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Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

ABSTRACT: The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3' untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegalovirus or the herpesvirus thymidine kinase promoter and with both human immunodeficiency virus- and murine leukemia virus-based vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of retroviral vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.

SUBMITTER: Zufferey R

PROVIDER: S-EPMC104046 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

Zufferey R R Donello J E JE Trono D D Hope T J TJ

Journal of virology 19990401 4

The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3' untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promote ...[more]

PMID: 10074136

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Project description:Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, beta(2)-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.

Project description:Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.

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Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

Publications

Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

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