Maria Laura Avantaggiati

Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo

We have constructed recombinant adenoviruses (Ad), with functional or defective E1a genes, which harbor either the hepatitis B (HB) virus s gene encoding the HB surface antigen, as well as the pre-S2 epitopes, or the bacterial gene encoding chloramphenicol acetyltransferase (CAT) under control of the Ad major late promoter (MLP). The recombinant viruses defective for E1a (Ad.MLP.S2 and Ad.CAT), which can be efficiently propagated only on 293 cells that complement this defect, and the nondefective (Ad.MLP.S2.E1A) recombinant were used to infect a wide spectrum of cells of different origin. The yields of HBs and CAT proteins obtained with these different recombinant viruses demonstrate no real advantage to using nondefective vectors, whatever the cell type infected. The injection into chimpanzees of Ad.MLP.S2 does not elicit the production of antibodies, but can immunologically prime the animals, resulting in a partial protection against HBV challenge.

Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX.

The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.

Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cMYC protooncogene at the transcriptional level

The products of the human hepatitis B virus (HBV) and woodchuck hepatitis B virus X genes (pXs) transactivate homologous and heterologous genes including the HBV-X and core promoters, the human immunodeficiency viruses 1 (HIV-1) and 2 (HIV-2) long terminal repeats and the beta interferon regulatory sequences. We report here that pX is also able to influence the expression of both extrachromosomal transfected c-myc regulatory sequences and endogenous c-myc gene. pX acts by increasing transcription of the c-myc gene and do not affect c-myc mRNAs stability. The presence of the first AUG of the X-ORFs is indeed necessary for the production of an active pX. The very carboxyterminus of the pX protein is dispensable for this transactivating activity and at least one domain important for its action is located between aminoacids 103 and 117.

Antibodies to hepatitis C virus in patients with hepatocellular carcinoma

To study the potential relationship between the hepatitis C virus (HCV), the major etiologic agent of parenterally transmitted non-A, non-B hepatitis, and the development of hepatocellular carcinoma (HCC), we tested the presence of anti-HCV antibodies in sera from a large panel of HCC patients of different racial and geographical origins. Anti-HCV antibodies were detected in 82 out of 114 (71.9%) HBsAg-negative HCC patients and in 15 out of 53 (28.3%) HBsAg-positive patients. No significant difference in the prevalence of anti-HCV antibodies was found in the HBsAg-negative HCC patients when they were divided according to presence of anti-HBs and/or anti-HBc antibodies, or absence of all hepatitis B virus (HBV) serological markers. The prevalence of anti-HCV antibodies was similar in HCC patients of Caucasian and African origin. No differences were noted when the patients were grouped according to sex. The mechanisms by which HCV might contribute to the development of HCC need to be further investigated. As for HBV infection, the necro-inflammation associated with HCV infection may induce cirrhosis, regeneration and eventually malignant transformation. The finding that few anti-HCV patients had HCC which is not superimposed on cirrhosis suggests that HCV could, however, exert some direct effect on the development of HCC.

The hepatitis B virus X protein transactivation of c-fos and c-myc proto-oncogenes is mediated by multiple transcription factors.

We have constructed two expression vectors in order to study the action of the HBV 17 Kd X protein on the c-fos and c-myc promoters. The results show that the promoters contain multiple elements that respond to X protein, suggesting involvement of multiple transcription factors. The exact mechanism of the interaction remains elusive, but our data allow speculation about the factors that may be influenced.

Truncated pre-S/S proteins transactivate multiple target sequences

In order to investigate the transactivational function of HBV truncated preS/S proteins we have constructed two sets of plasmids and have tested their transactivational potential on the c-myc regulatory sequences and the TPA-responsive element. We found that preS/S proteins only become transactivationally active when truncated at the carboxy terminal end. Furthermore, using immunofluorescence microscopy we determined that the proteins are located exclusively in the cytoplasm, apparently ruling out DNA binding and activation of factors in the nucleus.

The AP1 Transcription Factor as a Model to Study the Modulation of Intracellular Signalling Pathways by the Hepatitis B Virus Transactivator pX

The Hepatitis B Virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP-1. In this study we have analyzed the mechanisms of AP-1 induction by pX. The activation of API dependent transcription in HepG2 cells was associated with an increase in the AP-1 DNA-binding activity, that was not dependent on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on dimers formation between pX and the two proteins, thus suggesting the involvement of post-translational modifications of the transcription factor. The increased API binding observed in presence of pX was unaffected by the protein-kinase C (pKC) inhibitors Calphostin C and Sphingosine, and by the protein-kinase A (pKA) inhibitor HA1004, while it was almost completely blocked by Staurosporine, a potent and relatively non-specific protein-kinases inhibitor, suggesting that pKC- and pKA-independent phosphorylation events might play a role in the phenomenon.