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. 1999 Oct;73(10):8393-402.
doi: 10.1128/JVI.73.10.8393-8402.1999.

Identification of an internal ribosome entry segment in the 5' region of the mouse VL30 retrotransposon and its use in the development of retroviral vectors

M López-Lastra  1 , S Ulrici, C Gabus, J L Darlix
Affiliations

Identification of an internal ribosome entry segment in the 5' region of the mouse VL30 retrotransposon and its use in the development of retroviral vectors

M López-Lastra et al. J Virol. 1999 Oct.

Abstract

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5' region of VL30m could replace the 5' leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5' region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5' region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5' region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.

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Figures

FIG. 1
FIG. 1
Schematic representation of MLV-VL30m-lacZ monocistronic retroviral vectors. All MLV-VL30m vectors contain the 5′ LTR and the primer binding site of MLV. In vectors pVL30m-SJ E1 and pVL30m-SJ E2, different segments of the 5′ region of NLV-3 VL30 sequences were inserted upstream of the lacZ reporter cistron. The pMLV-LacZ+ vector, used as a positive control, contains the MLV Psi+ encapsidation sequence. pVL-SJE3, used as a negative control, contains no encapsidation sequence. Numbering is with respect to the VL30m RNA cap site (position +1).
FIG. 2
FIG. 2
Translation of VL30m bicistronic RNA in messenger-dependent RRL. (A) Schematic representation of the bicistronic plasmid constructs containing different portions of the VL30 5′ RNA located between the neo and lacZ genes under the control of the T7 promoter (Po T7) for in vitro expression. Numbering is with respect to the genomic RNA cap site (position +1). Po CMV, cytomegalovirus early promoter; KD, kilodaltons. (B) Translation of uncapped (−) and capped (+) bicistronic RNA in the Flexi-RRL system (Promega). After heat denaturation, 35S-labelled proteins were analyzed by SDS–15% PAGE. The positions of neomycin phosphotransferase (28 kDa) and the C-terminally truncated β-Gal protein (46 kDa) are indicated. Lanes 1 to 4, control RNAs containing the EMCV IRES (see Materials and Methods); lanes 7 to 10 RNAs containing different 3′ deletions in the putative VL30m IRES; lanes 5, 6, and 11 to 14, RNAs containing the 5′ region VL30m RNA or 5′ deletions of this sequence.
FIG. 3
FIG. 3
Effect of FMDV L protease on bicistronic-RNA translation. Bicistronic capped RNA was translated in the Flexi-RRL system (Promega) with (+) or without (−) L protease (PL). After heat denaturation, 35S-labelled proteins were analyzed by SDS–15% PAGE. The positions of neomycin phosphotransferase (28 kDa) and the C-terminally truncated β-Gal protein (46 kDa) are indicated. Lanes 1 and 2, control RNAs containing the EMCV IRES (see Materials and Methods); lanes 5 to 8, RNAs containing different 3′ deletions in the putative VL30m IRES; lanes 3, 4, and 9 to 12, RNAs containing the 5′ region VL30m RNA or 5′ deletions of this sequence.
FIG. 4
FIG. 4
Schematic representation of the bicistronic retroviral vectors. The VL30m-MLV-based retroviral vectors are built on a pBR322 backbone. VL30m corresponds to the 5′ RNA region of the mouse VL30 retrotransposon, VL30r corresponds to the 5′ untranslated region of HaMSV (8), and MLV E+ corresponds to the extended packaging region of MLV (5). Placental alkaline phosphatase (plap) and neomycin phosphotransferase (neo) were used as marker genes. The control vectors pEMCV-CBT4 and pRev-HW3 possess two IRESes (53, 84), the first from MLV (7, 87), which also directs packaging (E+), and the second from EMCV or REV-A (53). In all cases, numbering is with respect to the genomic RNA cap site (position +1).
FIG. 5
FIG. 5
Monitoring double transgene expression. Proteins extracted from transduced PLAP-positive neomycin-resistant NIH 3T3 cells were used to determine the level of expression of each transgene by vector constructs. (A) PLAP enzymatic activities were determined as described in Materials and Methods (53, 84). The mean values of alkaline phosphatase specific activities as well as the standard deviation for each set of experiments are shown. Data are the averages of values from three independent experiments. (B) Ten micrograms of total protein was loaded per lane and subjected to SDS–15% PAGE. Proteins were transferred to a polyvinylidene difluoride membrane and probed with a rabbit anti-neomycin phosphotransferase II antibody. The membrane was then incubated with a biotinylated anti-rabbit immunoglobulin G antibody and an avidin-peroxidase solution and, finally, developed by enhanced chemiluminescence. Lane 1, negative control (protein extract from nontransduced NIH 3T3 cells); lanes 2 through 6, protein extracts from cells transduced with the different retroviral vectors. The positions of molecular mass standards are shown on the left.
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References

    1. Adam M A, Ramesh N, Miller A D, Osborne W R A. Internal initiation of translation in retroviral vectors carrying picornavirus 5′ nontranslated regions. J Virol. 1991;65:4985–4990. - PMC - PubMed
    1. Adams S E, Rathjen P D, Stanway C A, Fulton S M, Malim M H, Wilson W, Ogden J, King L, Kingsman S M, Kingsman A J. Complete nucleotide sequence of a mouse VL30 retroelement. Mol Cell Biol. 1988;8:2989–2998. - PMC - PubMed
    1. Akiri G, Nahari D, Finkelstein Y, Le S Y, Elroy-Stein O, Levi B Z. Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription. Oncogene. 1998;17:227–236. - PubMed
    1. Attal J, Theron M C, Taboit F, Cajero-Juarez M, Kann G, Bolifraud P, Houdebine L M. The RU5 (′R′) region from human leukaemia viruses (HTLV-1) contains an internal ribosome entry site (IRES)-like sequence. FEBS LETT. 1996;392:220–224. - PubMed
    1. Bender M A, Palmer T D, Gelinas R E, Miller A D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987;61:1639–1646. - PMC - PubMed

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