II. Epidemiology: Clinical and Immunological Aspects Epidemiologic research has been and remains a main contributor to our understanding of the etiology of HCC. In the late 1970s, it became evident that chronic HBV infection was by far the major risk factor of liver cancer (Szmuness, 1978). These conclusions have rapidly invigorated the search for the molecular mechanisms linking HBV and HCC, and point out the importance of vaccination against HBV infection as the appropriate strategy to prevent HCC. Other carcinogenic factors frequently associated with HBV infection, like exposure to dietary aflatoxins and excessive alcohol intake, have also been implicated in human hepatocarcinogenesis (Bosch and Munoz, 1991). More recently, preliminary epidemiologic data also support a correlation between chronic infection with a quite different virus, the human hepatitis C virus (HCV), cirrhosis, and HCC (Saito et al., 1990). Early studies of the prevalence of HBV chronic carriers have been greatly facilitated by the presence of large amounts of empty viral particles, carrying the viral surface antigen (HBsAg) in the serum of many infected patients, that provide a stable and easily detectable marker of chronic hepatitis. Other serological markers, like the soluble antigen e related to the capsid (HBeAg) and the viral-associated DNA polymerase, allow the distinction, in many cases, of those patients who support active viral replication. The introduction of HBV DNA into the panel of commonly used HBV markers has been an important step in the improvement of diagnosis assays. DNA hybridization techniques and polymerase chain reaction (PCR) amplification procedures allow a more accurate, and even semiquantitative estimation of the rate of viral replication (Brechot et al., 1981a; Bonino et al., 1981; L a r d et al., 1988; Gerken et al., 1991b); these assays have played an essential role in the detection of HBV infections unrecognized by conventional assays (Brechot et al., 1985; Liang et al., 1991b) and in the identification of HBV variants lacking HBs antigenicity or presenting precore region defects (Thiers et al., 1988; Carman et al., 1989; Wands et al., 1986). However, epidemiologists must face major difficulties: the absence of clinical symptoms during most primary infections with HBV (inapparent or subclinical disease is the rule in young children) and the limited number worldwide of cancer registries, which sometimes do not discriminate between primary and secundary liver neoplasms (Bosch and Munoz, 1991). Within these constraints, the epidemiologic associa-


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tion has brought compelling evidence that chronic HBV infection plays an important role in development of HCC. The modes of spread of the virus, which differ in endemic and nonendemic regions, and the factors that influence the establishment of persistent infections and modulate the relative risk of cancer development, have been studied in great detail (reviewed by Beasley, 1987; Hollinger, 1990a), providing a valuable basis for biological investigations of the oncogenic properties of HBV. A. PREVALENCE OF HBV INFECTIONS AND MODESOF TRANSMISSION Persistent HBV infections are widely spread over the world, but are unevenly distributed, with prevalence rates ranging from 10 to 20% in coastal regions in China, in Southeast Asia, and subsaharan Africa, to less than 0.5% in northwest Europe, North America, and Australia. It has been recently estimated that worldwide there are nearly 300 million actively infective carriers of HBV markers (Ayoola et al., 1988), which is even more than in a previous estimation by Szmuness in 1978. Since 1985, however, a slight, gradual decrease in the incidence of hepatitis B has been observed in high endemic areas as well as in intermediate- and low-prevalence regions (Hollinger, 1990b; Goudeau, 1990). It may be attributed to the current hepatitis B control strategies, and particularly to the availability of new HBV vaccines. The unusual stability of infectious HBV virions, present mainly in the blood but also in other body fluids like saliva, urine, and semen, renders hepatitis B highly contagious. Transmission of viral hepatitis B can be achieved in many different ways, and varies greatly between regions of high and low endemicity. In endemic areas, the viral infection is most often acquired early in life, and the maintenance of an HBV carrier population has been correlated with perinatal transmission from infected mothers to their offspring, and with contact-associated transmissions during the first years of life. In low-risk areas, where parenteral transmission and sexual contacts appear to be the predominant modes of spread, hepatitis B is mostly confined to teenagers and adults. This epidemiologic distinction is essential for the establishment of control measures aimed to limit the spread of the virus; it is also important to understand the variations observed in the relative risk to develop persistent infections and eventually HCC among different populations. In highly endemic areas in Asia and Africa, as well as in endemic pockets that occur in low-prevalence regions within specific subgroups or limited areas (for instance, oriental immigrants in the United States,

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native Alaskans, and populations of Mediterranean countries in southern Europe and the Middle East), the majority of HBV infections occur in early childhood. Extensive epidemiologic studies have delineated the relative importance of vertical and horizontal spread, but the precise modes of maternal-neonatal transmission remain incompletely understood. This is mainly due to the failure of the currently used immunological methods (i.e., detection of HBsAg and HBeAg) to allow a direct appraisal of the HBV status in potentially infected infants. The hypothesis of true vertical transmission of HBV, raised by the presence of infectious virions in the semen and the finding of integrated HBV sequences in spermatozoa of patients with active hepatitis (Hadchouel et al., 1985),is difficult to confirm in humans. Although the prevalence of maternal-fetus transmission is poorly documented, it is generally considered to be very low, whereas perinatal transmission, occurring during labor or delivery or during the first months after birth, might be the predominant mechanism of viral spread in certain areas of the world, particularly in Asia (Beasley et al., 1977; Merrill et al., 1972; Schweitzer et al., 1973). However, transplacental transmission has been described on rare occasions (Beasley and Hwang, 1984; Stevens et al., 1984). Horizontal transmission of hepatitis B is considered as relatively common among siblings in the same household, or through child-tochild contact within nurseries in endemic regions (Hollinger 1990a; Whittle et al., 1983). In Africa, horizontal transmission during the first years of life may account for the majority of hepatitis B cases (Ayoola, 1988). Horizontal plus perinatal transmission may also coexist in other populations (Machado et al., 1988). The reasons for the seroepidemiological differences between modes of spread in several ethnic groups are now apparent: they reflect differences in the HBV status among HBsAg carriers in these populations. Women who are chronic carriers of HBeAg, a marker of productive HBV infection, almost invariably transmit the virus to their offspring (Okada et al., 1976), but perinatal transmission among HBsAg- antiHBeAg-positive women is much less frequent; infants with a high transplacental anti-HBeAg titer at delivery are at lower risk to become HBV carriers (Chen et al., 1991). The protective effect of maternal antibodies is prolonged for about 6 months; thereafter, the children become susceptible to HBV infection (Ayoola, 1988). The importance of the HBeAg/anti-HBeAg status in transmission is further emphasized in the establishment of persistent infections, as discussed later. The introduction of HBV vaccination as part of routine programs, already decided by several organizations and governments, should prove to rapidly decrease the incidence of hepatitis B in endemic re-


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gions. In Western, industrialized countries, major steps toward prevention of parenteral and contact-associated transmission were made recently through improved sensitivity in the screening of blood donors for HBsAg and relatively successful vaccination programs within a number of high-risk target groups like health care workers and paramedics, hemophiliacs, and persons living in institutions for the developmentally disabled (Hollinger, 1990b; Goudeau, 1990). Screening of pregnant women for the presence of HBsAg, and vaccination'of newborns from carrier mothers, should also further reduce the low rates of perinatal transmission. Persons at greater risk for contracting hepatitis B are now parenteral drug abusers, male homosexuals, and sexually active heterosexuals and the development of persistent infections is observed only in about 5% of primarily infected, immunocompetent adults (Aldershvile et al., 1980; McMahon et al., 1985). The differences observed in the mode of HBV transmission and in the rate of persistent infections between endemic and nonendemic countries are illustrated in Fig. 1. B. PROGRESSION TO CHRONICITY T h e establishment of persistent HBV infections remains incompletely understood; different mechanisms appear to be involved, most of them being related to the immunologic status of the host at the time of infection or to genetic heterogeneity of HBV. Most infections acquired at birth lead to a chronic carrier state; the risk drops rapidly within the first years of life and the frequency of chronicity decreases with increasing age at the time of infection (Beasley and Hwang, 1984; Hollinger, 1990a). A clear inverse correlation has been observed between increasing age and the outcome of clinical signs of acute hepatitis (McMahon et al., 1985) (Fig. 1). Immunocompetent adults are much less prone to the development of chronic hepatitis than young children, whose immune system is still immature. In adult life, increased risk of progression to chronicity is associated with immune deficiencies, as seen in renal transplant recipients, in patients with prior human immunodeficiency virus (HIV) infection, and in leukemic patients treated with chemotherapy (Degos et al., 1988; Taylor et al., 1988; Melegari et al., 1991). Hepatitis B virus replication in liver cells is associated with the production of three distinct antigens (HBsAg, HBcAg, and HBeAg) that elicit both cell-mediated and humoral immune responses (reviewed by Milich, 1988; Schodel et al., 1990). Anti-HBsAg is the main neutralizing antibody and its appearance signifies termination of HBV infection. HBsAg is also a potential target for the immune attack of infected hepatocytes by cytotoxic T lymphocytes ( C T L s ) . Studies of transgenic mice

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Endemic countries v e r t i c a l transmission (Asia)

Nonendemic countries

contact-associated transmission (Africa)

parenteral and sexual transmission

$. newborns infants

r

young children

chronic c a r r i e r s ( 10-20%o f the t o t a l population)

40%

I I

I

teenagers adults

3

5-10%

chronic c a r r i e r s (0.5%of the t o t a l

1 I

25%

cirrhosis I

hepatocellular carcinoma

I

hepatocel l u l a r carcinoma

FIG. 1 . Comparative description of the modes of HBV transmission, the rate of progression to chronicity, and the HCC incidence in endemic and nonendemic countries.

expressing HBsAg under the control of HBV or albumin promoter indicate that immune tolerance and the absence of liver disease results from in utero exposure to HBsAg (Babinet et al., 1985; Moriyama et al., 1990). Cytolysis of HBsAg-positive hepatocytes is observed in one of these models after adoptive transfer of spleen cells from immunized

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H B V A N D HEPATOCELLULAR CARCINOMA

congenic mice, indicating that cell-mediated immune response directed against the s antigen can be implicated in the pathogenesis of liver cell injury in human hepatitis B (Moriyama et al., 1990). It has recently been shown that tolerant animals can be induced to make antibodies against HBsAg group and subtype epitopes when injected with large amounts of HBsAg particles (Mancini et al., 1991). In humans, it can be correlated with the efficient humoral response of neonates to HBsAg carrier mothers following HBsAg vaccination (Beasley and Hwang, 1984). The predominant role of the HBeAgIanti-HBeAg status of the infected host in the outcome of chronic infections is now firmly established. The viral e antigen, first described in 1972 by Magnius and Espmark, has been identified with a processed product of the precore-core region, which differs from the capsid protein (HBcAg) by 10 additional amino-terminal residues originating from the pre-C region, after cotranslational cleavage of a signal peptide, and by a deletion of 34 amino acids from the carboxy terminus (Takahashi et al., 1983; Uy et al., 1986; Standring et al., 1988; Weimer et al., 1987) (Fig. 2). The t w o proteins share common antigenic determinants, but present distinct antigenic properties (Milich et al., 1978; Salfeld et al., 1989). These structural changes are also associated with different biological properties: the capsid proteins are linked to the viral genome through their phosphorylated carboxy-terminal region, self-assembled into core particles, and

--

pre C mRNA C mRNA

stop

HBV DNA 1814

2450

1901

174aa

precore precursor ( P25 1

212 aa

processingat signal sequewa

precore derivatlve (PZ)

HBe Ag ( pis-17e)

193aa

4

proteolyticcleavage 149-159 aa

FIG.2. The synthesis of HBV core antigen (HBcAg) and e antigen (HBeAg) in infected hepatocytes is controlled by different regulatory signals and by different posttranslational modifications.

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incorporated into the envelope of secreted virions, whereas the e antigen is presented on the hepatocyte membrane and excreted as a dimer into the bloodstream (Ou et al., 1968; Jean-Jean et al., 1989; Schlicht and Schaller, 1989). T h e question arises of the biological function of HBeAg, which appears to be dispensable for viral replication in an animal model (C. Chang et al., 1987; Schlicht et al., 1987; Pugh et al., 1989). It has been shown that HBeAg/HBcAg is the major target of the cellmediated immune response and plays an important part in liver damage during acute and chronic acute hepatitis B, as well as in virus clearance (Mondelli et al., 1982; Schlicht et al., 1991). Usually, seroconversion to anti-HBe represents a crucial step in the course of the disease, and signals extensive elimination of the virus. However, persistence of antiHBe has also been observed in a subset of HBV carriers with chronic liver disease (Bonino et al., 1981; Hadziyannis et al., 1983), and has recently been correlated with a genomic variation of HBV that prevents the secretion of HBeAg (Brunetto et al., 1989; Carman et al., 1989; Tong et al., 1990). Infection with HBV precore mutants, already detected in different patients from many parts of the world, now appears to prevail among persistent carriers with anti-HBe, showing that genetic variations are used by HBV as a strategy to evade the immune system and persist in the infected host (Okamoto et al., 1990; Bonino et al., 1991).HBe-defective HBV causes a form of hepatitis with severe pathogenicity and even fulminant hepatitis, but the factors involved in liver damage are still unknown (Brunetto et al., 1991; Carman et al., 1991; Liang et al., 1991a; Omata et al., 1991). It seems probable that different mutations arising in the pre-C sequence during long-term chronic HBV infections confer selective advantage to variant HBV and lead to the emergence of antiHBe-positive diseases (Tran et al., 1991). The spread of e-negative HBV mutants might be limited, in endemic regions, by the low rates of transmission from anti-HBeAg-positive mothers. In contrast, virtually all infants born to HBe-positive mothers become chronic carriers, and many of them remain HBe positive all their lives. A tolerogenic role of the e antigen in the establishment of these persistent infections has been recently proposed (Milich et al., 1990). The hypothesis that HBe may cross the placenta and induce tolerance in utero is supported by experimental data obtained in HBe-expressing transgenic mice; it is consistent with human epidemiological and serological observations. Other general mechanisms allowing a virus to persist in a host (Oldstone, 1989) may also operate in chronic hepatitis B. Avoidance of immune surveillance is achieved by “capping” of HBcAg by anti-HBc antibodies on the surface of infected hepatocytes (Mondelli et al., 1982).

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Abrogation of lymphocyte/macrophage function may result from HBV infection of the immunocytes-HBV DNA and antigens have been found in bone marrow cells and in peripheral blood lymphocytes (Elfassi et al., 1984; Pontisso el al., 1984; Pasquinelli et al., 1986; Laure et al., 1987), from selective killing of HBsAg-specific B cells by class I-restricted CTLs (Barnaba et al., 1990), and from deficient production or blunted action of a-interferon (INF), which reduces HLA class I protein synthesis and prevents recognition of infected cells by specific CTLs (Ikeda et al., 1986; Onji et al., 1989; Twu et al., 1988). In addition, males are at higher risk than females of becoming chronic carriers (Szmuness et al., 1978). The importance of steroid hormones in the development of chronicity has been established in clinical assays showing that treatment of acute hepatitis by steroids increases the frequency of subsequent persistent infections (Blum et al., 1969). The role of steroids in enhancing viral gene expression has been demonstrated in HBsAg-expressing transgenic mice (Farza et al., 1987). Multiple, interrelated mechanisms allowing for viral persistence may therefore contribute to the progression to chronicity. Progress in understanding these mechanisms would allow us to improve the treatment of chronic hepatitis B, a major health problem owing to the gravity of its sequellae, cirrhosis and primary liver cancer. C. EPIDEMIOLOGIC ASSOCIATION OF HBV WITH HEPATOCELLULAR CARCINOMA Primary liver cancer, mainly HCC, ranks among the most frequent cancers of males in many countries. In a recent estimation (Bosch and Munoz, 1991), it represents the eighth most common cancer, with about 250,000 new cases each year, 70% of which occur in Asia. Several lines of evidence associate chronic HBV infection with the development of HCC. 1. T h e incidence of HCC and the prevalence of HBV serological markers follow the same general geographic pattern of distribution. Hepatocellular carcinoma is common in regions where HBV is endemic, but comes far behind other types of cancer in regions where HBV infection is uncommon (Szmuness, 1978; Tabor, 1991; Hollinger, 1990a). 2. Serologic evidence of HBV infection is detected in about 70% of HCC patients in Africa, and more than 90% in mainland China, as compared with 10 to 20% of the total population residing in the same areas (Tabor, 1991). 3. A marked increase risk of HCC has been shown among HBsAg carriers, compared with noncarriers [risk factors up to 200 have been

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reported in different ethnic or social groups, using different methodologies of investigation (Beasley et al., 1981; Hall et al., 1985; Chen et al., 1991; Obata et al., 1980; Beasley and Hwang, 1991)]. The long period of chronic HBV infection that generally precedes the onset of liver tumors has led to the proposal that “the oncogenic sword of HBV is terrible, but not swift” (Ganem, 1990). It might be added that it is unevenly hanging over carrier individuals. Hepatocellular carcinoma usually develops after a 20- to 50-year period of chronic carriage, but it may also affect, although unfrequently, HBsAg-positive children under 12 years of age. In contrast, a large number of HBsAg carriers remain anaware of their carrier status before they die at old ages (Beasley and Hwang, 1984; Chen et al., 1991; Lok et al., 1991).Epidemiological data have clearly shown that HBV is causally related with the development of HCC, but also that there is a great variation in HCC incidence among different carrier populations. This variation may be attributed both to differences in the intrinsic properties of HBV infection patterns observed among chronic carriers, and to additional genetic and environmental determinants. Chronic infections resulting from material-neonatal transmission present a greater risk of HCC than those acquired as adults (Beasley and Hwang, 1984; Popper et al., 1987a). In the Far East, early detection of HCC is now frequent in asymptomatic carriers. Among HBsAg carriers infected at an early age, additional HCC risk has been associated with HBeAg carriage, with significant liver damage and a high level of antiHBcAg antibodies in chronic active hepatitis, and with the presence of cirrhosis (Beasley and Hwang, 1984; Chen et al., 1991). A gender discrepancy (males incur a two- to eight-fold elevated risk of developing HCC than do females) and familial tendency (familial clusters of HCC are common in Asia) have also been documented as factors involved in the frequency of tumor development (Obayashi et al., 1972; Lok et al., 1991). In addition, inconsistent geographical variations in HCC mortality and HBsAg prevalence have been observed in endemic regions, suggesting that other independent or cooperative factors might be implicated. In highly endemic regions, particularly in South Africa and in southern provinces of mainland China, an association of dietary aflatoxins and primary HCC has been recognized in several reports (Bosch and Munoz, 1991; Harris, 1990; Kew, 1990). The carcinogenic potential of aflatoxin B1 in liver cells is well known in many species (Newberne and Butler, 1969). Excessive alcohol intake also increases the risk of HCC, in HBV carriers as well as in cirrhotic males of advanced age in regions of


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low prevalence for HBV (Austin, 1991; Chen et al., 1991). However, the coexistence of HBV infection, often undetectable by conventional serologic assays, and/or of HCV infection in more than 90% of patients from various countries, has called in question the prevalence of chronic hepatitis induced by alcohol (BrCchot et al., 1985; Takase et al., 1991). T h e potential role of cigarette smoking and of long-term use of oral contraceptives is still debated (Austin, 1991). In addition, preliminary data indicate that infection with the human hepatitis delta virus (HDV), which causes extremely severe hepatic injury and cirrhosis, might be associated with a more rapid onset of liver tumors (Oliveri et al., 1991). It is noticeable, however, that HBV infection, alone or associated with cooperative factors, is clearly implicated in only 20% of HCC cases in low-endemic regions (North America and Europe) and in Japan. In this country, the incidence of liver cancer has been continuously increasing during the past decade, but the number of cases related to HBV remains constant. It seems now probable that infection with hepatitis C virus (HCV), a human RNA virus related to the Flaviviridae and Pestiviridae, plays an increasing part in the development of HCC in these regions, as well as in countries highly endemic for HBV, such as in China. With the development of HCV markers, evidence is now increasing for an association between HCV infection, cirrhosis, and HCC (Okuda, 1991). Some differences have been noted between HBV- and HCV-associated tumorigenic processes: HCV-related HCC develops after a longer incubation period and frequently presents more benign histological features. T h e relationship between cirrhosis and HCC appears to be complex and the degree of correlation varies with the etiology of cirrhosis. Macronodular cirrhosis precedes or accompanies a majority of HBVassociated HCC (over 80% in Asia and 40-60% in Africa), in children as well as at older ages (M. H. Chang el al., 1988). T h e risk of HCC is much lower in HBsAg-negative micronodular cirrhosis observed in alcohol hepatitis and in HCV infections (Beasley and Hwang, 1984; Kew and Popper, 1984; Craig el al., 1991). Whether cirrhosis and carcinogenesis result from the action of common factors, or whether increased risk of HCC can be related to some mechanism like regenerative stimulation, which occurs in cirrhotic livers, remains an unsolved problem.

Ill. Pathogenicity of Hepadnaviruses: Striking Similarities and Obvious Differences As in many infectious diseases in humans, progress in the study of viral hepatitis has been critically dependent on the development of animal models, consisting either in experimental systems like HBV-inoculated

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chimpanzees and mouse strains carrying viral transgenes, or in naturally occurring models. Hepatitis B virus is the prototype member of the hepadnaviruses family, which includes a small number of enveloped DNA viruses with liver tropism and restricted host range (Summers, 1981). These viruses share a common genetic organization and replication pathway (as discussed in Section IV) and the ability to induce acute and persistent infections in their natural hosts. Animal hepadnaviruses have been isolated from rodents and birds and form two groups that have largely diverged during evolution; they will be considered separately. A. MAMMALIAN HEPADNAVIRUSES

Mammalian viruses include woodchuck hepatitis virus (WHV),which infects eastern woodchucks (Mamnota monax) in several states on the eastern coast in North America (Summers et al., 1978),and ground squirrel hepatitis virus (GSHV), a virus present in Beechey ground squirrels (Spermophilus beecheyi) in a limited area bordering the Stanford campus in California, Richardson squirrels (Spermophilus richarhonyii) at Picture Butte in Alberta (Canada), and possibly tree squirrels (Marion et al., 1980; Minuk et al., 1986; Feitelson et al., 1986). All these species belong to the Sciuridae family of rodents, and derived about 10 million years ago from a common ancestor marmotini. The divergence time between WHV and GSHV has been estimated as 10,000years, suggesting that the evolution of the hepadnavirus family was independent of host-species divergence (Orito et al., 1989).Ground squirrel hepatitis virus can infect other members of the Sciuridae family, as shown by experimental transmission of GSHV to woodchucks and chipmunks, but not to other rodent species (Seeger et al., 1978, 1991; Ganem et al., 1982a; Trueba et d., 1985; Marion et al., 1983; Chomel et al., 1984). Hepadnaviruses might also be associated with the development of liver tumors in related species (Snyder, 1979). Several WHV and GSHV isolates have been cloned and their nucleotide and amino acid sequences do not differ more than those of two different HBV subtypes (Cohen et al., 1988; Etiemble et al., 1986; Galibert et al., 1982; Ganem et al., 1982b; Girones et al., 1989; Kodama et al., 1985; Seeger et al., 1984b; Siddiqui et al., 1981). Their surface and core antigens are highly cross-reactive, and also cross-react with the corresponding HBV antigens (Feitelson et al., 1981, 1982; Cote and Gerin, 1983; Gerlich et al., 1980). Chimpanzees immunized with WHV surface antigen (WHsAg)can be protected after challenge with HBV (Cote et al., 1986). However, substantial variations have been noted in the pathological properties of WHV and GSHV, not only in their natural hosts, but


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also in a common host, the experimentally infected newborn woodchuck (Ganem et al., 1982a; Marion et al., 1983; Popper et al., 1981; Seeger et al., 1991; Snyder and Summers, 1980). The mode of spread of mammalian hepadnaviruses in the wild has not been firmly established. Vertical transmission from carrier dams to their offspring might prevail, as suggested by the presence of WHV DNA in adult woodchuck ovaries and testis, and in livers and sera of several woodchuck fetal litters (Korba et al., 1988b; Kulonen and Millman, 1988). Experimental infection of newborn woodchucks with WHV progresses almost invariably to chronicity, whereas most animals infected at older ages develop acute hepatitis and efficient immune response that leads to viral clearance (Popper et al., 198713). Chronic WHV infection of woodchucks usually results in mild portal hepatitis, in which limited portal inflammation is associated with minimal liver damage and the absence of hepatocyte necrosis and fibrosis, mimicking the human “healthy” carrier state (Snyder and Summers, 1980; Popper et al., 1981, 1987b; Toshkov et al., 1990; M. A. Buendia, unpublished observations). Extensive studies of the tissue tropism of WHV in chronically infected woodchucks have shown that active viral replication occurs mainly in the liver, with high levels (500-2000 genome units per cell) of replicative intermediates and viral RNA in hepatocytes (Korba et al., 1988b). Abundant viral replicative forms and RNA transcripts have also been observed in the spleen, whereas nonreplicating WHV DNA has been found in peripheral blood lymphocytes (PBLs) and replicative forms in scattered foci of cells within the pancreas, kidney, thymus, and transiently in ovary and testis (Korba et al., 1986, 1987, 1988b, 1989a, 1990; Ogston et al., 1989). Studies of the natural history of WHV infections have revealed that lymphoid cells of the bone marrow are first infected, followed by the liver, spleen, PBLs, lymph nodes, and thymus (Korba et al., 1989a, 1990). In addition, WHV DNA replication can be induced in PBLs on in zritro activation with a mitogen (Korba et al., 1988a). These findings, which can be related to the presence of HBV DNA in human bone marrow and PBLs, address the question of the mechanisms targeting the oncogenic potential of hepadnaviruses almost exclusively toward liver cells. T h e clinical and histological signs of acute and chronic viral infection are even less apparent in GSHV carrier ground squirrels (Ganem et al., 1982a; Marion et al., 1980, 1983; Seeger et al., 1984a). T h e animals appear to be relatively healthy in captivity and, on histological examination, their livers show very mild portal hepatitis characterized by periportal inflammation and proliferation of bile ductules without evidence of hepatocellular necrosis o r significant disruption of hepatic architec-

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ture. Young animals are highly susceptible to horizontal infection, but neither clinical nor pathological signs of acute hepatitis have been demonstrated, despite the presence of high levels of viral replicative forms in the liver (600-6000 genome units per cell) and of abundant viral particles in the serum. Most primary infections transmitted by the percutaneous route are self-limited, the rate of persistence being as low as for HBV infections in adults. At variance with WHV-infected woodchucks, GSHV DNA has been detected only in livers of chronically infected ground squirrels. Finally, virtually all WHV carrier woodchucks succumb to HCC after 2-4 years. A much lower risk (5.4%) has been established between the development of HCC and past WHV infection with seroconversion to anti-woodchuck hepatitus surface antigen (anti-WHs) (Korba et al., 1989b; Popper et al., 1987b). Like human HCC, woodchuck HCCs are primarily of the well-differenciated trabecular type and occasionally of the pseudoacinar type, but they differ in their inability to produce metastasis. At the time HCCs are detected, pericarcinomatous livers show moderate or acute hepatitis, inflammation, proliferation of bile ducts and connective tissues, necrosis, and degeneration of hepatocytes. Ground-glass cells have been occasionally observed. These clinical and histological features are highly similar to those associated with chronic hepatitis B in humans and can be classified using the same criteria as medical pathologists. Whether these signs of necroinflammation and acute hepatitis precede the tumor onset by a short period of time or result from compression of adjacent liver tissues by expanding tumor masses has not been determined. A similar question has been raised about the sequence of events leading to cirrhosis and HCC (often detected simultaneously) in asymptomatic human carriers (Beasley and Hwang, 1984). In addition, regenerative hepatocellular nodules and hepatocellular adenomas, which are believed to represent precursor lesions to carcinoma, are characteristically observed in WHV-infected woodchuck livers as during experimental carcinogenesis induced by chemicals in rats and mice (Abe et al., 1988; Roth et at., 1985; Popper et al., 1981). The frequency of tumor incidence in humans and rodents is generally correlated with the fractional life span in a similar manner. The average life span of captive healthy woodchucks, which never develop primary liver tumors, is about 15 years. The viral-induced neoplastic process appears therefore relatively more rapid in woodchucks than in humans, suggesting that WHV might be a more oncogenic virus than HBV. Moreover, HCC occurs as well in captive woodchucks, infected at birth and kept under strictly controlled conditions, ruling out the contribution of exogenous carcinogenic cofactors (Popper et al., 198713).


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In sharp contrast, chronic GSHV infection induces a delayed onset of liver tumors, with a much lower incidence in naturally infected ground squirrels as well as in experimentally infected woodchucks (Marion et al., 1986, 1987; Seeger et al., 1991). Hepatocellular carcinoma develops only in about 50% of GSHV carrier animals after a 4- to 8-year latency period. A similar tumor incidence has recently been reported in a study of Richardson squirrels infected with a virus closely related to GSHV (Tennant et al., 1991). Furthermore, the occurrence of liver tumors in convalescent ground squirrels after seroconversion, and in aging animals showing no marker of past or ongoing GSHV infection, indicates a weaker epidemiologic association between GSHV carriage and HCC than in the M7HV/woodchuck model. Within the same period of time, the risk of HCC development has been estimated to be threefold higher in WHV-infected woodchucks than in GSHV-infected ground squirrels, a rather surprising observation considering that woodchucks and squirrels are members of the same Sciuridae family, and that WHV and GSHV are closely related to each other. The importance of viral and host determinants in the striking differences observed between the two systems has recently been investigated and it has been established that GSHV and WHV differ in oncogenic determinants that can affect the kinetics of HCC development (Seeger et al., 1991). Determining the precise nature of these oncogenic factors should be of crucial importance, not only for understanding viral-induced carcinogenesis in rodents, but also to get some insight into the mechanisms linking HBV infection and HCC development in humans. A main difference between human and rodent hepatitis B resides in the absence of associated cirrhosis in woodchuck and squirrel livers, even after prolonged viral infection. In the rodent species, hyperplastic nodules might play a role as a precursor lesion during transition to HCC. These nodules are clonal and present morphological and metabolic aberrations resembling those observed during chemically induced hepatocarcinogenesis in rats (Rogler et al., 1987; Toshkov et al., 1990). It is important to notice that rodents in general are not prone to the development of cirrhosis; the rat model of liver fibrosis, which reproduces some features of the human cirrhotic process, can be obtained only after treatment with carbon tetrachloride or long-term feeding with a high-fat diet containing ethanol (Hall et al., 1991; McLean et al., 1969; Tsukamoto et al., 1986). Species-specific factors like the rapid onset of hepatocytic proliferation following liver damage in rodents, rather than intrinsic properties of the different hepadnaviruses, might therefore account for this discrepancy. Similarly, the presence of high levels of viral replicative intermediates in woodchuck and squirrel livers at the

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tumor onset, as compared with the marked decline of HBV markers in human patients with HCC, might reflect a more rapid development of HCC following initiation and promotion in rodents (Farber, 1984). I n conclusion, clinical and histological studies have shown that both WHV and GSHV represent potent, albeit different, animal models for liver diseases and hepatocarcinogenesis in humans. Advantage might be taken of the substantial variations in oncogenic properties between the mammalian hepadnaviruses to approach the molecular mechanisms connecting HBV and HCC.

B. AVIANHEPADNAVIRUSES Duck hepatitis virus (DHBV) was first discovered in brown domestic ducks residing in China and thereafter in flocks of Pekin ducks and in other wild duck populations from most parts of the world; more recently, a related virus was isolated in gray herons in Germany (heron hepatitis virus, HHBV) (Zhou, 1980; Mason et al., 1980; Sprengel et al., 1988). These viruses are more distantly related to HBV in their genetic organization than are mammalian hepadnaviruses, but they share with other members of the hepadna group similar replication mechanisms and biological properties, and represent therefore interesting models. Experimental systems using infection of ducklings with cloned viral DNA, in uitro infection of primary duck hepatocytes, and transfection with cloned DNA of different cell lines capable of producing infectious virions have been successfully developed to study the hepadnaviral life cycle, as discussed in Section IV (Tuttleman et al., 1986b; Sprengel et al., 1984; Galle et al., 1988). Vertical transmission in DHBV-infected ducklings has been demonstrated as the prevalent mode of natural transmission in this model (Urban et al., 1985). Duck hepatitis virus is transmitted from the viremic dam to the yolk sac, probably through passive transfer with liver-derived yolk proteins, and then to the developing embryos. Viral replication starts in embryonic livers at 6-8 days of incubation (Urban et al., 1985; Tagawa et al., 1987). Like mammalian hepadnaviruses, DHBV is mainly hepatotropic, but extrahepatic viral replication has also been observed, particularly in kidney and pancreas (Halpern et al., 1983). Generally, chronic DHBV infection is not associated with apparent liver disease. Most infected duck livers produce high levels of viral replicative intermediates and excrete large amounts of viral particles into the circulating blood, but show only very mild hepatitis, which does not usually evolve to carcinogenesis (Marion et al., 1984). Variations in the severity of liver lesions and intensity of viremia have been noted among wild mallard and domestic Pekin DHBV infections (Lambert et al., 1991). The devel-


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opment of cirrhosis and HCC has been observed only in a particular Chinese flock of DHBV-infected ducks, originating from the province of Qidong in mainland China (Zhou, 1980; Yokosuka et al., 1985; Imazeki et al., 1988), but not in Pekin ducks infected in Western countries. This difference might be related to genetic variations among DHBV strains and/or among susceptible hosts. Alternatively, HCC development might not be strictly correlated with DHBV infection but rather be due to exposure to dietary afatoxins (L. Cova, personal communication), also demonstrated in the human food in the same area (Sun and Chu, 1984). Introduction of aflatoxin B1 in the diet of DHBV-infected Pekin ducks kept in laboratory facilities has also proven to play a critical part in the incidence of duck HCC (Uchida et al., 1988; Cova et al., 1990). The study of viral and chemical factors contributing to HCC development in ducks might help to clarify some of the basic mechanisms of liver carcinogenesis. IV. Hepadnavirus Genomes

Since the earliest studies of the nucleotide sequence of cloned HBV genomes and of the viral replication by reverse transcription of an RNA intermediate (Galibert et al., 1979; Pasek et al., 1979; Summers and Mason, 1982), there has been constant interest in the unique genetic organization and replicative pathway of hepatitis B viruses. Virological and molecular studies have outlined the structural organization of the HBV genome, its coding potential, the mode of transcription of individual viral genes, and their functional capacities; the main aspects of viral DNA replication and virion assembly within infected hepatocytes have been unraveled. This article does not attempt to give an exhaustive review of the physical and biological properties of hepadnaviruses, as several reviews have recently appeared (Tiollais et al., 1985; Howard, 1986; Ganem and Varmus, 1987; Robinson et al., 1987; Chisari et al., 1989a; Mason and Taylor, 1989; Schodel et al., 1989; Robinson, 1990; Schroder and Zentgraf, 1990; Tiollais and Buendia, 1991). Less is known about the viral-cellular interractions that control virus attachment, uncoating, and entry into susceptible cells (the cell-surface receptor for HBV has not been identified with certainty), as well as the regulation of viral gene transcription and the integration of viral DNA in the host cell genome. OF THE HBV GENOME A. GENETIC ORGANIZATION

Nucleotide and deduced amino acid sequences of cloned HBV DNA from different virus subtypes (adw, adr, ayw, ayr) have revealed a genome

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size of 3.2 kb and the presence of four open reading frames, localized on one viral strand in the same transcriptional orientation (Galibert et al., 1979; Pasek et al., 1979; Valenzuela et al., 1980; Fujiyama et al., 1983; Ono et al., 1983; Kobayashi and Koike, 1984; Bichko et al., 1985; Okamoto et al., 1986; Vaudin et al., 1988; Loncarevic d al., 1990) (Fig. 3). Two of them, the C and S regions, specify structural proteins of the virion core and surface (or envelope); the longest one, P, encodes a

FIG. 3. Genetic organization of the HBV genome. Four open reading frames encoding seven peptides are indicated by large arrows. Regulatory sequences [promoters, enhancers, and glucocorticoid responsive element (GRE)] are marked. Only the two major transcripts (corelpregenome and S mRNAs) are represented. DRl and DR2 are two directly repeated sequences of 1 1 bp at the 5’ extremities of the minus- and plus-strand DNA.


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polyprotein necessary for viral replication that binds to DNA at its amino terminus, and presents DNA polymerase, reverse transcriptase, and RNase H activities; the smallest, X, codes for a transcriptional transactivator. T h e entire viral genome is coding, even in two different reading frames on a large portion of the genome. More striking than the overlapping of the P gene with the other viral genes, a feature common to all structures presenting reverse transcriptase activity (Toh et al., 1983), is the constant and unusual overlapping of coding and regulatory sequences (promoters, enhancers, and termination signal). The genomes of different HBV subtypes differ mainly by single nucleotide substitutions or by addition of multiples of three nucleotide blocks, preserving the reading frames and leading primarily to conservative amino acid changes. Genomic variability among viruses of the same subtype have been characterized in different patients and also in the same patient at different times during chronic infection; annual mutation rates for viral DNA have been estimated at 2.6 X mutations/site/year or less, a low value compared to those of RNA viruses (Okamoto et al., 1987b). These genetic variations can be attributed to errors during synthesis of the minus-strand DNA by reverse transcriptase, an enzyme that lacks polymerase-associated proofreading functions. A number of mutations, however, lead to the outcome of HBV variants with modified immunological and pathological properties: altered immunological response has been correlated with HBV-related variants (Blum et al., 1991; Wands et al., 1986; Thiers et al., 1988; Tran et al., 1991), and severe liver injury with e-negative mutant-associated hepatitis (Raimondo et al., 1990; Liang et al., 1991a; Omata et al., 1991). Coinfection with different HBV isolates has been shown to induce homologous recombination between the different viral genomes, and defective viral particles carrying a deleted genome have been described (Gerken et al., 1991a; Okamoto et al., 1987a; Terri. et al., 1991), showing that HBV shares with other viruses, and notably with retroviruses, the property of developing defective variants in the natural host. In addition, the presence of an in-frame ATG upstream of the X translation initiation codon in some HBV subtypes, suggesting the existence of a pre-X open reading frame comparable to pre-S and pre-C, has been correlated with increased replication rate (Loncarevic et al., 1990). Although deleted viral forms have been observed in a human HCC, creating core/polymerase fusion proteins (Will et al., 1986), there is no experimental evidence that free defective HBV genomes might present oncogenic properties, or that particular HBV subtypes might be more oncogenic than others. Comparison of the HBV genome with those of animal hepadnaviruses reveals extensive homologies among mammalian hepadnaviruses, which

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share a basically identical genomic organization. The slightly longer size of rodent viral genomes results from additional sequences in the pre-S1 region, in the P gene immediately upstream of pre-S1, and in the carboxy-terminal part of the C gene (see Schodel et al., 1989). The best conserved regions are located in the C and S genes and in the viral DNA polymerase. The extent of homology is weaker in the pre-S1 region, which, however, retains the same general conformation and hydrophobicity profile. The rather strict host range of the different hepadnaviruses suggests that the cellular receptor for these viruses, which has not been fully characterized, might be poorly conserved during evolution. Accordingly, viral sequences implicated in the interaction of the virion with the cellular membrane are located in the amino-terminal part of the pre-S1 domain (Neurath et al., 1986), a variable region among hepadnaviral genomes. Finally, the internal domain of the X protein is the less conserved region. Two strong homology blocks in the amino- and carboxy-terminal parts of X might correspond to a conservative pressure for functional activity of the X trans-activator, and of the viral RNase H encoded by C-terminal P sequences, which overlap with the 5‘ end of X (Radziwill et al., 1990; Takada and Koike, 1990).Another striking feature is the shorter size of WHx and GSHx, which lack the homologous counterpart for the 9- 12 carboxy-terminal residues of HBx, abolishing the overlap with the pre-C region that occurs in all HBV subtypes, and creating a noncoding region of 11 nucleotides in GSHV. In WHV, the presence of an additional ATG upstream of the precore translation initiation codon has not been clearly correlated with the synthesis of a longer pre-C/C protein product. Sequence analysis of five WHV isolates has shown that the extent of nucleotide variation was lower than between different HBV subtypes, ranging from 0.5% among isolates from the same geographical area to 3.1 % among other isolates (Girones et al., 1989). Accordingly, the mutation rate of WHV DNA in a chronically infected woodchuck, inoculated with an infectious WHV clone has been estimated to be 2.3 x base substitutions/site/year, a value comparable to that found for HBV (Girones and Miller, 1989). Other studies have shown that a significant proportion of replicative intermediates in woodchucks is deleted and defective (Etiemble et al., 1988; Miller et al., 1990). Highly rearranged viral forms (“novel forms”) have also been described in chronic infections (Rogler and Summers, 1982). Replication defective WHV variants do not appear to play a part in the establishment of chronic infections (Miller et al., 1990). Whether they contribute to hepatocarcinogenesis has not been investigated, but it seems probable that novel forms can also integrate into the host genome (Hsu et al., 1988; Ogston et al., 1982).


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T h e avian hepadnavirus genomes are shorter (3.0 kb) and show marked divergence at the nucleotide and amino acid level with mammalian viruses (Mandart et al., 1984; Sprengel et al., 1988). An X open reading frame is missing and the avian C gene is significantly larger than its mammalian counterpart. Sequence comparisons between avian hepadnavirus genomes (DHBV and HHBV) have revealed significant genomic variations, as among HBV subtypes and among rodent viruses (Sprengel et al., 1988, 1991). The strict host range of DHBV and HHBV, which differ notably in the pre-S1 region, might allow the identification of pre-S sequences responsible for recognition of the cell-surface receptor for avian hepadnaviruses.

B. GENOME STRUCTURE AND REPLICATION The hepadnaviral genome, isolated from infectious extracellular virions (Dane particles), is made of two complementary DNA strands of different length, maintained in a circular configuration by base pairing at their 5’ extremities (Summers el al., 1975; Hruska et al., 1977; Sattler and Robinson, 1979) (Fig. 3). The viral replication pathway, virtually identical for all hepadnaviruses, takes place in the nucleus and cytoplasm of infected cells, and although it can be instructively compared with the retroviral life cycle, it is entirely extrachromosomal (Seeger et al., 1986; Will et al., 1987) (Fig. 4).On virus entry in hepatocytes, the partially double-stranded DNA is converted to doublestranded DNA and then to covalently closed circular DNA (cccDNA, also designated supercoiled DNA). This process, requiring removal of the primers covalently linked to 5’ DNA extremities and of the seven- to eight-base terminal redundancy of the minus strand, is achieved by a set of cellular enzymes that have not been clearly identified. cccDNA, localized primarily in the cell nucleus, serves as a template for viral transcription, a step that coincides with the multiplication of replicative forms. At this stage, several viral RNAs are produced: messenger RNAs specifying surface, pre-C/C, and X protein products, and the so-called “pregenome” RNA, identified as a replicative intermediate as well as the message for C and P genes (Summers and Mason, 1982; L. J. Chang et al., 1989; Schlicht et al., 1989). The contribution of viral proteins to subsequent steps of the replicative process has been largely delineated using mutational analysis of DHBV and in vitro culture systems. Briefly, the asymmetric synthesis of viral minus- and plus-strand DNA can be schematized as follows. 1. Packaging of selected genomic RNA into subviral cores, a cotranslational process mediated by the polymerase gene product, using

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+ strand envelope

- Strand

capsid

receptor

polyprotein polymerase

ccc DNA 3.5 m pmgenome RNA

L

- strand

d

2.4 kb mRNA

-3-

2.1 kbmRNA

3 .

large S middle

Nucleus 22 nm particle

0

J \

FIG. 4. Schematic illustration of the hepadnavirus life cycle in hepatocytes. "Polyprotein" designates the primary product of translation of the P gene, which encodes the terminal protein and the polymeraselreverse transcriptase. cccDNA, Covalently closed circular DNA.

a cis-acting encapsidation signal recently identified near DRl (Bartenschlager et al., 1990; Hirsch et al., 1990, 1991;Junker-Niepman et al., 1990; Ou et al., 1990; Roychoudhury et al., 1991) 2. Initiation of DNA synthesis at a DR1 repeat, probably in the 3' end

of pregenome RNA, using as a primer the N-terminal portion of the P gene polyprotein product that covalent binds to the 5' extremity of the growing minus-strand DNA (Molnar-Kimber et al., 1983, 1984; Bartenschlager and Schaller, 1988) 3. Degradation of the RNA template by the viral RNase H activity while complementary DNA is produced (Radziwill et al., 1990) 4. Initiation of plus-strand DNA synthesis at the DR2 repeat, using as


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a primer a capped oligoribonucleotide of 17 bases derived from the 5’ end of the pregenome RNA (Lien et al., 1986, 1987), and elongation of the plus-strand DNA by the viral DNA polymerase. Nucleocapsids containing replicati,ve intermediates at different stages of maturation, even as early as during synthesis of the minus-strand DNA, are then coated and excreted from infected cells, as indicated by the variable length of the short viral strand DNA and the presence of RNA/DNA duplexes in extracellular virions (Miller et al., 1984; Scotto et al., 1985). Finally, amplification of cccDNA through an intracellular process involving transport of newly synthesized viral intermediates to the nucleus allows the establishment of a pool of transcriptional templates in persistently infected cells (Tuttleman et al., 1986a). The viral pre-SIS proteins participate in the control of cccDN A copy numbers in persistent hepadnaviral infections (Summers et al., 1990, 1991).

C. VIRIONASSEMBLY In many chronic hepadnavirus infections, liver cells produce huge amounts of empty viral envelopes, that are assembled in spherical or tubular particles with a diameter of 22 nm and released into the bloodstream through the lumen of the endoplasmic reticulum. HBsAg particles are made of major and middle surface proteins (encoded by the S gene and the pre-S2IS region) embedded in cellular lipids in glycosylated and unglycosylated forms. They differ mainly from the envelope of infectious virions in the almost complete absence of large surface glycoproteins, encoded by the pre-S 1lpre-S2lS region (reviewed by Tiollais et al., 1985). Excretion through the membrane bilayer is governed by the major S protein, which contains three hydrophobic domains and topogenic signal sequences for directing transmembrane orientation and translocation of virions and HBsAg particles across the bilayer (Bruss and Ganem, 1991; Eble et al., 1986, 1987). The hypothetical transmembrane configuration of the middle S protein is shown in Fig. 5. Only the major S protein is required to form 22-nm particles, whereas all three envelope proteins are necessary for production of infectious virions (Persing et al., 1985; Ueda et al., 1991). Several lines of evidence indicate that the pre-S1 polypeptide plays a predominant role in the secretion of Dane particles and in the control of cccDNA amplification during persistent infections. Mutant viruses that do not produce the large S protein are unable to form infectious virions, whereas cccDNA and core particles carrying viral DNA accumulate intracellularly (Summers et al., 1991; Ueda et al., 1991). These mutants have probably lost

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.OW.

= ER-Signal

FIG. 5. Hypothetical conformation of the middle protein (encoded by the preS2IS region) in the endoplasmic reticulum bilayer. Truncation of this protein at residues 77 to 210 generates a novel transcriptional trans-activating activity. (From A. Kekule, Ph.D. thesis.)

the ability to envelope the newly synthesized nucleocapsids. Whether the absence of large S proteins modifies the conformation of budding envelope shells, preventing the envelope from encircling core particles, or whether unique sequences in the pre-Sl domain interact with the viral core has not been determined. It has been shown that retention of the large S protein in the endoplasmic reticulum is governed by signals within the myristilated amino-terminal pre-S1 domain (Kuroki et al., 1989; Persing et al., 1987; Prange et al., 1991). In the absence of a large excess of major and middle S proteins, the large S protein is retained in the endoplasmic reticulum and blocks efficient secretion of HBsAg particles (Chisari et al., 1986; McLachlan et al., 1987; Ou and Rutter, 1987; Persing et al., 1986; Standring et al., 1986). Furthermore, secretion of major and middle S proteins is inhibited in cells producing more than 20% of large S protein (Molnar-Kimber et al., 1988).Therefore, a tight regulation of the relative amounts of the three envelope proteins appears to be an important feature in viral assembly and in control of persistent hepadnaviral infections.

D. REGULATED EXPRESSION OF VIRALGENES In productive hepadnavirus infections, a strict balance in the amount of individual viral gene products is necessary for active viral replication


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and release of infectious virions as well as for survival of infected host cells. Despite the small size of the HBV genome and a very compact organization of coding sequences, the expression of the different viral genes is subjected to a complex regulation at various levels, both transcriptionally and posttranscriptionally. Each individual HBV gene is controlled by an independent set of regulatory signals, probably acting in cooperation with HBV elements that coordinate the relative level of viral gene expression. All HBV regulatory signals are contained within coding sequences, and for some of them in regions encoding two different polypeptides from overlapping reading frames. In chronically infected livers and in cell lines supporting active viral replication, two major HBV transcripts of molecular size 3.5 and 2.1 kb are produced from cccDNA template at roughly similar levels (Cattaneo et al., 1984; Gough, 1983) and minor 2.4- and 0.8-kb transcripts have been described (Ou and Rutter, 1985; Siddiqui et al., 1986, 1987; Treinin and Laub, 1987). These polyadenylated RNAs are colinear with the viral genome and complementary to the DNA minus strand. They have heterogeneous 5‘ ends and a common termination site in the core gene, downstream of a variant polyadenylation signal shared by all hepadnaviruses. The transcription patterns of WHV and GSHV in chronically infected livers are strikingly similar, whereas that of DHBV differs mainly by higher levels of 2.4-kb RNA (Buscher et al., 1985; Enders et al., 1985; Moroy et al., 1985). Two distinct 3.5-kb RNAs serving different functions start around the pre-C initiator codon (see Fig. 2), the longer one specifying the pre-C/C polypeptide (HBeAg) and the shorter the nucleocapsid protein (HBcAg) and the viral P gene products. The shorter RNA species, designated “pregenome,” is also implicated in the viral replicative process (Seeger et al., 1987; Will et al., 1987). The 3.5-kb RNAs are longer than genome size by about 120 nucleotides, and their synthesis requires RNA polymerase reading through the polyadenylation signal without cleavage at the first passage, a feature probably associated with a strong secondary structure at the 5’ end of the nascent transcript as in other retroid elements (Russnak and Ganem, 1990). In contrast with that of retroviruses, translation of the hepadnaviral polymerase from the 3.5-kb pregenome RNA does not involve ribosomal frame shifting, but more probably de n o w translational initiation (L. J. Chang et al., 1989, 1990; Ou et al., 1990; Schlicht et al., 1989).The liver-specific promoter for 3.5kb RNAs is made of a basal AT-rich element and of upstream promoter sequences that bind different nuclear factors (Lopez-Cabrera et al., 1990; Yaginuma and Koike, 1989). Two enhancer elements stimulate transcription from the core/pregenome promoter: EN I, positioned

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about 450 bp upstream of the core promoter and EN 11, located in the X gene coding region (Shaul et at., 1985; Yee, 1989) (see Figure 3). The HBV enhancers I and I1 bind multiple liver-specific factors as well as ubiquitous transcriptional activators and exhibit a preferred activity in differentiated hepatocytes or hepatoma-derived cell lines (Ben-Levy et al., 1989; Jameel and Siddiqui, 1986; Lopez-Cabrera et al., 1991; Shaul and Ben-Levy, 1987; Y. Wang et al., 1990; Yuh and Ting, 1990). Enhancer I stimulates expression of all viral genes (Antonucci and Rutter, 1989; Hu and Siddiqui, 1991). A strong enhancer element has also been identified upstream of the pregenome start site of DHBV (CrescenzoChaigne et al., 1991). Recently, two spliced RNA species of about 2 kb, which share 5'- and 3'-terminal sequences with the 3.5-kb RNAs and present different coding capacities, have been described (Chen et al., 1989; Su et al., 1989; Suzuki et at., 1989). These relatively abundant transcripts appear to be dispensible for HBV replication, but they are detected in most natural infections, suggesting that they may serve some biological function(s) (Wu et al., 1991). One of the spliced RNAs has been shown to be efficiently packaged and reverse transcribed in vivo, giving rise to defective viral particles (Terre et al., 1991). The major 2. l-kb and minor 2.4-kb RNAs direct the synthesis of the three envelope proteins (Cattaneo et al., 1983).Expression of the hepatitis B surface antigen gene has been extensively studied in cultured cells (Pourcel et al., 1982; Roossinck et al., 1986; Siddiqui et al., 1986; Standring et al., 1984) and in transgenic mouse models (Babinet et al., 1985; Burk et al., 1988; DeLoia et al., 1989).It presents one of the most striking examples of the efficient organization of the compact HBV genome, in that it involves a complex set of regulatory elements embedded in two overlapping coding sequences. The major and middle surface proteins are synthesized from the major 2.1-kb transcripts, which initiate heterogeneously 5' and 3' of the pre-S2 ATG codon. The longer 2.1-kb species encode both the major and middle S proteins, but the shorter one encodes only the major S. The levels of expression of the pre-S2/S and S genes are controlled by a dual promoter located in a 200-bp sequence of the pre-S2 region, and by differential use of the pre-S2 and S translational initiator codons. The pre-S2/S promoter, highly active in a variety of cultured hepatoma cells, has no TATA motif and shares homology with the simian virus 40 (SV40) origin-late promoter (Cattaneo et al., 1983). Transcription from this promoter is regulated by a complex interplay between positive and negative elements and is stimulated by the downstream glucocorticoid responsive element (GRE) and by the viral enhancer I (Bulla and Siddiqui, 1988; H. K. Chang et al., 1987; De-


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Medina et al., 1988; Farza et al., 1988; Raney et al., 1989, 1991; Shaul et al., 198613; Tur-Kaspa et al., 1986). In contrast, the pre-S1 promoter is markedly weaker and shows a strict cell-type specificity attributed to the binding of liver-specific transcription factors, like HNF- 1. This might account partially for the hepatotropism of HBV (Bulla and Siddiqui, 1989; Chang and Ting, 1989; H. K. Chang et al., 1989; Courtois et al., 1988; Nakao et al., 1989; Raney et al., 1990; Zhou and Yen, 1991). Finally, the production of a 0.9-kb mRNA encoding the viral X gene product is better seen in cultured cells transfected with viral subgenomic fragments than in natural HBV infections, indicating that the HBX promoter might be down regulated during the viral replicative process, which is associated with very low concentrations of X protein in infected human livers (Asselsbergs et al., 1986; Gough and Murray, 1982; Saito et al., 1986; Treinin and Laub, 1987). V. Potential Oncogenic Properties of Viral Proteins

It has been admitted for a long time that prolonged expression of viral genes might have no direct cytotoxic effects on the infected hepatocytes. Transfection of cultured cells with HBV DNA has not been usually associated with tumorigenic conversion, and most transgenic lines of mice bearing the full-length HBV genome or subgenomic constructs never show any sign of liver cell injury. However, recent studies in particular experimental systems have indicated that abnormal overexpression of different viral proteins, including the surface proteins and the X gene product in native or modified forms, might play a part in malignant transformation of infected hepatocytes. A. SURFACE GLYCOPROTEINS In natural HBV infections, the production of infectious virions and HBsAg particles depends on a tight regulation of the relative levels of the three envelope glycoproteins, as shown in Section IV,C. Neither liver lesions nor HCC have been observed in any of the published transgenic lineages that produce the middle and major surface proteins from HBVderived regulatory sequences (Araki et al., 1989; Babinet et al., 1985; Burk et al., 1988; Farza et al., 1988). However, it has been shown that the appearance and rate of production of preneoplasic nodules and primary tumors following carcinogen administration are slightly increased in HBsAg-positive transgenic mouse livers as compared to negative littermates, suggesting that HBsAg expression might enhance the effects of the hepatocarcinogens (Dragani et al., 1989).When the endogenous preS1 promoter is replaced by an exogenous promoter (the metallothionein

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or the albumin promoter) the production of roughly equimolar ratios of large S protein with respect to middle and major S leads to intracellular accumulation of nonsecretable filamentous envelope particles within the endoplasmic reticulum of transgenic mouse hepatocytes (Chisari et al., 1986, 1987). This provokes histological and ultrastructural features of “ground-glass” hepatocytes, which have been described in some cases of chronic human liver diseases and are considered to be typical of chronic hepatitis B (Gerber et al., 1974; Stein et al., 1972), ultimately killing the cells. I n the transgenic mouse lineages, mild persistent hepatitis was followed by the development of regenerative nodules and eventually HCCs by 12 months of age (Chisari et al., 1989b; Dunsford et al., 1990). T h e preneoplastic nodules and tumors display a marked reduction in transgene expression, suggesting that hepatocytes that express low levels of the large S polypeptide would have a selective survival advantage. Exogenous, chemical cofactors are not required for tumorigenic induction in this model, but exposure of adult transgenic mice to hepatocarcinogens produced more rapid and extensive development of preneoplastic lesions and HCC, under conditions that do not alter the liver morphology of nontransgenic controls (Teeter et al., 1990). These data show that inappropriate expression of the large S protein has the potential to be directly cytotoxic to the hepatocyte and may initiate a cascade of events that ultimately progress to malignant transformation, although the molecular mechanism connecting viral and host factors in this process has not been elucidated. Studies of integrated HBV sequences in human liver tumors have also suggested a possible role for abnormal expression of rearranged viral S genes in HCC development. It has been shown that deletion of the carboxy-terminal region of the S protein generates a novel transcriptional trans-activation activity (Caselmann et al., 1990; Kekul6 et al., 1990) (see Fig. 5). Integrated HBV sequences from a human tumor and a hepatoma-derived cell line, as well as different constructs bearing similarly truncated pre-S2/S sequences, can stimulate the SV40 promoter in transient transfection assays; trans-activation occurs at the transcriptional level and is dependent on the SV40 enhancer. The c-myc P2 promoter is also activated in trans. These findings support the hypothesis that accidental 3’ truncation of integrated pre-S2/S genes could be a causative factor in HBV-associated oncogenesis. B. HBx: A TRANSCRIPTIONAL TRANS-ACTIVATOR

T h e smallest of the four HBV open reading frames was initially designated “ X because it was unclear at that time (and remained as such for


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several years) that it might encode a protein produced during HBV infection, and that this potential protein might have a predictable function in the viral life cycle (Galibert et al., 1979). Examination of the nucleotide and amino acid sequences of HBx has led to the prediction of a regulatory function for the deduced X polypeptide and has revealed a codon usage similar to that used in eukaryotic cell genes, suggesting that X might have been transduced by the HBV genome (Miller and Robinson, 1986). The strong conservation of X sequences among HBV subtypes (Lo et al., 1988) and the presence of homologous reading frames in the WHV and GSHV genomes suggest that their products may be of importance for viral replication. The absence of an X open reading frame in the avian hepadnavirus genomes, despite some weak sequence homologies in the DHBV core gene (Feitelson and Miller, 1988), raises questions about an essential contribution of this protein to the viral life cycle. Evidence for expression of the HBV X gene has been obtained by Moriarty et al. (1985) and by.Kay et al. (1985),who reported that the sera of HBV-related HCC patients recognize synthetic peptides made on X sequences. Expression of the X reading frame in prokaryotic and eukaryotic cells, using various vectors, has allowed the identification a 16.5kDa polypeptide that reacts with serum samples from a number of HBVinfected individuals (Elfassi et al., 1986; Meyers et al., 1986; Pfaff et al., 1987; Schek et al., 1991b). Anti-HBx antibodies have been detected in a minor proportion of acutely infected patients, about 3-4 weeks after the onset of clinical signs, and more frequently in chronic HBsAg carriers showing markers of active viral replication. Very few patients show antiHBx antibodies after seroconversion to anti-HBs and at the time HCC develop (Levrero et al., 1991). However, conflicting results have been obtained regarding the association of anti-HBx antibodies with other viral markers and with HCC. These problems may be related to the weak antigenicity of HBx or to its sequestration into cellular compartments that render it inaccessible to the host immune system. The hepatitis B x antigen (HBxAg) has been detected in livers and sera of HBsAg carriers and has been correlated with ongoing viral replication and chronic liver disease (Haruna et al., 1991; Levrero et al., 1990; Wang et al., 1991).The X protein is localized mainly in the cytoplasm of in vivo-infected cells, at or near the plasma membrane and at the nuclear periphery (Levrero et al., 1990; Vitvitski et al., 1988; Wang et al., 1991; Zentgraf et al., 1990). The X protein has been detected in the nuclear compartment only in transfected cell lines (Hohne et al., 1990; Seifer et al., 1990). The recent finding that the X gene product can trans-activate transcription from a number of HBV and heterologous promoters is of

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considerable importance in defining its role in viral replication and in pathogenesis. T h e X trans-activator stimulates transcription from the the C, S, or X promoters coupled to HBV enhancer (Colgrove et al., 1989; Raney et al., 1990; Spandau and Lee, 1988), from heterologous viral promoters like the SV40 early promoter, the HSV-thymidine kinase (TK) promoter and the HIV-1 long terminal repeat (LTR) (Spandau and Lee, 1988; Twu and Robinson, 1989; Twu et al., 1989; Zahm et al., 1988), and from cellular promoters including @-interferon, HLA-DR, and c - m y promoters (Hu et al., 1990; Twu and Schloemer, 198’7; A. KekukC, personal communication). Trans-activation of RNA polymerase I11 promoters has also been reported (Aufiero and Schneider, 1990). Conflicting results on X-mediated trans-activation of several cis-acting regulatory sequences in different transfected cell lines might be related to protein interactions between X and cellular factors. Indeed, the X protein has been shown to interact, directly or indirectly, with transcriptional activators such as AP1, AP2, CREB, ATF2, NFKBand possibly Spl (Maguire et al., 1991; Seto et al., 1990; Twu et al., 1989; A. Kekule, personal communication). T h e subcellular localization of the X polypeptide makes it unlikely that it might directly bind to its DNA target sequences; activation of cellular and viral genes, which occurs at the level of transcription (Colgrove et al., 1989), might be mediated via the protein kinase C (PKC) signal transduction pathway, as for tumor promoters (A. Kekuke, personal communication). It has been shown that HBx can be phosphorylated in vivo and that it displays an intrinsic serinel threonine protein kinase activity (Schek et al., 1991a; Wu et al., 1990), two features also shared by a number of proteins implicated in intracellular signalling pathways. HBx might therefore directly activate cellular transcription factors. Although present at very low levels in chronically infected livers, the X protein has been shown to stimulate the production of viral particles in transient transfection assays (Yaginuma et al., 1987). However, it has not been established that the expression of cellular genes showing Xresponsive promoters is stimulated in HBV-infected hepatocytes in vivo. More clues to the possible role of HBx in HBV-associated pathogenesis have been provided by three recent lines of studies, including both in nitro and in vivo studies and direct analysis of human liver and HCC biopsy samples. It has been shown that high levels of X expression may induce malignant transformation of certain cultured cells, like the NIH 3T3 cell line (Shirakata et al., 1989), immortalized hepatocytes expressing the SV40 large tumor antigen (Hohne et al., 1990), and primary embryo fibroblasts cotransfected with X and ras expression vectors (A. Kekuld, per-


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sonal communication). Studies of transgenic mice carrying the X reading frame controlled by its natural HBV enhancer/promoter sequences or by heterologous liver-specific promoters have given rise to conflicting results. In two lines of mice derived from the inbred CDl strain, carrying a 1.15-kb HBV fragment (spanning the enhancer, the complete X coding region, and the polyadenylation signal), the development of preneoplastic lesions has been observed in the liver, followed by malignant carcinomas at 8-10 months of age (Kim et al., 1991). In contrast, other transgenes, in which the X coding domain was placed under the control of the a-l-antitrypsin or the antithrombin-3 regulatory region, failed to induce serious liver damage in ICR x B6C3F1 or C57BL/6 X SJL/J transgenic animals, although X mRNAs were detected in liver tissues (Lee et al., 1990; P. Briand, personal communication). Analysis of integrated viral sequences in tumor DNA has shed new light on one of the mechanisms leading to overexpression of HBx in chronically infected livers and in HCCs. It has been shown that HBV sequences are frequently interrupted between the viral direct repeats DR1 and DR2 on integration into host cell DNA (see Section VI,A) and that overproduction of hybrid viral/host transcripts may result from HBV DNA integration in a hepatoma cell line (Freytag von Loringhoven et al., 1985; Ou and Rutter, 1985). T h e presence of viral/host transcripts containing a 3'truncated version of the X coding region fused with flanking cellular sequences and retaining trans-activating capacity was first described in a human HCC (Wollersheim et al., 1988). Moreover, enhanced trans-activating capacity of the integrated X gene product has been related to the substitution of viral carboxy-terminal residues by cellular amino acids (Koshy and Wells, 1991). Trans-activating ability of similarly truncated X products made from fusion of integrated HBV sequences with adjacent cell DNA has also been shown in many chronic hepatitis tissues (Takada and Koike, 1990). This suggests that the integrated X gene might be essential for maintaining the tumor phenotype that develops at the early stages of carcinogenesis. Consistent with this model, viral/host junctions have been mapped in the carboxy-terminal region of X in a majority of human HCCs (Nagaya et al., 1987; Shih et al., 1987). Further studies are now necessary to better delineate the contribution of X gene product to malignant transformation in persistent HBV infections. VI. Integrated State of Viral DNA in Chronic Infections and Hepatocellular Carcinoma

The hepadnavirus replication pathway in infected cells has been shown to take place within nuclear and cytoplasmic compartments (see

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Section IV,B) and more recent studies have indicated that it can be entirely cytoplasmic (Zhou and Standring, 1991). Unlike retroviruses, this process does not require viral DNA integration in the host cell genome. However, hepadnaviruses share with other retroelements of common evolutionary origin-transposons and retroviruses (Xiong and Eickbush, 1990)-the ability to integrate their DNA into cellular chromosomes. The molecular events leading to the invasion of cell DNA by hepadnaviral DNA have not been fully elucidated. The main question is whether viral integrations might play a part in the virally induced transformation process, either by conferring a selective growth advantage to targeted cells, leading to the onset of preneoplastic nodules, or by providing an additional step in tumor progression. Whereas insertional activation of protooncogenes now emerges as a common event in WHV-induced woodchuck HCC, a related mechanism has been observed only in rare examples of human HCCs, in which different activities for integrated HBV sequences have been proposed. A. INTEGRATED SEQUENCES: PHYSICAL AND FUNCTIONAL ASPECTS Analyses of Southern profiles have allowed the identification and primary characterization of integrated HBV sequences in established hepatorna cell lines and in about 80% of human HCCs (BrCchot et al., 1980, 1981b, 1982; Chakraborty et al., 1980; Edman et al., 1980; Koshy et al., 1981; Shafritz et al., 1981; for reviews, see Tiollais et al., 1985; Matsubara and Tokino, 1990). Hepatitis B virus DNA integrations occur at early stages in natural acute infections and in experimental infections of cultured cells (Scotto et al., 1983; Lugassy et al., 1987; Ochiya et al., 1989; Yaginuma et al., 1987). As a result of multiple integrations in chronic hepatitis tissues (Boender et al., 1985; Brechot et al., 1981b; Shafritz et al., 1981; Tanaka et al., 1988),integrated HBV sequences have been detected in most HBV-related HCCs that arise from clonal outgrowth of one or a few transformed liver cells (see Matsubara and Tokino, 1990). Single HBV insertions are common in childhood HCCs but are rather uncommon later in life, suggesting that multiple integrations occurring during the course of long-standing HBV infections might accumulate within single cells (Chang et al., 1991), as also indicated by sequence divergence among HBV inserts in the same tumor (Imai et al., 1987). Integrated WHV sequences have been similarly detected in woodchuck liver and in a majority of woodchuck HCCs (Ogston et al., 1982; Rogler and Summers, 1984; Hsu et al., 1990), but


20 1

viral integrations appear to be less frequent in GSHV- and DHBV-related tumors (Marion et al., 1986; Yokosuka et al., 1985; L. Cova, personal communication). Studies of the organization of cloned HBV inserts in liver tissues and HCCs have shown that HBV sequences are fragmented and rearranged and that integration and recombination sites are dispersed over the viral genome, indicating that HBV integration does not occur through a unique mechanism, as in the case of other retroelements and retroviruses (Dejean et al., 1984; Koch et al., 1984b; Shaul et al., 1984; Ziemer et al., 1985). T h e absence of complete genomes in virtually all HBV inserts, which consist either of linear subgenomic fragments or of rearranged fragments in different orientations, indicates that these integrated sequences cannot serve as a template for viral replication. Rearrangements of integrated HBV sequences may take place during the integration process as well as after the formation of viral inserts (Mizusawa et al., 1985; Nagaya et al., 1987; Tokino et al., 1987). Integrated forms made of a continuous genome or subgenomic fragment, which are frequent in HCC and hepatitis tissues from children (Yaginuma et al., 1987), are believed to represent primary products of integration. They are of particular interest in the study of the molecular mechanisms responsible for HBV DNA integration. Highly preferred integration sites have been mapped in the HBV genome within the “cohesive ends” region, that lies between two 1 l-bp direct repeats (DRl and DR2) highly conserved among hepadnaviruses (Koshy et al.. 1983; Nagaya et al., 1987). A narrow region encompassing DR1 has been shown to be particularly prone to recombination (Hino et al., 1989; Nakamura et al., 1988; Shih et al., 1987; Yaginuma et al., 1987). This region coincides with a short terminal redundancy of the minusstrand DNA, which confers a triple-stranded structure to the circular viral genome (Seeger et al., 1986; Will et al., 1987). Integration sites are tightly clustered both at the 5‘ and 3’ ends of minus-strand DNA, suggesting that replication intermediates and specially relaxed circular DNA might be preferential preintegration substrates (Nagaya et al., 1987; Shih Pt al., 1987). Invasion of cellular DNA by single-stranded HBV DNA, using mainly free 3‘ ends, might take place through a mechanism of illegitimate recombination, also suggested by frequent patch homology between HBV and cellular sequences at the recombination break points (Matsubara and Tokino, 1990). Although different minor changes in flanking cellular DNA have been associated with viral integration (both microdeletions and short duplications have been described) (Berger and Shaul, 1987; Dejean et al., 1986; Hino et al., 1989; Nakamura et al., 1988; Yaginuma et al., 1985), more precise mechanisms have been proposed. T h e recombination-proficient region spanning

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DRl is located close to a U5-like sequence highly conserved between hepadnaviruses, suggesting that sequences necessary for precise recombination with cellular DNA have been retained from a common ancestor with retroviruses, despite the absence of a gene coding for an integrase in the HBV genome (Miller and Robinson, 1986). A specific mechanism based on sequence polarity in DR1 and DR2 and on deletion of 1-2 bp at the 5' end of the viral DRs has been proposed (Dejean et al., 1984), but collection of data on the structure of about 40 HBV inserts has not confirmed this model. Mapping of a set of preferred topoisomerase I (TopoI) sites near DR1 and DR2 and in nitro studies of WHV DNA integration into cloned cellular DNA have sustained the hypothesis that TopoI might promote illegitimate recombination of hepadnavirus DNA in viuo (Wang and Rogler, 1991). A role for the eukaryotic enzyme TopoI has been proposed not only in nonhomologous recombinations that lead to integration in other DNA virus systems (Bullock et al., 1984, 1985) but also in the life cycle of different retroviruses (Priel et al., 1991). The mechanisms underlying HBV DNA integration remain to be fully identified, but it seems probable that similar events lead to the integration of all hepadnaviruses, although only a limited number of WHV and DHBV insertions and no integrated forms of GSHV have been analyzed until now (Fourel et al., 1990; Hsu et al., 1988; Imazeki et al., 1988; Ogston et al., 1982; Rogler and Summers, 1984). As a consequence of the viral integration process, sequences of the S and X genes and of the enhancer I element are almost systematically present in HBV inserts, whereas those of the C gene are less frequently represented. It has been shown that the pre-SP/S promoter was transcriptionally active in its integrated form in human and woodchuck HCCs (Caselmann et al., 1990; Freytag von Loringhoven et al., 1985; Ou and Rutter, 1985; Y. Wei and M. A. Buendia, unpublished results) and that HBsAg might be produced from viral inserts (Dejean et al., 1984; Zhou et al., 1987). Highly rearranged HBV inserts show virus junctions scattered throughout the viral genome, and in some of them, recombination break points have been mapped in the S coding region (Nagaya et al., 1987). It has been recently shown that truncation of the S gene between residues 77 and 22 1 confers a transcriptional activation activity to the mutated pre-SPIS products (Caselmann et al., 1990; Kekule et al., 1990). T h e shorter pre-SP/S protein lacks carboxy-terminal signals for translocation through the endoplasmic reticulum membrane and should be retained in the bilayer. Activation of the c-my oncogene promoter, demonstrated in in vitro assays, might result from an indirect transacting action of the truncated viral proteins (Kekule et al., 1990). Whether this o r some related mechanism participates in liver cell trans-


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formation remains to be determined (see Section V,A). Other studies have shown that an important percentage of viral junctions is localized in the carboxy-terminal part of the viral X gene, predicting a fusion of the X open reading frame to flanking cellular sequences in a way that might preserve the functional capacity of the X trans-activator. Evidence for transcriptional activity at integrated X sequences has been provided in tumors and chronically infected livers (Hilger et al., 1991; Miyaki et al., 1968; Takada and Koike, 1990; Wollesheim et al., 1988) and might be correlated with the detection of HBxAg in a number of human HCCs (Moriarty et al., 1985; Kay et al., 1985). It has also been shown that a number of viral X/cell fusion peptides harbor transcriptional activation activity. The contribution of downstream cellular sequences to activated expression and/or to enhanced trans-activating capacities of the integrated HBV sequences has been suggested in two cases (Freytag von Lorinhoven et al., 1985; Wollesheim et al., 1988). These data indicate that abnormal expression of integrated and truncated X gene might play a part in HBV-associated oncogenesis, by deregulating the normal expression of cellular genes in trans (see Section V,B). B. CELLULAR TARGETS FOR VIRALINTEGRATION IN HUMAN HEPATOCELLULAR CARCINOMA Studies of different viral insertions in many human HCCs have revealed that integration can take place at multiple sites on various chromosomes (25 insertion sites have been mapped on 16 different human chromosomes) (Matsubara and Tokino, 1990). These studies failed to demonstrate the presence of any known dominant oncogene or tumor suppressor gene in the immediate vicinity of any integration site. It has been reasoned that integration of HBV DNA occurs at random in the human genome and that it has no direct mutagenic effect on growth control genes in most cases. However, contrary to a widely held opinion, integration of retroelements and viruses might not be entirely random; it has been shown that the possible sites for retroviral integration in eukaryotic DNA are numerous but not unlimited (about 500-1000) (Shih et al., 1988). It has been proposed that simple repetitive elements are hot spots for HBV insertion in the human genome (Berger and Shaul, 1987). Indeed, Alu-type repeats, minisatellite-like, satellite 111, or VNTR sequences have frequently been identified near HBV insertion sites (Berger and Shaul, 1987; Nagaya et al., 1987; Shaul et al., 1986a), suggesting that chromosomal regions accessible to specific families of mobile repeated sequences are also preferential targets for HBV insertion. A small cellular DNA compartment (H3) characterized by a base

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composition close to that of HBV DNA and a high concentration of Alu repeats has been designated as a major target for stable HBV integration (Zerial et al., 1986). It has been shown that HBV DNA integration may enhance chromosomal instability: in many tumors, large inverted duplications, deletions, amplifications, or chromosomal translocations have been associated with HBV insertions, suggesting that this process may function as a random mutagen, promoting chromosomal defects in hepatocytes (Hatada et al., 1988; Hino et al., 1986; Koch et al., 1984a; Mizusawa et al., 1985; Rogler et al., 1985; Tokino et al., 1987; Yaginuma et al., 1985). It has also been shown that HBV DNA promotes homologous recombination at a distance from the insertion site (Hino et al., 1991). However, a role for most of these chromosomal abnormalities has not been assigned as yet, although in a few cases the p53 or hstl loci have been altered as a sequel to HBV integration in the same chromosomal region (Hatada et al., 1988; Slagle et al., 1991; Zhou et al., 1988). Evidence for a direct cis-acting promoter insertion mechanism has been provided in two independent HCCs (Dejean et al., 1986; de The et al., 1987; Dejean and de The, 1990; J. Wang et al., 1990). These investigators have analyzed early tumors that developed in noncirrhotic livers from clonal proliferation of a cell containing a single specific viral integration. In one case, the HBV insertion occurred in an exon of the retinoic acid receptor P gene (RARP) and fused the amino-terminal domain of the viral pre-S1 gene to the DNA-binding and hormonebinding domains of RARP (Dejean et al., 1986). Retinoic acid and retinoids are vitamin A-derived substances that have striking effects on differentiation and proliferation in a large variety of systems (Brockes, 1990). Retinoic acid receptors are members of the steroid thyroid hormone receptors family, which includes the c-er6A protooncogene (Graf and Berg, 1983). Recently, the chromosomal translocation t( 15;17) fusing the RARa gene to a cellular gene termed PML has been implicated in acute promyelocytic leukemias (de The et al., 1990, 1991). In the human HCC, it seems most probable that inappropriate activation of RARP resulted in expression of a chimeric HBV/RARP protein at greater levels than that of native RARP protein, and participated in the tumorigenic process. In the second case, HBV sequences were found to be integrated in an intron of the human cyclin A gene, resulting in the production of spliced HBV/cyclin A fusion mRNAs initiated at the preS2/S promoter (J. Wang et al., 1990; C. Brechot, personal communication). In the deduced polypeptide, the amino-terminal domain of cyclin A, a target for proteolytic degradation of cyclin A at the end of the M phase, was replaced by residues of the viral S region. Cyclins are impor-

H B V AND HEPATOCELLULAR CARCINOMA

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tant in the control of cell division, and disruption of the cyclin A gene by viral insertion probably contributed to oncogenesis. It should be interesting to further analyze other tumors that, like in the two cases reported above, are at an early stage of cancer development to better delineate the relative contribution of insertional mutagenesis and other mechanisms, associated or not with HBV DNA insertion, in the virally induced transformation pathway. C. INSERTIONAL ACTIVATION OF myc FAMILY GENES I N WOODCHUCK HEPATOCELLULAR CARCINOMA T h e availability of naturally occurring animal models for HBVinduced liver disease and cancer has been largely exploited for a better understanding of the viral/host interactions, as shown in Section I11 and IV. In the WHV/woodchuck model particularly, the natural history of viral infections, the presence and state of viral DNA, and the patterns of viral gene expression have been extensively investigated. Experimental inoculation of newborn woodchucks with infectious virions has given conclusive information on the oncogenic activity of WHV: this virus now appears to be the most potent inducer of liver cancer among the hepadnavirus group (Popper et al., 1987b; Seeger et al., 1991). A search for transcriptional activation of already known protooncogenes and for viral insertion sites in tumor cell DNA has revealed that WHV acts as an insertional mutagen, activating myc family genes (c-myc or N-myc) in more than one-half of the tumors examined (Fourel et al., 1990; Hsu et al., 1988; Moroy et al., 1986; Y. Wei, A. Ponzetto, P. Tiollais, and M. A. Buendia, unpublished results). Analysis of the mutated c-myc alleles in two individual tumors has shown integration of WHV sequences in the vicinity of the c-myc coding domain, either 5' of the first exon or in the 3'-untranslated region (Hsu et al., 1988). Deregulated expression of the oncogene driven by its normal promoters resulted from deletion or displacement of c-myc regulatory regions known to exert a negative effect on c-myc expression, and their replacement by viral sequences encompassing the enhancer I element. Such a mechanism is highly reminiscent of that previously reported for c-myc activation in murine T cell lymphomas induced by murine leukemia viruses (MuLV) (Corcoran et al., 1984; Selten et al., 1984). Evidence for a direct role of WHV DNA integration into c-myc in hepatocyte transformation has been provided by the development of hepatocellular carcinoma in transgenic lines of mice bearing WHV and myc sequences from the mutated allele of a woodchuck HCC (J. Etiemble, C. Babinet, P. Tiollais, and M. A. Buendia, unpublished results). Transient ex-

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pression of the transgene in liver cells after birth was correlated with the onset of primary liver tumors at 5-12 months of age in 100% of cases. More frequently observed was the insertional activation of N-myc genes. In contrast with human and mouse, the woodchuck genome contains two distinct N-myc genes: N-myc 1, the homolog of known mammalian N-myc genes similarly organized into three exons, and N-myc 2, a functional processed pseudogene or “retroposon,”which has retained extensive coding and transforming homology with parental N-myc (Fourel et al., 1990). In woodchuck HCCs, N-myc 2 represents by far the most frequent target for WHV DNA integrations. As shown in Fig. 6, in about one-half of cases viral inserts were detected either upstream of the gene or in a short sequence of the 3’-untranslated region, also identified as a unique hot spot for retroviral insertions into the murine N-myc gene in T cell lymphomas (Dolcetti et al., 1989; Setoguchi et al., 1989; van Lohuizen et al., 1989). Activated expression of the N-myc 2 retroposed oncogene, frequently correlated with overexpression of c-fos and c-jun,

N-myc2

t

If

t 5

9

7

N-mycl

t 1

c-myc

t 1

t 1

t 1

FIG. 6. Insertion sites of WHV DNA in the c-myc and N-my genes in 49 woodchuck HCCs. The processed N-my2 oncogene, generated by retrotransposition of the parental woodchuck N-myc gene (N-mycl), is specific to the Sciuridae family of rodents. The number of individual WHV insertions found in each region of the myc genes in different woodchuck HCCs is indicated in bold characters. Insertional activation of myc family genes was observed in about 50% of the tumors analyzed.


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was observed in a large majority of woodchuck HCCs (Hsu et al., 1990). Significantly enhanced expression of the human N-myc, c-fos, or c-jun protooncogenes has been observed only occasionally in HBV-associated HCCs (M. A. Buendia, unpublished results), underlying the importance of host cell factors in the differences observed between the tumorigenic processes induced by hepadnaviruses in humans and woodchucks. Further evidence that myc family genes are predominantly implicated in rodent liver tumors associated with hepadnavirus infection comes from other studies of woodchuck and ground squirrel HCCs. In two independent woodchuck tumors, a genetic rearrangement fusing the coding domain of c-myc with the promoter and 5’-translated sequences of a cellular locus termed ‘‘hcr”has been observed (Etiemble et al., 1989; Moroy et al., 1986, 1989; 0. Hino, personal communication). The colocalization of c-myc and hcr on the same woodchuck chromosome (Y, Mizuno, personal communication) suggests that a large intrachromosomal deletion is responsible for the rearrangement, which apparently does not involve the direct interaction of integrated viral sequences. In a recent study of ground squirrel HCCs, frequent amplifications of c-myc were found in tumor cell DNA (6 of 14 cases examined) and were associated with enhanced expression of the oncogene (C. Transy, G. Fourel, P. Marion, and M. A. Buendia, unpublished results). Integration of GSHV DNA into host cell genome, which occurs only rarely in squirrel tumors, has not been correlated with the observed genetic alterations of c-myc. It is interesting to note that such alterations have also been described in some cases of chemically induced liver tumors in rodents (Chandar et al., 1989; Hayashi et al., 1984; Tashiro et al., 1986). Although amplification of c-myc has been observed on rare occasions in HBV-positive human liver tumors (Trowbridge et al., 1988; M. A. Buendia, unpublished results), there is no experimental demonstration, until now, that deregulated expression of myc genes might be generally associated with HBV-induced tumorigenesis in human livers, by any known cis- or trans-acting mechanism. The strategy used by WHV in liver cell transformation now appears strikingly comparable to that of some nonacute retroviruses, like Moloney murine leukemia virus (MoMuLV), which induce disease (usually leukemias) slowly, emphasizing the described similarities between hepadnaviruses and retroviruses (Wain-Hobson, 1984; Miller and Robinson, 1986). These conclusions ask two different, albeit related, questions. What are the factors driving the oncogenic potential of WHV exclusively toward hepatocytes, as we know that this virus can infect a wide variety of woodchuck tissues? How can the apparent differences in

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the strategies of the closely related mammalian hepadnaviruses be explained at the molecular level? To address these issues and identify the genomic variations responsible for such discrepancies, it would probably be instructive to reexamine the retroviral genetic elements influencing disease specificity and latency of different MuLVs (Li et al., 1987; Portis et al., 1991; Thiesen et al., 1988) or the oncogenic properties of human papilloma virus (HPV) strains in different tissues (Miinger et al., 1989; Romanczuk et al., 1991; zur Hausen and Schneider, 1987). VII. Genetic Alterations in HBV-Related Hepatocellular Carcinoma

Different genetic alterations, which cannot be clearly associated to a direct effect of viral infection, have been observed in human HCCs. These somatic changes include allele losses on several chromosomal regions, mutation and activation of cellular genes showing oncogenic potential, and deletion or mutation of a tumor suppressor gene. Search for activated oncogenes using the NIH3T3 cell transformation assay has not been conclusive for most HCC DNAs analyzed. In rare cases, a transforming DNA called “Eca” has been obtained in the transformation assays (Ochiya et al., 1986). This novel oncogene, located on human chromosome 2, is expressed at a proliferative stage in fetal liver, and its activation in liver cancer has not been associated with gross rearrangements of the gene (Matsubara et al., 1987; Shiozawa et al., 1988). Conflicting results have been obtained with human HCC DNA samples using the NIH3T3 cells transformation assay. Barbacid and co-workers have failed to isolate a transforming gene, whereas others report a high incidence of activated N-ras gene (Pulciani et al., 1982; Gu et al., 1986). Indeed, a very low incidence of point mutations in the c-Ha-rm, c-Ki-ras, and N-rm genes has been described in liver tumors (Ogata et al., 1991; Tsuda et al., 1989). Loss of heterozygosity on the distal l p region and on chromosomes 4q, 1 lp, 13q, and 16q appears frequently in human liver tumors, and it has been suggested that these parts of the human genome might contain some genes whose functional loss might be involved in hepatocellular carcinogenesis (Buetow et al., 1989; Pasquinelli et al., 1988; Simon et al., 1991; Tsuda et al., 1990; Wang and Rogler, 1988). Because the largescale chromosomal alterations that arise in cancer cells occur infrequently in normal cells, it is probable that control mechanisms that safeguard chromosomal integrity are abrogated in the development of malignancy (Wright et ul., 1990). These changes might represent secondary events linked to tumor progression and reflect a general property of


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transformed cells; whether HBV DNA integration, which has been shown to promote genetic instability (Hino et al., 1991), contributes to these events has not been elucidated. Allele loss of the short arm of chromosome 17, which includes the p53 gene, has been commonly observed in human HCCs and hepatomaderived cell lines (Slagle et al., 1991; Fujimori et al., 1991). Studies of the p53 gene at the DNA, RNA, and protein levels have revealed abnormal structure and expression in most established HCC cell lines; these alterations, including partial deletions or DNA rearrangements and abnormal expression patterns, might not occur as a late in uitro event and were not correlated with integration of HBV DNA (Bressac et al., 1990). Although viral insertions in chromosome 17p have been described in some liver tumors (Hino et al., 1986; Tokino et al., 1987; Zhou et al., 1988), it seems most probable that the genetic alterations observed in a majority of human HCCk are not due to a direct action of the virus. The wild-type p53 gene seems to regulate negatively the cellular growth and was therefore designated as a tumor suppressor gene or “antioncogene” (see Levine et al., 1991). Mutant forms of p53 frequently gain a growthstimulatory function, and genetic alterations of the gene, a common feature in human neoplasms, generally consist in the deletion of one p53 allele and in the mutation of the second allele. Point mutations that would alter the functional properties of p53 have recently been reported: liver-specific hotspot mutations at the third base of codon 249, consisting of G + T transversions, have been observed in more than 50% of liver cancers from patients residing in South Africa and in the Qidong province of China (Bressac et al., 1991; Hsu et al., 1991). Other studies providing similar results have also shown that two-thirds of the patients with the specific mutation had lost the remaining p53 allele (B. Slagle, personal communication). In these countries, exposure to high levels of dietary aflatoxin is well documented (see Section II,C) and the G T transversions are known to be induced by aflatoxin B 1 (Puisieux et al., 1991). However, this carcinogen also binds other G residues, particularly at codon 248, a mutational hotspot in other tumors but not in HCC. This suggests that additional factors might contribute to the selective targeting to Godon 249. Activation of aflatoxin B1 to metabolites having mutagenic o r DNA-binding activities is mediated by several forms of the human hepatic cytochrome P450 (Aoyama et al., 1991). Whether HBV infection plays part in the induction of p53 mutations is not clear. T h e p53 mutation at codon 249 has not been observed in HBV-related HCCs from patients that did not accumulate high exposure to aflatoxin B 1 (Hayward et al., 1991; Ozturk et al., 1991), or only in rare cases (M. Y. Lai, personal communication). Further studies have

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identified point mutations in other p53 codons in 5 of 55 tumors from Taiwan, a region highly endemic for HBV but not for aflatoxin (M. Y. Lai, personal communications). However, a link between HBV infection and inactivation of a tumor-suppressor gene has not been established.

VIII. Conclusions The exponential relationship of HCC incidence to age indicates that multiple steps, probably involving independent genetic lesions, are required as in other human cancers. In particular, the long latency of HCC development after the initial HBV infection may be interpreted as a sign of an indirect action of the virus: a long-term toxic effect of viral gene products and/or the immune response against infected hepatocytes would trigger continuous necrosis and cell regeneration, which would in turn favor the accumulation of genetic alterations (Chisari et al., 1989b). In this model, productive HBV infections might potentiate the action of exogenous carcinogenic factors, like aflatoxins and alcohol. It might also be speculated that the latency period depends on the occurrence of a decisive HBV integration event, which would promote genetic instability or lead to cis- or trans-activation of relevant genes (Caselmann et al., 1990; Dejean et al., 1986; Hino et al., 1991; Kekule et al., 1990; Wollesheim et al., 1988;J. Wang et al., 1990).Recent investigations of the functional and pathological properties of HBV gene products and of the consequences of HBV integration in the liver DNA suggest that various and probably cooperative mechanisms may operate in the development of liver cancer, and that HBV may share with other human oncogenic viruses a number of basic strategies. The HBV genome encodes at least seven different polypeptides; none of them seems to act as a strong, dominant oncogene, but several lines of evidence indicate that the surface glycoproteins and the viral X trans-activator might participate in carcinogenesis, in a native or modified state. In this respect, it is noteworthy that in oncogenic viruses such as human T cell lymphotropic virus type I (HTLV-I), Epstein-Barr virus (EBV),and human papilloma virus (HPV) 16 and 18, transforming capacity cannot be dissociated from transcriptional trans-activation activity of viral gene products (Cohen et al., 1991; Hawley-Nelson et al., 1989; Henderson et al., 1991; Inoue et al., 1986; Fuji et al., 1991; Kieff and Liebowitz, 1990; Munger et al., 1989). Comparative analyses of the different viral trans-activators might help to guide future work in this field. Studies of mammalian HBV-related viruses have revealed strikingly different mechanisms that might be irrelevant to elucidate the molecular basis of HBV-induced tumorigenesis. However, they have revealed that activation of myc family genes lies at


21 1

the meeting point of the oncogenic pathways triggered by hepadnavirus infections in rodents, as for several human oncogenic viruses. Human papilloma virus infection has been correlated with amplifications and rearrangements of c-myc in cervical tumors and in squamous cell carcinoma of the anus and insertional mutagenesis of c-my or N - m y by HPV DNA has been occasionally observed (Couturier et al., 1991; Crook et al., 1991; Ocadiz et al., 1987); EBV has been associated with the development of Burkitt lymphomas, a tumor characterized by chromosomal translocations joining c-my to different immunoglobulin gene regions (reviewed by Magrath, 1990). So far, a role for HBV in activating the cm y oncogene, suggested by in uitro assays, has not been established in uivo and the identification of the cellular effectors in the HBV-induced transformation pathway remains the main unsolved question. ACKNOWLEDGMENTS I am grateful to C. Brechot, P. Briand, L. Cova, 0. Hino, A. Kekule, and Y. Mizuno for communicating recent unpublished data and to F. Chisari, K. Matsubara, M. Roggendorf, H. Schaller, C. Schroder, C. Seeger, B. Slagle, and H. Will for sending preprints. I also wish to thank M. Robertson for reading the manuscript, P. Tiollais and my collaborators for their critical comments. and L. M. Da for secretarial assistance.

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Hepatocellular carcinoma and hepatitis B surface protein.

Hepatitis B virus and hepatocellular carcinoma.

Clinical aspects and epidemiology of hepatitis B and C viruses in hepatocellular carcinoma in Japan.

Hepatocellular carcinoma, hepatitis B and measles.

Genetic variability of hepatitis B and C viruses in Brazilian patients with and without hepatocellular carcinoma.

Hepatitis B virus infection and primary hepatocellular carcinoma.

Autophagy and microRNA in hepatitis B virus-related hepatocellular carcinoma.

Relation between aflatoxin, hepatitis-B virus, and hepatocellular carcinoma.

Hepatitis B virus infection and primary hepatocellular carcinoma.

Hepatocellular carcinoma and hepatitis B virus markers in Europe.

Myofibroblasts in hepatitis B related cirrhosis and hepatocellular carcinoma.

Hepatitis B virus infection and primary hepatocellular carcinoma.

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma.

Key role of hepatitis B virus mutation in chronic hepatitis B development to hepatocellular carcinoma.

Does hepatitis B seroconversion affect survival outcome in patients with hepatitis B related hepatocellular carcinoma?

Host nucleotide polymorphism in hepatitis B virus-associated hepatocellular carcinoma.

Management of hepatitis B virus infection during treatment for hepatitis B virus-related hepatocellular carcinoma.

Hepatocellular carcinoma in children with hepatitis B surface antigen.

[Incidence of hepatocellular carcinoma in chronic infection with hepatitis B].

Risk scores for hepatocellular carcinoma in chronic hepatitis B.

Dermatomyositis associated with hepatitis B virus-related hepatocellular carcinoma.

Hepatitis B virus antigens in human primary hepatocellular carcinoma tissues.

Long noncoding RNAs in hepatitis B virus-related hepatocellular carcinoma.

Alcohol-dysregulated microRNAs in hepatitis B virus-related hepatocellular carcinoma.