Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1.

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.

Construction of adenoviral and retroviral vectors coexpressing the genes encoding the hepatitis B surface antigen and B7-1 protein.

Recombinant retroviral (re-Rv) and adenoviral (re-Ad) vectors for delivery of two foreign genes were constructed, using the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) which mediates initiation of cap-independent translation. The first gene encoded the hepatitis B surface antigen (HBsAg) and the second encoded human or murine B7-1 molecule, a cell surface protein which is a costimulator for T cell activation. The EMCV IRES sequence was placed between the first and second coding sequences to form a dicistronic DNA fragment. In Rv vectors, the dicistronic fragment was inserted between the 5' long terminal repeat (LTR) and an internal promoter for the neomycin (neo) gene, so that the transcription initiated from the 5' LTR would generate a dicistronic mRNA for the HBsAg and B7-1 molecules. For Ad vectors, the dicistronic fragment was inserted between a cytomegalovirus promoter and a polyA signal to form a transcription cassette. This transcription cassette was inserted into the early region 1 of Ad5 genome to form a replication-defective re-Ad vector, or into early region 3 to form replication-competent vectors. Human cell line A549 infected with the re-Rv vectors or with the re-Ad vectors synthesized and secreted HBsAg at comparable levels, while the B7-1 molecules were detected at the surface of the infected cells, indicating both foreign genes carried by the Rv and Ad vectors were expressed efficiently.

Costimulatory protein B7-1 enhances the cytotoxic T cell response and antibody response to hepatitis B surface antigen.

There is a need for more effective therapy for chronic virus infections. A principle natural mechanism for elimination of virus-infected host cells is activation of viral antigen-specific cytotoxic T lymphocytes (CTL). In an effort to develop methods of inducing virus-specific CTL responses that might be utilized in therapy of virus infections, we have investigated the effect of B7, a costimulatory factor for T-cell activation. In this study we show that delivery of genes encoding human B7-1 and a viral antigen in the same recombinant viral vector to cells of mice induces a greater viral antigen-specific CTL response than does similar delivery of the viral antigen gene alone. Two recombinant adenovirus vectors were constructed with the foreign genes inserted in the early region 3. One of them (Ad1312) directed expression of the surface antigen gene of hepatitis B virus (HBS); the other (Ad1310) directed coexpression of HBS and human B7-1 (CD80) by means of an internal ribosomal entry site placed between the two coding sequences. When inoculated into BALB/c mice, both vectors induced a viral surface antigen-specific CTL response. The response induced by Ad1310 was stronger than that by Adl312 as measured by a chromium release assay for CTL activity and limiting dilution analysis for CTL precursor frequency, indicating that the B7-1 gene co-delivered with the HBS gene had an enhancing effect on the CTL response against surface antigen. Ad1310 also induced a higher titer of antibody against surface antigen than did Ad1312. This result suggests that expression of a costimulatory protein and a viral antigen in the same cells in vivo induces stronger immune responses than expression of the antigen alone. This could be a novel strategy for development of both preventive and therapeutic vaccines against infectious agents.

State of the p53 gene in hepatocellular carcinomas of ground squirrels and woodchucks with past and ongoing infection with hepadnaviruses.

Infection with hepadnaviruses and exposure to dietary aflatoxin are considered major risk factors in the development of hepatocellular carcinoma (HCC) both in humans and in animals. Recently, a broad range of mutations in the p53 tumor suppressor gene has been reported in human HCCs, predominantly from hepatitis B virus carriers in areas with either high or low levels of exposure to dietary aflatoxin. To determine whether p53 mutations are common to HCCs of hosts infected with related hepadnaviruses with and without treatment with aflatoxin, we studied the occurrence of mutations in the p53 gene in HCCs of ground squirrels and woodchucks with history of infection with ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus, respectively. Sequencing of wild type p53 genes from ground squirrels and woodchucks revealed remarkable homology between the two species with only a few amino acid differences in exons 4, 8, and 9. Using direct polymerase chain reaction sequencing, we analyzed the state of the p53 gene (exons 4-9) in 20 HCCs from ground squirrels (2 uninfected, 7 with past, and 11 with ongoing infection with GSHV) and in 11 HCCs from woodchucks persistently infected with woodchuck hepatitis virus. Five GSHV carrier and two uninfected ground squirrels received i.p. administration of aflatoxin B1. We detected only one mutation in the p53 gene of the tested animals. This mutation was located in codon 176 of exon 5 in the HCC of a GSHV-positive ground squirrel treated with aflatoxin. Mutation was caused by a G to T transversion in the second position of the codon, resulting in the replacement of cysteine with phenylalanine, and was accompanied by a tumor-specific loss of heterozygosity. p53 allelic amino acid variation with sequences coding for aspartic acid or asparagine was present in codon 61 in the variable region of exon 4 in both HCCs and nonneoplastic tissues of ground squirrels. In view of the considerably lower apparent rate of mutations in comparison to human HCCs, we suggest a less important role for aflatoxin in the induction of p53 mutations in HCCs of ground squirrels. Alternatively, etiological factors other than p53 mutations may be of greater significance in the development of HCC in ground squirrels and woodchucks.

Selected mutations of the duck hepatitis B virus P gene RNase H domain affect both RNA packaging and priming of minus-strand DNA synthesis.

The genome of all hepadnaviruses has an open reading frame called the P gene, which encodes a polypeptide of 90 to 97 kDa. The product or products of this P gene are involved in multiple functions of the viral life cycle. These functions include a priming activity which initiates minus-strand DNA synthesis, a polymerase activity which synthesizes DNA by using either RNA or DNA templates (reverse transcriptase), a nuclease activity which degrades the RNA strand of RNA-DNA hybrids (RNase H), and involvement in packaging the RNA pregenome into nucleocapsids. In a previous study, we found that a single point mutation at position 711 in the duck hepatitis B virus (DHBV) P gene product RNase H domain prevented viral RNA packaging. In the present experiments, we have mutated additional conserved amino acids in the DHBV RNase H domain and examined the ability of viral genomes containing these mutations to package RNA and replicate viral DNA. Charged and sulfur group amino acids adjacent to Cys-711 were mutated. None of these mutants was defective in either RNA packaging or viral replication. We also tested a number of mutations on the basis of common elements in the crystal structures of Escherichia coli and human immunodeficiency virus reverse transcriptase RNase H enzymes and on the basis of the similarities of their amino acid sequences to those of the RNase H domains of DHBV and HBV. Our results revealed that the entire beta 4 strand and amino acids Leu-712, Leu-697, and Val-719 in the putative hydrophobic cores of the beta 4, alpha A, and alpha B regions, respectively, are involved in pregenomic RNA encapsidation. This suggests that the basic structure of the RNase H domain in the DHBV P gene product is required for viral RNA packaging. We used the in vitro DHBV minus-strand DNA priming system developed by Wang and Seeger (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992) to test the effect of RNase H packaging mutations on P gene product enzymatic activity. While all packaging-defective mutants tested maintained DNA priming activity, levels were decreased 5- to 20-fold compared with that of the wild-type genome. This observation suggests that the hepadnavirus RNase H domain plays a role in optimizing priming of minus-strand DNA synthesis.

Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.

Molecular events in the pathogenesis of hepadnavirus-associated hepatocellular carcinoma.

Chronic hepadnavirus infection is associated with hepatocellular carcinoma (HCC) in natural hosts such as humans, woodchucks, and Beechey ground squirrels. Several possible oncogenic mechanisms have been identified, including a potential role of the hepadnavirus x (hbx) gene, which transactivates transcription regulated by certain cis-acting sequences, e.g. regulatory sequences of the hepatitis B virus (HBV) and heterologous regulatory sequences of other viruses and cellular genes. The oncogenic potential of hbx is suggested by the observation of HCCs in hbx transgenic mice, the oncogenic transformation of cells expressing hbx in culture, and the transactivation of oncogenes c-myc and c-jun by hbx. Cis-activation of cellular oncogenes N-myc and c-myc by viral promoter insertion has been a common finding in woodchuck hepatitis virus (WHV)-associated HCCs of woodchucks. No such cis-activation of any cellular gene has been shown in virus-associated HCCs of ground squirrels or humans. Amplification and overexpression of the c-myc gene has been a common finding in HCCs of ground squirrels, and is rare in woodchuck or human HCCs. Point mutations in the p53 gene and allelic deletion of p53 have been common findings in human HCCs, but have not been found in HCCs in woodchucks and have been found rarely in ground squirrels. How each of these genetic changes in the different hosts contributes to HCC remains to be determined, but apparently different changes in different HCCs of hepadnavirus-infected hosts suggest that several separate genetic events may contribute to the development of HCC. These events may differ in each host, and some may not result from a direct virus-specific mechanism. Chronic hepadnavirus infection is often associated with chronic necroinflammatory liver disease and cirrhosis, a pathologic process common to several other risk factors for HCC. This suggests that this pathologic process (necroinflammatory disease) may be hepatocarcinogenic regardless of the inciting agent. Thus hepadnavirus infection may play an important role in the development of HCC by causing chronic hepatitis and HCC with the same mechanisms by which other risk factors for HCC cause chronic necroinflammatory liver disease and HCC.

Presence of viral replicative intermediates in the liver and serum of patients infected with hepatitis C virus.

Hepatitis C virus (HCV) is a positive-polarity, single-stranded RNA virus, distantly related to the pestivirus and flavivirus genera. These viruses replicate through the formation of a minus-strand RNA intermediate, which encodes the positive-strand genome, which is subsequently encapsidated, enveloped, and released from infected cells. Minus-strand RNA is not found in the mature, circulating virions of flaviviruses. In an attempt to study the relative amounts of viral plus and minus strand in the liver and serum of HCV-infected individuals, we have developed a technique to amplify specifically each of the viral strands using a modified reverse transcriptase/polymerase chain reaction protocol on extracted RNA. Liver tumor and nontumor tissue from a patient with C-100-3 antibody was analyzed using this technique. In both cases, viral plus and minus strands were detected, although the plus-strand signal was several fold stronger than minus-strand signal by Southern hybridization. Sera from 11 C-100-3 antibody-positive patients with abnormal serum AIT levels were similarly analyzed. In all cases viral plus strand was detected, and in 10 of 11 cases viral minus strand was detected. The minus-strand signal was always much weaker than the plus-strand signal and the ratio of plus strand to minus strand varied among patients. No correlation was found between the level of minus strand detected or its ratio to plus strand with the level of serum transaminases or any other clinical parameter.

Activation of protooncogene c-jun by the X protein of hepatitis B virus.

A specific viral oncogenic mechanism has not been shown for hepatitis B virus (HBV), although persistent HBV infection has been strongly associated with the development of hepatocellular carcinoma (HCC). Most HCCs in HBV carriers contain integrated viral sequences in host DNA and this raises the question of whether such integrations ever contribute to oncogenesis. HBV does contain a gene (designated the hbx gene) which encodes a transcriptional trans-activator protein capable of activating homologous and heterologous regulatory sequences. Hbx has been detected in some human HCC with HBV integrations and the expressed hbx protein appears to have transcriptional transactivating activity. These findings raise the possibility that hbx expression could contribute to hepatocarcinogenesis by activating cellular genes that could contribute to oncogenicity. The possibility that the hbx protein may activate certain protooncogenes was investigated and we found that hbx can activate the protooncogene c-jun promoter. c-Jun was found to be expressed at a very low level in normal liver tissue but at high levels in HCCs of HBV-infected patients.

The role of hepatitis B virus in the development of primary hepatocellular carcinoma: Part I.

Chronic infections with hepatitis B virus (HBV) of humans and animal hepadnavirus infections in their natural hosts are strongly associated with primary hepatocellular carcinoma (HCC). Although viral integrations are found in cells of many HCC, no general viral-specific hepatocarcinogenic mechanism for hepadnaviruses has been identified. In approximately one half of HCC in woodchuck hepatitis virus (WHV) infected woodchucks, viral integrations near the c-myc or N-myc genes have been reported which result in enhanced expression of the respective gene. Such host gene-specific insertional mutagenesis has not been found in HCC of other hepadnavirus infected hosts. Thus in humans, ground squirrels and ducks hepadnaviral integrations appear to be at different host chromosomal DNA sites in each HCC and few integrations have been found within or near any cellular gene. Other possible hepadnavirus-specific carcinogenic mechanisms that are being investigated include transactivation of cellular gene expression by an hepadnavirus gene product (e.g. the X-gene), and mutation of host genes by unknown hepadnavirus-specific mechanisms. It should be noted, however, that chronic hepadnavirus infection is associated with chronic necroinflammatory liver disease with hepatocellular necrosis and regeneration (sometimes leading to cirrhosis in humans), a pathological process that is common to numerous other risk factors for HCC. This suggests the possibility that this pathological process is hepatocarcinogenic irrespective of the inciting agent and the role of hepadnavirus infection is no different from that of other risk factors in causing chronic necroinflammatory liver disease.

Frequent amplification of c-myc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus.

Persistent infection with hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC) in humans. HCC has also been observed in animals chronically infected with two other hepadnaviruses: ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV). A distinctive feature of WHV is the early onset of woodchuck tumors, which may be correlated with a direct role of the virus as an insertional mutagen of myc genes: c-myc, N-myc, and predominantly the woodchuck N-myc2 retroposon. In the present study, we searched for integrated GSHV DNA and genetic alterations of myc genes in ground squirrel HCCs. Viral integration into host DNA was detected in only 3/14 squirrel tumors and did not result in insertional activation of myc genes, despite the presence of a squirrel locus homologous to the woodchuck N-myc2 gene. This suggests that GSHV may differ from WHV in its reduced ability to induce mutagenic integration events. However, the high frequency of c-myc amplification (6/14) observed in ground squirrel HCCs indicates that myc genes might be preferential effectors in the tumorigenic processes associated with rodent hepadnaviruses, a feature not reported so far in HBV-induced carcinogenesis. Together with previous observations, our results suggest that hepadnaviruses, despite close genetic and biological properties, may use different pathways in the genesis of liver cancer.

Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging.

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.

Effects of purine nucleoside analogues with a cyclobutane ring and erythromycin A oxime derivatives on duck hepatitis B virus replication in vivo and in cell culture and HIV-1 in cell culture.

The effects on duck hepatitis B virus (DHBV) replication of specific analogues of two classes of chemical compounds not previously tested against hepadnaviruses are described. One is erythromycin A-9-methyloxime (EMO) and other oxime derivatives of erythromycin A, and the other is purine nucleoside analogues (cyclobut A and cyclobut G) with cyclobutane rings. Viral replication was assessed by measuring serum levels of DHBV DNA in infected ducklings and DHBV DNA in infected primary duck hepatocyte cultures. Administration of EMO 15 mg/kg of body weight IM to infected ducklings resulted in a rapid fall in DHBV DNA levels during therapy and a return to pretreatment levels after EMO administration was stopped. There was local toxicity at injection sites with muscle necrosis in some animals. When 100 mg/kg EMO was administered by gastric tube no such viral response was observed. The difference in virus response to EMO 15mg/kg IM and 100 mg/kg by gastric tube was not due to failure to achieve comparable blood and tissue levels of EMO administered by the different routes. The results suggest an indirect effect dependent on IM injection of EMO rather than a direct antiviral effect of the compound. Administration of cyclobut G or cyclobut A at 70 mg/kg IM led to a rapid reduction of DHBV DNA to undetectable levels in serum, and in only 1 of 4 animals did DHBV DNA became detectable again within 10 days after stopping the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of glove combinations in reducing cell culture infection after glove puncture with needles contaminated with human immunodeficiency virus type 1.

OBJECTIVE: To study the effect of various latex and treated glove combinations in reducing the frequency of human immunodeficiency virus (HIV) infection of tissue culture cells after puncture by surgical needles contaminated with infectious human immunodeficiency virus type 1 (HIV-1). DESIGN: One, two, or three layers of sterile latex glove material, or two latex layers with intermediate cotton or Kevlar (with or without the virucidal compound nonoxynol-9) were used to cover 24-well cell culture dishes containing MT2 cells in cell culture medium. Surgical needles wet with cell culture medium containing HIV-1 (HTLV IIIA strain) were passed through the glove materials into the culture medium in the wells of the culture dishes. The culture medium in each well was then assayed biweekly for HIV-1 p24 antigen as a test for infection of cells in the well. RESULTS: The rate of HIV-1 infection of cell cultures after glove puncture was greater than 90% with a single latex surgical glove barrier, 23% to 60% with double or triple layers of latex gloves, less than 8% with an intermediate cotton glove impregnated with 4% nonoxynol-9, 6% with an intermediate Kevlar glove, and 0% with an intermediate Kevlar glove impregnated with nonoxynol-9. CONCLUSIONS: An intermediate glove of Kevlar or of Kevlar or cotton impregnated with virucidal compound nonoxynol-9 between standard latex gloves may improve surgical glove safety, compared with latex gloves alone with respect to needlestick transmission of HIV-1. The experimental model used may permit rapid investigation of other glove systems as barriers to the transfer of infectious agents through gloves by needlestick.

Peptide mapping of neutralizing and nonneutralizing epitopes of duck hepatitis B virus pre-S polypeptide.

Antibodies to the envelope proteins of duck hepatitis B virus neutralize viral infection in vitro. Using a library of murine monoclonal antibodies (Mabs) against the envelope proteins, we previously identified four neutralizing and two non-neutralizing epitopes on the pre-S region of the large envelope proteins. In this study we report the localization of all but one of these epitopes at the amino acid level. All but 28 nucleotides of the pre-S and S genes were cloned in pUC vectors and expressed in Escherichia coli. All Mabs in this study reacted with the expressed gene products in Western blots. Deletion mutants of the pre-S region were generated and their expressed products tested on Western blots for reactivity with the Mabs. Of the three epitopes involved in neutralization, the epitope found to be immunodominant in convalescent ducks was localized to nine amino acids of the middle portion of the pre-S gene product, while a second epitope was mapped to nine amino acids upstream of the immunodominant epitope and the third epitope to seven amino acids adjacent to the S gene. One of the two non-neutralizing epitopes was located between the two groups of neutralizing epitopes while the other mapped to the same region as one of the neutralizing epitopes. Our data indicate that several regions of the pre-S polypeptide may play a role in neutralization of hepadnaviruses.