[Show abstract][Hide abstract] ABSTRACT: With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1alpha and HNF 4alpha, two transcription factors essential for HBV gene expression and replication. CONCLUSION: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.
[Show abstract][Hide abstract] ABSTRACT: Adenovirus type 12 (Ad12) propagation in hamster BHK21 cells is blocked prior to viral DNA replication. The amounts of Ad12 DNA in the nuclei or cytoplasm of hamster cells are about 2 orders of magnitude (2 h postinfection [p.i.]) and 4 to 5 orders of magnitude (48 h p.i.) lower than in permissive human cells. Cell line BHK21-hCAR is transgenic for and expresses the human coxsackie- and adenovirus receptor (hCAR) gene. Nuclear uptake of Ad12 DNA in BHK21-hCAR cells is markedly increased compared to that in naïve BHK21 cells. Ad12 elicits a cytopathic effect in BHK21-hCAR cells but not in BHK21 cells. Quantitative PCR or [(3)H]thymidine labeling followed by zone velocity sedimentation fails to detect Ad12 DNA replication in BHK21 or BHK21-hCAR cells. Newly assembled Ad12 virions cannot be detected. Thus, the block in Ad12 DNA replication in hamster cells is not released by enhanced nuclear import of Ad12 DNA.
Journal of Virology 05/2008; 82(8):4159-63. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induction of heme oxygenase-1 (HO-1) has been shown to be beneficial in immune-mediated liver damage. We now investigate the effects of HO-1 induction in models of human hepatitis B virus (HBV) infection.
Adenoviral transfer of an HBV 1.3 genome into wild-type mice was used as a model for acute hepatitis B. HBV transgenic animals were used as a model for chronic HBV infection. HBV replication was assessed by HBV viremia, antigenemia, and Southern blotting, liver damage was assessed by serum alanine aminotransferase activities and histopathology of liver sections. To investigate HO-1 effects on HBV replication at a molecular level, stably HBV-transfected hepatoma cells were used. HBV gene expression, protein stability, transcription, and replication were determined. HO-1 was induced by either cobalt-protoporphyrin-IX or over expressed by adenoviral gene transfer.
In the acute hepatitis B model, liver injury was reduced significantly after HO-1 induction. In addition, HO-1 showed a pronounced antiviral effect, which was confirmed in stably HBV-transfected hepatoma cells and in persistently HBV replicating transgenic mice. We showed that HO-1 induction repressed HBV replication directly in hepatocytes at a posttranscriptional step by reducing stability of HBV core protein and thus blocking refill of nuclear HBV covalently closed circular (ccc)DNA. Small interfering RNA directed against HO-1 proved that this effect depended on the expression level of HO-1.
Besides its hepatoprotective effect, HO-1 showed a pronounced antiviral activity in HBV infection. Therefore, induction of HO-1 might be a novel therapeutic option for inflammatory flares of hepatitis B.
[Show abstract][Hide abstract] ABSTRACT: The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.
Journal of Virology 03/2005; 79(4):2404-12. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ad12 oncogenesis in hamsters has been studied in detail to provide the following new data in this tumor model. Cells in the Ad12-induced tumors, often thought to be of neuronal origin, actually exhibit mesenchymal and neuronal characteristics and are probably of an undifferentiated derivation. Their intraperitoneal spread upon intramuscular injection of Ad12 adds another important new aspect. Differences in the integration patterns among the tumors suggest clonal origins from individual transformation events. Ad12 gene expression in the tumors is determined, at least in part, by the patterns of DNA methylation imprinted de novo upon the integrated Ad12 genomes. Differential Ad12 gene expression patterns, which have previously not been described in tumors, are an important parameter in Ad12 oncogenesis. The availability of cellular DNA arrays has opened up unprecedented possibilities to document changes in cellular transcription patterns, particularly of cancer-specific genes. These patterns exhibit differences and similarities among the different Ad12-induced tumors. Among the cellular genes, which are expressed in the Ad12-induced tumors, many are cancer-specific. We pursue the hypothesis that these alterations in cellular transcription patterns as a consequence of viral DNA integration and expression play an essential role in Ad12 oncogenesis.
Current topics in microbiology and immunology 02/2004; 273:215-44. · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intramuscular (i.m.) injection of human adenovirus type 12 (Ad12) into newborn Syrian hamsters caused widespread dissemination of up to 15 tumors over the entire peritoneal cavity in 70-90% of the animals within 30-50 days. Subcutaneous (s.c.) injections led to local tumor formation only. Independent of location, tumor histology revealed Homer-Wright rosette-like structures typical for primitive neuroectodermal tumors (PNET). All tumor cells showed markers indicative of neuroectodermal and mesenchymal derivations. Each Ad12-induced tumor cell carried multiple copies of integrated Ad12 genomes at one chromosomal site which was different for each tumor. For Ad12 tumor induction in hamsters, the patterns of Ad12 viral and cellular gene expression were important and were affected by changes in DNA methylation, both in the integrated Ad12 DNA and the cellular genome. By applying the bisulfite protocol, the de novo DNA methylation in the integrated Ad12 genomes was determined. These patterns were complex, characterized by regional initiation and by excluding genome segments in the E1A and E1B promoters. In all tumors, the Ad12 segments E1A, E1B, E2A, parts of E3 and E4 were similarly transcribed, as shown by the RT-PCR and DNA microarray methods. Changes in the transcription of a large number of cellular genes was assessed by using mouse gene microarrays encompassing about 1980 different mouse genes with 87-96% homology to hamster genes. Similarities and differences existed in the transcription of cellular genes of different functional classes among the different Ad12-induced tumors. These alterations in cellular gene transcription may be an important parameter in the oncogenic transformation by Ad12.
Virus Research 01/2004; 98(1):45-56. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human adenovirus type 12 (Ad12) induces undifferentiated tumors in newborn Syrian hamsters, and this tumor model has been investigated in detail in our laboratory. One of the characteristics of the Ad12-hamster cell system is a strictly abortive infection cycle. In this chapter, we summarize previous and more recent results of studies on the interaction of Ad12 with the nonpermissive BHK21 hamster cell line. The block of Ad12 replication lies before viral DNA replication and late gene transcription which cannot be detected with the most sensitive techniques. Ad12 adsorption, cellular uptake and transport of the viral DNA to the nucleus are less efficient in the nonpermissive hamster cells than in permissive human cells. However, most of the early functions of the Ad12 genome are expressed in BHK21 cells, though at a low level. In the downstream region, the first exon, of the major late promoter (MLP) of Ad12 DNA, a mitigator element of 33 nucleotide pairs in length has been identified which contributes to the inactivity of the MLP in hamster cells and its markedly decreased activity in human cells. The E1 functions of Ad2 or Ad5 are capable of partly complementing the Ad12 deficiencies in hamster cells in that Ad12 viral DNA replication and late gene transcription can proceed, e.g. in a BHK hamster cell line, BHK297-C131,which carries in an integrated form and constitutively expresses the E1 region of Ad5 DNA. Nevertheless, the late Ad12 mRNAs, which are synthesized in this system with the authentic nucleotide sequence, fail to be translated to structural viral proteins. Hence, infectious virions are not produced in the partly complementing system. Probably there is also a translational block for late Ad12 mRNAs in hamster cells. We have recently shown that the overexpression of the Ad12 preterminal protein (pTP) gene or of the E1A gene facilitates the synthesis of full-length, authentic Ad12 DNA in BHK21 cells infected with Ad12. Apparently the pTP has a hitherto unknown function in eliciting full cycles of Ad12 DNA replication even in nonpermissive BHK21 cells when sufficient levels of Ad12 pTP are produced. We pursue the possibility that the completely abortive infection cycle of Ad12 in hamster cells ensures the survival of Ad12-induced hamster tumor cells which all carry, integrated in their genomes, multiple copies of Ad12 DNA. In this way, the viral genomes are immortalized and expanded in a huge number of tumor cells.
Current topics in microbiology and immunology 02/2003; 272:415-40. · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction of human adenovirus type 12 (Ad12) with Syrian hamster cells is remarkable in that there is a block of viral DNA replication and late gene transcription. We have screened several cellular factors known to play a role in adenovirus replication for their possible contributions to the interactions of Ad12 in the abortive BHK21 hamster cell system. (1) Western blot analyses of total protein extracts from Ad12- or Ad2-infected BHK21 cells do not reveal a significant difference in the accumulation of NFIII protein at different times after infection. Transcriptional levels of the NFIII gene in BHK21 cells are not altered upon the abortive infection with Ad12 or the productive infection with Ad2. The amount of NFIII protein is markedly reduced in nuclear extracts from BHK21 cells as compared with extracts from C131 hamster cells or human HeLa cells. A presumptive defect in NFIII transport to the nuclei rather than overall reduced NFIII gene transcription might explain the low abundance of NFIII in the nuclei of uninfected or Ad12-infected BHK21 cells. The productive infection of BHK21 or C131 cells with Ad2 leads to an increase in the NFIII concentration in the nuclei of infected cells, late after infection to a decrease; (2) NFI levels in the nuclei of mock-infected or Ad2- or Ad12-infected BHK21 cells are comparable with those in HeLa or in C131 cells. Thus, deficiencies in NFI may not play a role in the abortive system; (3) The absence of morphological alterations in PML protein domains from globular to track-like structures in the nuclei of Ad12-infected hamster cells correlates with the inability of Ad12 DNA to replicate in BHK21 cells. In BHK21 cells, the E4-ORF3 of Ad12 DNA is only weakly transcribed and only small amounts of the gene product are synthesized. In Ad12-infected C131 cells, which allow the replication of Ad12 DNA, the E4-ORF3 of Ad12 DNA is expressed, and track-like PML protein structures are observed. Transfection of the 12-E4-ORF3-EGFP construct leads to the expression of both the green fluorescent protein (GFP) and of the 12-E4-ORF3 gene product in 20-30% of the transfected BHK21 cells and elicits the morphological reorganization of the PML protein structures in the successfully transfected BHK21 cells. Similar results are obtained upon transfection of the 2-E4-ORF3 construct. Untransfected cells or cells transfected with the empty pIRES2-pEGFP vector carry the globular PML protein phenotype; (4) The expression of the 12-E4-ORF3-EGFP and/or of the NFIII-EGFP constructs upon transfection following Ad12-infection of BHK21 cells fails to promote Ad12 DNA replication. Hence, the formation of track-like PML protein structures in BHK21 cells by itself is not a sufficient precondition for Ad12 DNA replication in this abortive system. The data demonstrate that the expression of NFI, NFIII, and/or the conversion of the PML domains do not suffice to elicit Ad12 DNA replication in the abortive hamster cell system.
Virus Research 01/2002; 81(1-2):1-16. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP.
Journal of Virology 12/2001; 75(21):10041-53. · 5.08 Impact Factor