Biomedical subjects
Publications and source records attributed to M Perricaudet.
We have evaluated the use of Adenovirus as a new vector for gene transfer in vivo. The capacity of this virus to express a foreign gene even in the absence of viral and cellular replication makes it attractive to deliver a gene to quiescent cells. We show here that organs like lung, liver and muscles are relevant to this strategy.
The Epstein-Barr virus (EBV) is a ubiquitous Herpes virus which causes infectious mononucleosis and is associated with such different neoplasms as Burkitt's lymphoma and nasopharyngeal carcinoma. EBV latently infects its target cells; nevertheless, evidence of viral replication in NPC tumours has been uncovered. Among the EBV transactivators, the ZEBRA protein plays a crucial role in switching the virus from a latent to a productive mode. ZEBRA protein was produced using a eukaryotic expression vector: the open reading frame containing the BZFLI cDNA has previously been inserted down-stream from the adenovirus major late promoter leading to expression of a 38-kDa nuclear protein. We performed serological studies by employing ZEBRA protein expressed in human cells for immunofluorescence and Western-blot assays. We were able to detect IgG anti-ZEBRA antibodies (IgG/ZEBRA) in 87% of NPC patients. These antibodies were absent in control sera; IgG/ZEBRA antibodies can be proposed as a useful marker for diagnosis of NPC tumors.
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.
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.
The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.
The modulation of nuclear antigen expression from the Epstein-Barr virus (EBV) genome in virus-infected cells is crucial to the establishment and maintenance of the immortalized state. The EBV nuclear antigen (EBNA) genes are all transcribed from either of two promoters whose alternate usage is not understood. We have studied the EBNA transcriptional domain by using various promoter constructs to transfect a variety of EBV positive cell lines and then measured promoter activity by RNase protection analysis and nuclear run-on transcription assay. These experiments have shown that at least two distinct and novel DNA sequence elements are located in the intronic region situated between both promoters and that their presence results in a 100-fold stimulation of transcription initiated from the upstream promoter. One of these elements is shown to bind nuclear proteins from an EBV-positive cell line. In addition, an important sequence homology is noted between this element and the core sequences of a number of well known viral enhancers. Taken together, the results show that EBNA transcription is controlled by upstream and intronic elements in a regulatory domain that is at least 7 kb long.
Patients with AIDS and AIDS-related complex often show symptoms of Epstein-Barr virus (EBV) reactivation. Several EBV-encoded trans-acting factors activate the EBV lytic cycle, and one, ZEBRA (BamHI Z EBV replication activator; also called EB1), switches EBV from its latent to productive cycle. Indirect immunofluorescence studies were done using human cells transfected with a recombinant DNA-harboring cDNA sequence spanning BZLF1 (BamHI Z left frame 1) that was inserted downstream of the adenovirus major late promoter. IgG anti-ZEBRA antibodies were detected in a high proportion of asymptomatic HIV carriers and in AIDS patients but were absent in healthy control individuals. The presence of anti-ZEBRA antibodies in the sera of HIV-positive patients favors the hypothesis that EBV reactivates in such subjects. This finding may be of practical importance in the prognostication of AIDS development.
The immortalization in vitro of human B cells by the Epstein-Barr virus (EBV) is associated with the expression of a restricted group of nuclear proteins (EBNA). EBNA transcription is driven from either of two mutually exclusive promoters whose modulation is likely to involve an important host/virus interaction. Our experiments on the EBNA transcriptional domain have led us to identify several new transcription elements which are located on the DNA sequence between the two promoters. These elements stimulate the activity of the upstream EBNA promoter by a factor of at least one hundred. One of these elements may also be involved in the in vivo reactivation of EBV in certain cases.
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Recently a small number of Epstein-Barr viral (EBV) genes, characteristically expressed in latently infected, growth-transformed B-lymphocytes, have been cloned and several have been transiently expressed by DNA transfection. Here we demonstrate production of stable human cell lines containing episomal EBV vectors and expressing EBV nuclear antigen 3 from the adenovirus major late promoter or the mouse metallothionein promoter, which retains metal-regulation in the episomal state. This system has proved useful in an analysis of the role of these and other EBV genes implicated in immortalization and/or oncogenic transformation of human cells.
All mammalian hepatitis B virus genomes contain an open reading frame X (X-ORF) of unknown function which could encode a protein of 17 kDa. Using a plasmid containing the entire X-ORF preceded by the adenovirus type 2 major late promoter and its tripartite leader sequence efficient expression of the HBV X-gene was achieved. The X protein of 17 kDa was characterized by immunoblotting and immunoprecipitated with an antiserum prepared against a X fusion protein produced in E. coli. By cell fractionation and indirect immunofluorescence the X-protein was found at least in part associated with nuclei. Human cell extracts containing the X protein have been used to screen human sera for anti-HBx antibodies. Such antibodies were detected in sera from patients with active chronic hepatitis with ongoing viral replication. The efficient expression of the HBV X protein obtained will facilitate its functional analysis.
Mutant mice of the Spf-ash strain have an inherited defect in ornithine transcarbamylase (OTC) protein synthesis, and were used to ascertain the potential of recombinant adenoviruses for introducing and expressing the normal gene lacking in these mice. These OTC mutant mice are characterized by a reduction in the amount of OTC activity, resulting in hyperammonemia, pronounced orotic aciduria, growth retardation, and sparse fur until weaning. A recombinant adenovirus that harbors the rat OTC cDNA under the control of the viral major late promoter (MLP) was constructed and injected into such newborn mice. The effect of the virus was analyzed by monitoring the hepatic OTC enzyme during several months after the injection. An increase in OTC activity was detected and was accompanied by a diminution of orotic acid in the urine. The observation of MLP-OTC mRNA transcripts over 1 year following the injection attests to the relatively long-term presence of the transferred gene. In those mice showing the greatest OTC activity, a normalization of the fur was also observed. The experiments reported here document the feasibility of using adenovirus for the direct delivery in vivo of a gene to restore an impaired metabolism.
The gene encoding the pseudorabies virus glycoprotein gp50 was cloned at the very left end of the genome of adenovirus type 5 to give a recombinant adenovirus (Ad-gp50) defective for the E1A gene. Ad-gp50 expressed high levels of gp50 in cells which either complemented (293 cells) or did not complement (Vero and HeLa cells) the E1A gene. Surprisingly, over an extended period, higher levels of gp50 were produced in HeLa cells which lack the E1A gene. Rabbits and mice inoculated with Ad-gp50 showed a strong antibody response against gp50. Some of them were protected from a virulent challenge with pseudorabies virus.
Recombinant plasmids containing sequences from the BamHI-E rightward reading frames 2a and 2b (BERF2a and 2b) of the Epstein-Barr virus (EBV) genome were isolated from a library of cDNA clones which had been previously made from the EBV B95-8 lymphoblastoid cell line (M. Bodescot, O. Brison, and M. Perricaudet, Nucleic Acids Res. 14:7103-7114, 1986). The characterization of these clones in combination with RNase mapping experiments led to the identification of one leftward and several rightward transcripts traversing the EBV-determined nuclear antigen EBNA3B coding region. One cDNA (T7) contains a continuous open reading frame generated by the splicing together of BERF2a and BERF2b. The T7 clone was used to reconstruct a complete fused BERF2a/2b open reading frame in an adenovirus-based expression vector. Western immunoblotting and immunofluorescence experiments using human 293 cells showed that the recombinant plasmid is capable of expressing a protein with a size, immunological characteristics, and a subcellular localization indistinguishable from those of native B95-8 EBNA3B.
We have previously demonstrated that the Adenovirus 2 (Ad2) E4 promoter is activated by an E1A gene product through an inducible enhancer. We now show that several DNA-protein complexes can be identified by gel-shift assay; the formation of one of these complexes involves the two core sequences previously found critical to the promoter activity.
The gene which encodes the Epstein-Barr gp 220/340 was inserted into a eukaryotic expression vector. A cDNA clone corresponding to the mature mRNA coding for gp 220 was isolated from an Epstein-Barr virus cDNA library and inserted in the same expression vector, enabling us to identify the precise location of the intron within the gp 220/340 coding sequence. The recombinant plasmids direct the expression of membrane proteins detected by immunofluorescence experiments using an anti-gp 220/340 monoclonal antibody in transfected human cells. The region of the gp 220/340 gene encoding the domain for membrane anchorage was removed from the two recombinant plasmids and the sequence containing the intron produced secreted forms of both truncated gp 220 and gp 340 whereas only the former was obtained with the intronless sequence.
The C gene of the hepatitis B virus, which contains two in-phase initiation codons delimiting the pre-C sequence and the C region, directs the synthesis of the major protein of the capsid (HBcAg) and of a precore protein which upon processing results in the secretion of the HBeAg. We used an adenovirus-based vector to study in the human 293 cell line the C gene products, the intermediates of the precore protein processing and the kind of protease involved in this processing. The synthesis of the 21-kDa HBcAg polypeptide was dependent on the deletion of the pre-C sequence suggesting that a pre-C mRNA is not used for the synthesis of the major capsid protein. With the construct containing the complete C gene, two proteins of 25 and 22 kDa were detected intracellularly, corresponding to the unprocessed and partially processed precore protein, respectively. In addition, a 15-kDa protein (HBeAg) was secreted in the culture medium. Using pepstatin, an inhibitor specific for aspartyl proteinases, reduction of HBeAg secretion and accumulation of the 22-kDa processing intermediate were observed, suggesting the involvement of an aspartyl proteinase in the conversion of the 22-kDa protein into HBeAg.
cDNAs synthesized from the 40S RNA genome of rubella virus were cloned into the lambda gt10 bacteriophage. A clone (pRV # 1475) which contains a truncated version of the E1 envelope glycoprotein (amino acid residues 17-481) and the 3' non-coding region was constructed from two overlapping cDNAs. pRV # 1475 was inserted into a eukaryotic expression vector under the control of the cytomegalovirus immediate early promoter. After transfection of 293 cells, a protein with intact antigenic domains could be detected.