Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-alpha genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.     

Infectious, linear, unintegrated DNA of Moloney murine leukemia virus

A closed circular, double-stranded infectious DNA of Moloney leukemia virus has been described previously. The present report characterizes a second type of infectious, unintegrated viral DNA which is linear, largely double stranded, and of mass comparable to that of the closed circular viral DNA. The linear form is of nonpermuted sequence, and SalI endonuclease cleaves at one site 45% from one end.     

Woodchuck hepatitis virus enhancer I and enhancer II are both involved in N-myc2 activation in woodchuck liver tumors

Direct activation of the N-myc2 oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3' of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.     

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.     

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.     

In vitro transformation by hepatitis B virus DNA

There is strong epidemiological evidence that the hepatitis B virus (HBV) contributes to the development of hepatocellular carcinoma (HCC). In several immortalized cell lines, an in vitro transforming activity of HBV DNA and expression vectors for the viral protein X (HBx) has now been demonstrated. Furthermore, it appears as if still unknown parts of the HBV genome other than HBx contribute to the transforming activity of HBV DNA in vitro. Only one of several studies found that HBx-transgenic mouse lines develop HCC. A mouse line transgenic for the large surface protein of HBV develops HCC due to concomitant necroinflammatory infection. Growing evidence shows the importance of recombination of integrated viral DNA and cellular DNA for HCC development. A direct transforming potential of one of these viral integrates has been demonstrated. Chemical carcinogens are more effective in HBV-containing cell lines or transgenic mice.     

Distance and end configuration effects on VDJP-mediated DNA joining

We have previously reported the cloning of a protein, VDJP, that is capable of binding the nonamer element of the V(D)J Recombinational Signal Sequence (RSS) as well as joining linear DNA fragments containing RSS elements in vitro. We show here that the linearized DNA molecules must contain a 5' extension or blunt end in order to be joined by VDJP. DNAs with 3' extensions are not efficiently joined by VDJP. Furthermore, the joining activity of DNAs with 5' extensions is significantly increased as the distance between the end and the RSS decreases. It is not yet clear what role VDJP plays in vivo, because our assay may not mimic exactly the in vivo DNA intermediates.     

Role of surgical interventions in the treatment of patients with trophoblastic tumor resistant to chemotherapy

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.     

Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.     

Clinical hepatitis after transplantation of hepatitis C virus-positive kidneys: HLA-DR3 as a risk factor for the development of posttransplant hepatitis

BACKGROUND: Exposure to hepatitis C virus (HCV) and subsequent infection after renal transplantation lead to significant clinical hepatitis in approximately 50% of graft recipients. METHODS: One hundred thirty-two consecutive renal allotransplant patients, who underwent transplantation of kidneys from HCV-positive cadaveric donors, were studied to investigate the relationship between donor and recipient HLA type and the risk of developing clinical hepatitis. Specific attention was directed toward the DR3 and DR4 alleles, as these had previously been associated with worse prognoses in autoimmune and viral hepatitis. RESULTS: Overall, 42% of patients receiving kidneys from donors seropositive for HCV developed clinical hepatitis. This was unrelated to preoperative recipient HCV serum reactivity (P=0.65). Patients receiving kidneys from seropositive donors with HCV RNA as detected by PCR were more likely to develop hepatitis than those receiving kidneys from PCR-negative donors (56% vs. 11%; P=0.005). The presence of the DR3 allele was associated with a significant risk of clinical hepatitis (P=0.025); 80% of DR3-positive recipients (n=34) progressed to hepatitis compared with 42% of DR3-negative patients. No other recipient HLA type was significantly related to prognosis. All patients receiving a donated kidney that expressed the B41 allele developed hepatitis, compared with 55% of recipients of non-B41 grafts (P=0.039). No association between the development of clinical hepatitis and HLA compatibility was found. CONCLUSIONS: These results suggest that both HLA type and viral presence as assayed by polymerase chain reaction, influence the risk of disease progression after transplantation of HCV-positive kidneys. Application of these associations may decrease the relative risk of a recipient contracting HCV hepatitis after cadaveric renal transplantation.     

The GDPAL region of the pre-S1 envelope protein is important for morphogenesis of woodchuck hepatitis virus

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.     

A method for producing recombinant baculovirus expression vectors at high frequency

A system has been developed that can generate recombinant baculovirus expression vectors at frequencies approaching 100%. This system provides a selection for recombinant viruses by using the essential gene downstream of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin expression locus. Two AcMNPV derivatives were constructed in which the expression locus and part of the downstream gene are flanked by restriction sites. The parental viruses are viable; however, restriction of the viral DNAs removes an essential piece of the viral genome. Transfer vectors carry a copy of the missing sequences downstream from the site into which foreign genes are inserted for expression; hence, recombination between a transfer vector and the restricted viral DNA can restore the integrity of the essential gene. Such recombination events also transfer any foreign gene present in the expression locus of the transfer vector to the viral genome. Recombinant viruses therefore have a selective advantage over nonrecombinant viral DNAs. Consequently, a high proportion of the viruses obtained by co-transfecting transfer vector DNA and restricted viral DNA of one of these new viruses expresses the target gene from the transfer vector. This system greatly reduces the time needed to make recombinant baculovirus expression vectors.     

Directed integration of minute virus of mice DNA into episomes

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.     

T-Cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.