The Secondary Structure of the R Region of a Murine Leukemia Virus Is Important for Stimulation of Long Terminal Repeat-Driven Gene Expression

Cupelli, Lisa A.; Okenquist, Sharon A.; Trubetskoy, Alla; Lenz, Jack

doi:10.1128/jvi.72.10.7807-7814.1998

Cited by 26 publications

(21 citation statements)

References 52 publications

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“…Primer extension analysis indicated that the RSL affected the levels of cytoplasmic RNA. Nuclear run-on assays indicated that deletion of the RSL had a small effect on transcriptional initiation and no effect on RNA polymerase processivity (15,16). Thus, the main effect of the RSL was on one or more steps that occurred after the template was transcribed by RNA polymerase.…”

mentioning

confidence: 99%

“…We previously showed that R region sequences of MuLV SL3 affected expression of a reporter gene under the control of a viral LTR (15,16). R region sequences from Moloney MuLV or feline leukemia virus could substitute for the SL3 element.…”

mentioning

confidence: 99%

“…R region sequences from Moloney MuLV or feline leukemia virus could substitute for the SL3 element. Thus, the R region element appears to be a general feature of the mammalian type C genus of retroviruses (16). The crucial sequences were mapped to the first 28 nucleotides of the 68nucleotide R region of SL3.…”

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confidence: 99%

“…Phylogenetic analysis indicated that the sequences important for the maintenance of this secondary structure were conserved among mammalian type C retroviruses. Mutations that dis-rupted secondary structure decreased the level of expression of a reporter gene under the control of viral LTR sequences about 10-fold in transient expression assays and 20-fold in fibroblasts stably transformed with the LTR reporter plasmids (16). A compensatory mutant in which the nucleotide sequence of the stem was different but the predicted secondary structure was maintained had most of the activity of the wildtype LTR (16).…”

mentioning

confidence: 99%

“…Mutations that dis-rupted secondary structure decreased the level of expression of a reporter gene under the control of viral LTR sequences about 10-fold in transient expression assays and 20-fold in fibroblasts stably transformed with the LTR reporter plasmids (16). A compensatory mutant in which the nucleotide sequence of the stem was different but the predicted secondary structure was maintained had most of the activity of the wildtype LTR (16). Thus, the stem-loop structure was important for the maximum activity of the SL3 LTR and presumably for the LTRs of other type C mammalian retroviruses.…”

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confidence: 99%

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R Region Sequences in the Long Terminal Repeat of a Murine Retrovirus Specifically Increase Expression of Unspliced RNAs

Trubetskoy

Okenquist

Lenz

1999

J Virol

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A stem-loop structure at the 5′ end of the R region of the long terminal repeat (LTR) of the murine leukemia virus SL3 and other type C mammalian retroviruses is important for maximum levels of expression of a reporter gene under the control of the viral LTR. This element, termed the R region stem-loop (RSL), has a small effect on transcriptional initiation and no effect on RNA polymerase processivity. Its major effect is on posttranscriptional processing of LTR-driven transcripts. Here we tested whether the RSL affected the production of RNAs from a full-length SL3 genome. Mutation of the RSL in the 5′ LTR of SL3 reduced the cytoplasmic levels of full-length viral transcripts but not those of spliced, env mRNA transcripts. Thus, the RSL specifically affected the cytoplasmic levels of the unspliced viral RNA. To test further whether the effect was specific for unspliced transcripts, a system was devised in which the expression of a reporter gene under the control of the viral LTR was tested in the presence or absence of an intron. Mutation of the RSL resulted in only about a twofold decline in the level of reporter gene expression when the transcripts contained an intron. However, when the intron was removed, mutation of the RSL reduced expression of the reporter gene about 10- to 60-fold in various cell lines. The secondary structure of the RSL was essential for its activity on the intronless transcript. Thus, the RSL appears to be important for the cytoplasmic accumulation of unspliced viral RNA and unspliced RNA from chimeric transcription units under the control of the viral LTR.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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R Region Sequences in the Long Terminal Repeat of a Murine Retrovirus Specifically Increase Expression of Unspliced RNAs

Trubetskoy

Okenquist

Lenz

1999

J Virol

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Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy

Vigna¹,

Naldini²

2000

J. Gene Med.

307

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Lentiviral vectors are tools for gene transfer derived from lentiviruses. From their first application to now they have been strongly developed in design, in biosafety and in their ability of transgene expression into target cells. Primate and non-primate derived lentiviral vectors are now available and with both types of systems a lot of studies tuned to improve their performances in a large number of tissues are ongoing. Here we review the state of the art of lentiviral vector systems discussing their potential for gene therapy.

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Lentiviral Vectors for Enhanced Gene Expression in Human Hematopoietic Cells

Ramezani¹,

2000

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Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer-promoter to transcribe the gene of interest, are not yet optimized for efficient expression in human hematopoietic stem/progenitor cells (HSPCs). As a first step toward this goal, we constructed self-inactivating derivatives of the HIV-1-based transfer vector pHR' containing the enhanced green fluorescent protein (GFP) gene as reporter and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including the murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape leukemia virus (GALV) LTR, the human elongation factor 1alpha (EF1alpha) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. In contrast to results obtained in human embryonic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV promoter expressed GFP at the highest levels, significantly higher levels of GFP expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1alpha promoter, and the CAG promoter in the human HSPC line KG1a. Removal of the WPRE indicated that it stimulated GFP expression from all of the vectors in KG1a cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1alpha and CAG promoters (<1.5-fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PGK promoter were the most efficient at transducing primary human CD34+ cord blood progenitors under the conditions employed. High-level GFP expression in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basis for the rational design of improved delivery vehicles for human HSPC gene transfer applications.

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The Secondary Structure of the R Region of a Murine Leukemia Virus Is Important for Stimulation of Long Terminal Repeat-Driven Gene Expression

Cited by 26 publications

References 52 publications

R Region Sequences in the Long Terminal Repeat of a Murine Retrovirus Specifically Increase Expression of Unspliced RNAs

R Region Sequences in the Long Terminal Repeat of a Murine Retrovirus Specifically Increase Expression of Unspliced RNAs

Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy

Lentiviral Vectors for Enhanced Gene Expression in Human Hematopoietic Cells

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