Construction and characterization of pentacistronic retrovirus vectors

Pablo de Felipe{dagger} and Marta Izquierdo

Departamento de Bioquímica y Biología Molecular-Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco, 28049 Madrid, Spain

Correspondence
Marta Izquierdo
mizquierdo{at}cbm.uam.es


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
The picornavirus foot-and-mouth disease virus 2A sequence was combined with three different internal ribosome entry segments to construct and characterize three independent pentacistronic retroviruses of different sizes. Efficient co-expression of the five proteins was successful and titres obtained for these pentacistronic virus vectors (final genome size ~7·9 kb) were comparable to those of vector systems with shorter genomes. Other vectors constructed that exceeded the genome length of the wild-type virus suffered frequent deletions.

{dagger}Present address: Centre for Biomolecular Sciences, North Haugh, University of St Andrews, St Andrews, Fife KY16 9ST, UK.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
The ability of retrovirus vectors to deliver genes to a variety of dividing cell types in vitro and in vivo has been applied in basic research and gene therapy largely over the last 20 years. As the interest in gene therapy is expanding from single gene disorders to multiple gene diseases and combined gene therapies, new reliable polycistronic vectors capable of transducing several genes together are required. Tetracistronic retrovirus vectors derived from Moloney murine leukaemia virus (MoMLV) are currently available (de Felipe & Izquierdo, 2000). These vectors carry two different internal ribosome entry sites (IRESs) (Adam et al., 1991; Ghattas et al., 1991; Morgan et al., 1992; Martínez-Salas, 1999; Hellen & Sarnow, 2001) from encephalomyocarditis virus (ECMV) and avian reticuloendotheliosis virus type A (REV-A) in combination with the autocleavable picornavirus foot-and-mouth disease virus (FMDV) 2A sequence (Ryan & Drew, 1994; Luke & Ryan, 2001). No internal promoters that may interfere with the potent 5' LTRp (long terminal repeat promoter) of the provirus are present in the recombinants. We have now constructed three pentacistronic retrovirus vectors, Penta-7.9, Penta-8.5 and Penta-9.3, each of which shares the same basic structure but are of increasing length. We wanted to know if it was possible to increase the size of the retrovirus vector from that of the wild-type genome (8·3 kb) (Weiss et al., 1985). We show here that only recombinants that preserve the size of, or are smaller than, the wild-type genome, are viable, stable and efficient at co-expressing all of the genes carried and can maintain a high virus titre.

The three pentacistronic retrovirus vectors of increasing lengths (Fig. 1) were made by standard genetic-engineering techniques and were maintained as retrovirus plasmids. The autocleavable FMDV {Delta}1D2A sequence and three different IRESs were used to link various selection genes (pac, neo, hygro, tk) and/or reporter genes (egfp, luc, plap). Penta-7.9 derives from Penta-8.5 and was engineered last. Penta-8.5 and Penta-9.3 derive from Tetra-1 and Tetra-2, described previously (de Felipe & Izquierdo, 2000), introducing a BglII FMDV IRES-luc cassette at the unique BglII site between the neo gene and the EMCV IRES. The FMDV IRES-luc cassette came from plasmid pBiC (Martínez-Salas et al., 1993), filling the BfmI single-stranded ends with Klenow and ligating it to the also filled BglII site of pSXLC-TK (Sugimoto et al., 1994). The construction of Penta-7.9 was achieved by deleting the luc gene from Penta-8.5 and replacing it with the hygro gene via the BglII site downstream of the FMDV IRES. The hygro gene comes from pBabeHygro (Morgenstern & Land, 1990) and was amplified by PCR incorporating BamHI and BglII sites at both ends of the gene. The hygro gene and the junction between FMDV IRES and hygro were verified by standard sequencing procedures.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1. Genetic structure of the pentacistronic vectors used in this study and a summary of the gene expression results from the NIH 3T3 cell clones tested. Four clones of 7·9 kb in length (top), three of 8·5 kb in length (middle) and three of 9·3 kb in length (bottom) were analysed. The following abbreviations and symbols were used: LTR, long terminal repeat; E/D/IRESM, encapsidation/dimerization/IRES sequences of MoMLV, also called the {Psi} region; pac, puromycin N-acetyl transferase; {Delta}1D2AF, FMDV {Delta}1D2A sequence; 2AF, FMDV 2A sequence; egfp, enhanced green fluorescence protein; plap, human placental alkaline phosphatase; IRESR, REV-A IRES; neo, neomycin-phosphotransferase II; IRESF, FMDV IRES; hygro, hygromycin B phosphotransferase; luc, luciferase; IRESE, EMCV IRES; HSV1tk, herpes simplex virus type 1 thymidine kinase; +, positive expression, as determined by the corresponding assay; -, negative expression; and ND, not determined.

 
All vectors share the same structure (Fig. 1) and the final length of each one (7·9, 8·5 and 9·3 kb) is given by the sizes of the genes carried: luc (1797 bp) substitutes for hygro (998 bp) in Penta-8.5 and plap (1574 bp) substitutes for egfp (719 bp) in Penta-9.3.

Plasmid retrovirus vectors were used to transiently transfect ecotropic packaging {Psi}CRE mouse cells and their supernatants to infect amphotropic {Psi}CRIP cells, as described by Miller & Rosman (1989). Cells were selected with 2 µg puromycin ml-1 for 6 weeks to obtain a population of resistant cells. Titres were estimated by infecting NIH 3T3 cells as described previously (Izquierdo et al., 1997). In all experiments, a plate of uninfected NIH 3T3 cells was placed under the same selection conditions as the infected plates to ensure that a lethal dose of antibiotic selection was used. Cells in the {Psi}CRIP-infected plates used to harvest the supernatant were counted after collection and the titres expressed as transducing units (TU) ml-1 per cell. The luciferase activity assay was performed as described by Martín et al. (2000) with minor modifications (1·3x105 cells were used and 10 µl of the total 100 µl of cell extract was taken to measure luciferase activity). To estimate plap expression, cells were fixed and stained with BCIP/NBT, as described previously (de Felipe & Izquierdo, 2000).

Supernatants from {Psi}CRIP populations showed titres of about 2x105 TU ml-1 for Penta-7.9 (equivalent to 0·02 TU ml-1 per cell) and somewhat surprisingly, titres were higher in the larger vectors (0·36 and 0·15 TU ml-1 per cell for Penta-8.5 and Penta-9.3, respectively). Expression for all of the reporter genes was positive in cell lawns from Penta-7.9 but low or negative for the last two genes in lawns from Penta-8.5. Expression was also very weak or negative for all genes in Penta-9.3, except for the selection gene pac (data not shown). These results suggested the presence of rearrangements in the larger vectors. No replication-competent retroviruses were detected (de Felipe & Izquierdo, 2000) using the same supernatants from producer {Psi}CRIP cells.

As lawns may represent a mixture of different integration events, it is always preferable to work with individual clones descendant from a single cell. Retrovirus-containing supernatants from producer {Psi}CRIP cells were used to infect NIH 3T3 fibroblasts. These cells were selected in medium containing 2 µg puromycin ml-1 and surviving colonies were picked and expanded for gene expression analysis. Four NIH 3T3 cell clones harbouring the vector Penta-7.9 were obtained and studied. Expression of egfp was monitored by flow cytometry (Fig. 2a) under the conditions described previously (de Felipe & Izquierdo, 2000).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2. Gene expression from NIH 3T3 cell clones infected with retrovirus Penta-7.9. (a) EGFP expression measured by flow cytometry. Vertical lines show the setting of the electronic gate used to distinguish between EGFP-negative and -positive cells. Percentage of total cells and mean values of fluorescence are also shown. (b) Resistance to 2 µg puromycin ml-1, 1 mg G418 ml-1, 150 µg hygromycin ml-1 and the combination of the three, expressed as per cent cell survival. Survival in the presence of puromycin was considered arbitrarily as 100 %. Cells grown without drug selection were considered as 100 % survival. Under these conditions, it is not unusual to obtain survival values in the drug selection samples higher than in controls. (c) Sensitivity to 1 µg GCV ml-1, expressed as per cent cell survival. Values for cells preselected in puromycin and without GCV were considered as 100 %.

 
Only clones 1 and 3 were positive for expression. To detect the expression of other drug-resistance markers, clones were plated and selected for 7 days with 2 µg puromycin ml-1, 1 mg G418 ml-1 and 150 µg hygromycin ml-1, either alone or in combination. Colonies 1, 2 and 3 showed resistance to the four situations. The combination of the three drugs reduced cell viability to 50 %; this could be attributed to difficulties in expressing three resistant proteins at levels high enough to resist the combined triple selection. Despite this, we were able to obtain cell lines that were cultured for 4 weeks in each case (Fig. 2b). Clone 4 was resistant only to puromycin. Finally, puromycin-resistant cell clones were plated and maintained in media containing either no drug or 1 µg GCV (ganciclovir) ml-1 for 4 days. Again, the first three clones showed a good sensitivity to GCV (Fig. 2c).

Individual cell clones containing vectors Penta-8.5 and Penta-9.3 were also isolated by resistance to 2 µg puromycin ml-1 and the expression of the first, third and fifth genes was assayed. These results are summarized in Fig. 1 by positive or negative symbols under each gene and corresponding clone number. The three NIH 3T3 clones isolated after infection with Penta-8.5 displayed the same pattern of gene expression as that with the {Psi}CRIP population (lawn): resistance to puromycin and G418 (and the combination of both drugs), but absolute lack of GCV sensitivity. In clone number 2, full analysis was carried out and expression of egfp was detected, while no luciferase activity was monitored. A final group of three clones infected with Penta-9.3 showed only resistance to puromycin. The expression of plap was not determined in these clones as the lawn estimation for the gene was negative.

To verify the structural integrity of the proviruses, a long PCR was carried out on genomic DNA extracted from the 10 clones isolated (Fig. 3). The extraction of genomic DNA and the long PCR procedures were carried out as described previously (de Felipe & Izquierdo, 2000), except that elongation times were extended to 20 min. The amplified region begins at the centre of the pac gene and finishes at the 3' LTR. The experiment enables us to correlate deficiencies in gene expression with major rearrangement in the vectors (involving changes in the size of the bands amplified by PCR). Clones 1, 2 and 3 from Penta-7.9 show a band that matches with the expected size fragment of 6·0 kb, but clone 4 seemed to have suffered a large deletion and produced a small band of about 1·6 kb. The only functional gene coded in this deleted provirus is pac, as the expression of all other genes is not detected. More difficult to explain is clone 2, in which a deletion is not apparent (within the detection limits of the electrophoretic analysis), but the second gene, egfp, is not expressed. Sequencing revealed a run of nine thymidine residues between the FMDV 2A gene and the ATG codon of egfp, a situation known to create a hot spot for frameshift mutations in retroviruses (Pathak & Temin, 1990; Burns & Temin, 1994). Two different PCR bands amplified from the genome of clone 2 showed 10 thymidine residues instead of the original nine, thereby creating a frameshift mutation.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3. PCR from genomic DNA of NIH 3T3 cell clones infected with pentacistronic retrovirus vectors. A molecular mass marker (kb) is shown on the right. Genomic DNA from NIH 3T3 cells was used as a negative control, as well as a PCR reaction without DNA.

 
Large deletions were detected in all Penta-8.5 and Penta-9.3 virus clones (Fig. 3), explaining the lack of expression of reporter genes downstream of the selection marker and the high titres observed. These results suggest that it may be risky to go close to or over the natural 8·3 kb size of the MoMLV genome. Nevertheless, previous reports show acceptable stabilities when short fragments of less than 1·5 kb are introduced into the full 8·3 kb retrovirus genome (Logg et al., 2001a, b). Highly manipulated viruses such as the ones we present here may be more susceptible to size limitations.

Despite the aforementioned restrictions, we have demonstrated that is possible to introduce five foreign cistrons into a retrovirus. Naturally, as the number of cistrons increases, the genes cloned have to become smaller. In our vectors, all the genes carried are smaller than 2 kb; therefore, it may be difficult to acquire substantial additional space. To our knowledge, these are the first functional pentacistronic retrovirus vectors constructed to date that allow the expression of five independent proteins from a single transcription unit. There are reports of pentacistronic vectors using herpes simplex viruses (Krisky et al., 1998) or vaccinia virus (Carroll et al., 1998). However, these vectors, unlike the ones reported here, use several promoters along their large viral genomes. In the present study, we have constructed complex pentacistronic vectors able to co-express five independent genes provided that the size of the wild-type genome is maintained. These results increase our knowledge about the capability and limitations of retroviruses as polycistronic gene vehicles.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Comunidad de Madrid, 8.6/21/98, Ministry of Culture and Education, PM98-0007, and Plan Nacional de I+D, 2FD97-1401. The Centro de Biología Molecular Severo Ochoa is also the recipient of an institutional grant from the Fundación Ramón Areces. P. F. has been supported by a FPI grant from the Comunidad Autónoma de Madrid (Consejería de Educación y Cultura). We gratefully acknowledge: Jean-Michel Heard for the packaging cell lines {Psi}CRIP and {Psi}CRE; Hartmut Land for the plasmids pBabePuro and pBabeHygro; Ira Pastan for plasmid pSXLC-TK; Martin Ryan for sequences 2A and {Delta}1D2A of FMDV; Marcelo López-Lastra for pREV-HW3; Juan A. de Carlos for plasmid pEGFP-N1; and Encarnación Martínez-Salas for FMDV IRES and pBiC. Thanks are also due to Marta Vaz for technical assistance.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Adam, M. A., Ramesh, N., Miller, A. D. & Osborne, W. R. (1991). Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions. J Virol 65, 4985–4990.[Medline]

Burns, D. P. & Temin, H. M. (1994). High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication. J Virol 68, 4196–4203.[Abstract]

Carroll, M. W., Overwijk, W. W., Surman, D. R., Tsung, K., Moss, B. & Restifo, N. P. (1998). Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen. J Natl Cancer Inst 90, 1881–1887.[Abstract/Free Full Text]

de Felipe, P. & Izquierdo, M. (2000). Tricistronic and tetracistronic retroviral vectors for gene transfer. Hum Gene Ther 11, 1921–1931.[CrossRef][Medline]

Ghattas, I. R., Sanes, J. R. & Majors, J. E. (1991). The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol 11, 5848–5859.[Medline]

Hellen, C. U. T. & Sarnow, P. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 15, 1593–1612.[Free Full Text]

Izquierdo, M., Cortés, M. L., Martín, V., de Felipe, P., Izquierdo, J. M., Pérez-Higueras, A., Paz, J. F., Isla, A. & Blázquez, M. G. (1997). Gene therapy in brain tumours: implication of the size of glioblastoma on its curability. Acta Neurochir Suppl 68, 111–117.[Medline]

Krisky, D. M., Marconi, P. C., Oligino, T. J., Rouse, R. J., Fink, D. J., Cohen, J. B., Watkins, S. C. & Glorioso, J. C. (1998). Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications. Gene Ther 5, 1517–1530.[CrossRef][Medline]

Logg, C. R., Tai, C.-K., Logg, A., Anderson, W. F. & Kasahara, N. (2001a). A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors. Hum Gene Ther 12, 921–932.[CrossRef][Medline]

Logg, C. R., Logg, A., Tai, C.-K., Cannon, P. M. & Kasahara, N. (2001b). Genomic stability of murine leukemia viruses containing insertions at the Env-3' untranslated region boundary. J Virol 75, 6989–6998.[Abstract/Free Full Text]

Luke, G. A. & Ryan, M. D. (2001). Translating the message. Biologist (London) 48, 79–82.[Medline]

Martín, V., Cortés, M. L., de Felipe, P., Farsetti, A., Calcaterra, N. B. & Izquierdo, M. (2000). Cancer gene therapy by thyroid hormone-mediated expression of toxin genes. Cancer Res 60, 3218–3224.[Abstract/Free Full Text]

Martínez-Salas, E. (1999). Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol 10, 458–464.[CrossRef][Medline]

Martínez-Salas, E., Saiz, J. C., Davila, M., Belsham, G. J. & Domingo, E. (1993). A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo. J Virol 67, 3748–3755.[Abstract]

Miller, A. D. & Rosman, G. J. (1989). Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–988.[Medline]

Morgan, R. A., Couture, L., Elroy-Stein, O., Ragheb, J., Moss, B. & Anderson, W. F. (1992). Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res 20, 1293–1299.[Abstract]

Morgenstern, J. P. & Land, H. (1990). Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18, 3587–3596.[Abstract]

Pathak, V. K. & Temin, H. M. (1990). Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: substitutions, frameshifts, and hypermutations. Proc Natl Acad Sci U S A 87, 6019–6023.[Abstract]

Ryan, M. D. & Drew, J. (1994). Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 13, 928–933.[Abstract]

Sugimoto, Y., Aksentijevich, I., Gottesman, M. M. & Pastan, I. (1994). Efficient expression of drug-selectable genes in retroviral vectors under control of an internal ribosome entry site. Biotechnology 12, 694–698.[Medline]

Weiss, R., Teich, N., Varmus, H. & Coffin, J. (editors) (1985). In RNA Tumor Viruses, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Received 22 November 2002; accepted 22 January 2003.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Felipe, P. d.
Articles by Izquierdo, M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Felipe, P. d.
Articles by Izquierdo, M.
Agricola
Articles by Felipe, P. d.
Articles by Izquierdo, M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS