Woodchuck hepatitis virus posttranscriptional regulatory element

Transcription of the major viral proteins is mediated by four promoters which are partially regulated by HBV enhancers I and II. Both enhancers are liver specific, although enhancer I retains lower activity levels in some nonhepatic cells 25 , 33 , HBV enhancer I maps upstream of the X open reading frame and consists of a modulatory domain, a core enhancer domain, and a basal X promoter domain 3 , 30 ; enhancer II maps to the core promoter region and is thought to influence levels of genomic RNA The transcriptional regulatory elements of WHV are not as thoroughly characterized.

Recent studies have shown that WHV enhancer II is a strongly liver-specific enhancer that regulates the production of pregenomic RNAs, which is an important rate-limiting step of hepadnavirus replication 6 , Surprisingly, the WHV region homologous to HBV enhancer I lacks enhancer activity in the three human liver cell lines tested 2 , 6 , This region failed to activate transcription of the four viral promoters as well as a heterologous thymidine kinase tk promoter.

The authors suggest that either the human liver cells do not express the required transcription factors or major differences exist in the transcriptional control of HBV and WHV 2. A single HBVPRE subelement displays a low level of posttranscriptional activity, and both subelements function cooperatively when duplicated. The subelements most likely represent distinct binding sites for cellular RNA binding proteins.

An additional element has been found in the intronless tk gene of herpes simplex virus type 1 All of these cis -acting elements are essential for the cytoplasmic localization of viral RNA and, with the exception of the complex retrovirus elements, are thought to interact with cellular RNA binding proteins.

The cytomegalovirus CMV surface expression construct was synthesized by amplifying nucleotides to from GenBank accession no. The pDM vector system has been previously described To construct the pDM reporter derivatives, base oligonucleotides were synthesized and used to PCR amplify the fragment of interest from the DNA template.

The mutant and the WPRE activities were identical data not shown. The fragments were then ligated with the corresponding fragment into the pDM vector. Before the cells were transfected, the medium was removed and the DNA-CaPO 4 mix was added directly to the naked cells.

After 10 min, 5 ml of medium was placed back onto the cells. The medium was changed 16 h after transfection. The cells were harvested 36 to 48 h later. Luciferase activity was determined by standard methods. The medium was changed approximately 16 h after transfection. The spent medium was harvested 48 h later. The spent medium from duplicate transfections was assayed for the presence of surface antigen with an Ausria II kit Abbott Laboratories and quantitated in a gamma counter.

As an indicator of transfection efficiencies, the medium was also assayed for the presence of secreted alkaline phosphatase. The lysates were spun briefly to pellet insoluble cell debris. Substrate and products were resolved by thin-layer chromatography and quantitated by a PhosphorImager Molecular Dynamics. In this study, the nucleotide numbering schemes of accession no. Consequently, homologous nucleotides are offset by bases.

The posttranscriptional activity of an element will be discussed as a percentage of the WPRE activity. CV1 cells were used to avoid any liver-specific transcriptional effects of enhancer I.

The results Fig. Surface protein expression was increased 6. The shaded bars represent the mean counts per minute from the media of duplicate transfections of CV1 cells. The large black arrow represents the immediate early CMV promoter upstream of the HBV surface protein open reading frame.

A schematic representation of the pGL3 vector is shown at the top. The black arrows represent the simian virus 40 SV40 promoter; the luciferase gene is labeled. The shaded bars represent mean luciferase activities of triplicate transfections.

RLU, relative light units. N2 - The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy.

AB - The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy.

Overview Fingerprint. Abstract The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Link to publication in Scopus. Link to the citations in Scopus. 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. Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy.

Hum Gene Ther ; 10 : — EMBO Rep ; 6 : — Lentiviral vectors for enhanced gene expression in human hematopoietic cells. High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors.

Blood ; 96 : — Ailles LE, Naldini L. HIVderived lentiviral vectors. Curr Top Microbiol Immunol ; : 31— Self-inactivating retroviral vectors with improved RNA processing. Gene Therapy ; 11 : — B-cell-specific transgene expression using a self-inactivating retroviral vector with human CD19 promoter and viral post-transcriptional regulatory element.

The enigmatic X gene of hepatitis B virus. J Virol ; 78 : — Hepadnavirus integration: mechanisms of activation of the N-myc2 retrotransposon in woodchuck liver tumors. J Virol ; 66 : — The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication. J Virol ; 79 : — Analysis of the X gene promoter of woodchuck hepatitis virus.

Virology ; : — Woodchuck hepatitis virus enhancer I and enhancer II are both involved in N-myc2 activation in woodchuck liver tumors. Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice. Mol Ther ; 12 : — Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element WPRE.

Gene Therapy ; 12 : 3—4. Retroviral vector backbone immunogenicity: identification of cytotoxic T-cell epitopes in retroviral vector-packaging sequences. Gene Therapy ; 12 : — Inhibition of human immunodeficiency virus type 1 entry in cells expressing gpderived peptides. Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine-DNA-methyltransferase in bone marrow cells. Mol Ther , Oct 10; Epub ahead of print. A third-generation lentivirus vector with a conditional packaging system.

Novel retroviral vectors for efficient expression of the multidrug resistance mdr-1 gene in early hematopoietic cells. J Virol ; 69 : — Download references. We thank R Seyd and M Id for excellent technical assistance.