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Short Communication |
1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 3E2, Canada
2 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E2, Canada
Correspondence
Sadhna Joshi
sadhna.joshi.sukhwal{at}utoronto.ca
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ABSTRACT |
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MAIN TEXT |
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; Jones et al., 2005). Five Ll.LtrB intron insertion sites have been identified within the DNA of human immunodeficiency virus type 1 (HIV-1) (Guo et al., 2000). Intron insertion at two of these sites, located at nt 4021 and 4069 within the sense DNA strand of the integrase-coding region of the HIV-1 pol gene, was shown to occur at high frequency. Therefore, we used the introns (I4021s and I4069s) targeted against these two sites (Fig. 1a) to address a number of key questions regarding the therapeutic application of this mobile group II intron.
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). We modified these plasmids to confer a selectable phenotype by cloning the T7 promoter and the neo gene, conferring kanamycin (Km) resistance, into intron domain IV as follows. An XhoI–XhoI fragment containing the T7 promoter along with Bsp120I and SalI sites was cloned at the SalI sites within intron domain IV into pACD-I4021s and pACD-I4069s (Guo et al., 2000). A SalI–Bsp120I fragment of pMoTN (Magli et al., 1987) containing the Shine–Dalgarno sequence and the neo gene was then cloned at these sites to obtain pACD-I4021sN and pACD-I4069sN.
As intron insertion has not yet been demonstrated in mammalian chromosomes, we bypassed this step by allowing intron insertion in an infectious HIV-1 provirus DNA clone in Escherichia coli. To obtain the intron-inserted HIV-1 proviral DNA clones (Fig. 1a
), E. coli HMS174(DE3) cells were co-transformed with pACD-I4021sN or pACD-I4069sN (CmRKmR) together with pHIV (an infectious HIV-1 provirus DNA clone, originally referred to as pNL4-3; ApR) (Adachi et al., 1986) (Fig. 2). The cells were induced with 1 µM IPTG for 1 h at 37 °C to allow insertion of I4021sN and I4069sN introns at nt 4021 and 4069, respectively, within the integrase-coding region. The cells were washed and cultured overnight in lysogeny broth containing ampicillin (Ap) and chloramphenicol (Cm). Plasmids were extracted from these cells and digested with SacII to degrade pACD-I4021sN and pACD-I4069sN. E. coli DH5
cells were transformed with the DNA after digestion with SacII, and ApRKmRCmS colonies were identified as they contained only pHIV-I4021sN or pHIV-I4069sN. Plasmid DNA from these colonies was extracted and analysed.
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). On day 4 post-transfection, total RNA from these cells was incubated with RNase-free DNase and analysed by RT-PCR to test for group II intron-inserted HIV-1 RNA production. Reverse transcription was performed using primer IIS-3' (5'-AGGCGGCCTTAACTGTAG-3'). cDNAs were then PCR-amplified using the primer pair IIS-5' (5'-ATGGGTTGGTCAGTGCTG-3')/IIS-3' or DV-5' (5'-GCCGTATACTCCGAGAGG-3')/IIS-3'.
RT-PCR amplification of HIV-1 RNA in the control pHIV-transfected 293T sample and PCR amplification of pHIV DNA were carried out using the IIS-5'/IIS-3' primer pair, giving rise to a product of 423 bp (Fig. 3a, lanes 1 and 4). This primer pair was designed to hybridize with the HIV-1 sequences flanking the intron insertion sites. RT-PCR amplification of the I4021sN intron-inserted HIV-1 RNA in the pHIV-I4021sN-transfected 293T sample and PCR amplification of pHIV-I4021sN by the DV-5'/IIS-3' primer pair gave rise to products of 275 bp (Fig. 3a, lanes 2 and 5). RT-PCR amplification of the I4069sN intron-inserted HIV-1 RNA in the pHIV-I4069sN-transfected 293T sample and PCR amplification of pHIV-I4069sN DNA by the same primer pair gave rise to products of 227 bp (Fig. 3a, lanes 3 and 6). This primer pair was designed to hybridize within intron domain V and HIV-1 RNA further downstream of the intron insertion sites. As a control, RT-PCR amplification of endogenous β-actin mRNA was performed using the primer pair β-actin-5' (5'-GCTCGTCGTCGACAACGGCTC-3') and β-actin-3' (5'-CAAACATGATCTGGGTCATCTTCTC-3') (Fig. 3b, lanes 1–3).
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As shown in Fig. 1(b), all viral RNAs and proteins produced in pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells were expected to be wild type, except for the group II intron-inserted HIV-1 RNA and the Gag–Pol precursor (Fig. 1b). Therefore, progeny viruses should have been produced in the pHIV-I4021sN- and pHIV-I4069sN-transfected cells. However, these viruses were expected to be non-infectious because of the increased length of the intron-inserted HIV-1 RNA (11.1 kb instead of 9.2 kb, which may prevent encapsidation) and/or the absence of a functional integrase (which should prevent integration, even if the RNA is packaged and reverse-transcribed). As the insertion sites for the two introns are located in the integrase-coding region, a 91 aa (aa 1–80 of the integrase +11 aa from the I4021sN intron) or 107 aa (aa 1–96 of the integrase +11 aa from the I4069sN intron) truncated integrase should be produced in the pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells, respectively (Fig. 1c); the full-length integrase is 298 aa.
Progeny viruses from pHIV-, pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells were tested for HIV-1 RNA and group II intron-inserted HIV-1 RNA packaging by RT-PCR. To this end, the virion RNA was treated with RNase-free DNase and reverse-transcribed using the IIS-3' primer, followed by PCR using the IIS-Up-5' (5'-TTTGCAGGATTCGGGATTAG-3'; designed upstream of the IIS-5' primer)/IIS-3' primer pair and the DV-5'/IIS-3' primer pair. When the IIS-Up-5'/IIS-3' primer pair was used, a 613 bp product was detected following RT-PCR analysis of virion RNA from the progeny of pHIV-transfected cells (Fig. 3c, lane 1). RT-PCR amplification of virion RNA from the progeny of pHIV-I4021sN- or pHIV-I4069sN-transfected cells using the same primer pair would have given rise to products of >2 kb, which would not have been detected under the RT-PCR conditions used in this experiment (Fig. 3c, lanes 2 and 3). However, when the DV-5'/IIS-3' primer pair was used, products of 275 and 227 bp were detected following RT-PCR analysis of virion RNA from the progeny of pHIV-I4021sN- and pHIV-I4069sN-transfected cells, respectively (Fig. 3d, lanes 2 and 3). These results demonstrated that the group II intron-inserted HIV-1 RNAs were packaged.
The progeny viruses (
10 ng p24 equivalent) from the pHIV-, pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells were then used to infect a human CD4+ T lymphoid (PM1) cell line, as described previously (Liem et al., 1993). As the group II intron-inserted HIV-1 RNA was packaged, reverse transcription should have occurred. To detect the reverse-transcribed HIV-1 and group II intron-inserted HIV-1 dsDNA, genomic DNA was extracted at 1 h post-inoculation and analysed by PCR using the primer pair long terminal repeat (LTR)-5' (5'-GAGAGCTGCATCCGGAGTAC-3') and LTR-3' (5'-AGGCAAGCTTTATTGAGGCTTAAGC-3') to amplify the LTR region. A 220 bp product was amplified from the genomic DNA of PM1 cells infected with the progeny from pHIV-, pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells (Fig. 3e, lanes 1–3). As expected, no PCR product was amplified from the genomic DNA of uninfected PM1 cells (Fig. 3e, lane 4).
We then tested for the presence of integrated provirus DNA by PCR analysis of genomic DNA isolated on day 8 post-infection from uninfected and infected PM1 cells. PCRs were performed using the primer pair Tat-5' (5'-ATATCATATGTAATACGACTCACTATAGGGCGAATACTTGGGCAGGAGTGGAAGC-3') and Tat-3' (5'-GATCTATGCATGAGCCAG-3') to detect a 424 bp region within the tat-coding region of both HIV-1 and group II intron-inserted HIV-1 provirus DNA. No provirus DNA could be detected in PM1 cells infected with the progeny from pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells (Fig. 3f, lanes 2 and 3). As expected, a 424 bp product resulting from HIV-1 provirus DNA amplification was detected in control PM1 cells infected with the progeny from the pHIV-transfected 293T cells (Fig. 3f, lane 1). No PCR product was detected in the uninfected PM1 sample (Fig. 3f, lane 4).
The presence of reverse-transcribed HIV-I4021sN and HIV-I4069sN dsDNA accompanied by the absence of integrated provirus DNA indicated that the 91 and 107 aa integrases (in the progeny from pHIV-I4021sN- and pHIV-I4069sN-transfected 293T cells) were non-functional. These results further indicate that, even if wild-type HIV-1 dsDNA was generated as a result of deletions during reverse transcription (Menendez-Arias, 2002), it could not integrate due to the absence of a functional integrase.
Infected PM1 cells were then tested for progeny virus production in cell-culture supernatants collected from pHIV-, pHIV-I4021sN- or pHIV-I4069sN-infected PM1 cells by measuring the amount of p24 using an HIV-1 p24 Antigen EIA kit. As expected, virus production (>600 ng ml–1), extensive cell death and syncytia were observed by day 10 post-infection in control PM1 cells infected with the progeny virus from pHIV-transfected 293T cells. PM1 cells infected with progeny virus from pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells were healthy with no cell death, no syncytia and no progeny virus production for the duration of the experiment (up to 62 days in two independent experiments). PM1 cells are highly permissive to HIV-1 replication. Therefore, if any wild-type HIV-1 had been produced as a result of group II intron self-splicing in the pHIV-I4021sN- or pHIV-I4069sN-transfected 293T cells, progeny should have been detected.
A concern with sense DNA-targeting introns is that their splicing from the HIV-1 transcripts could enable the group II intron-inserted HIV-1 provirus DNA to complete a normal virus life cycle. Our results indicate that self-splicing of the particular introns used here is not a concern for inhibition of HIV-1 replication. It should be noted that a modified LtrA protein, absent in our experiments, would be present in a gene therapy setting. Therefore, to avoid splicing of sense DNA-targeting group II introns, this protein should be expressed in an inducible manner.
In conclusion, we have demonstrated here for the first time that group II intron insertions are stable and that the Ll.LtrB-derived I4021sN and I4069sN introns can be used to confer ‘complete’ inhibition of HIV-1 replication at the intended step. If one were to extrapolate our findings to a gene therapy setting, the results obtained from 293T cells transfected with the HIV-1 provirus DNA that contained a group II intron could be considered similar to the results one would obtain from gene-modified HIV-1-infected cells that allow intron insertion at 100 % frequency. Even then, the gene-modified cells would not have provided any therapeutic benefit, as they would have produced the same amount of progeny virus as the unmodified cells. The only difference observed would have been that the progeny virus produced from gene-modified cells would be non-infectious. We believe that a gene therapy based on a strategy such as this would not be beneficial, as it would not confer a survival advantage to the gene-modified cells. Therefore, the group II introns used in the present study must be further modified to inhibit HIV-1 replication in the gene-modified cells. We have now generated group II introns that either would prevent transcription from the provirus DNA containing the group II intron or would cleave the transcripts soon after they are produced. In addition, for a group II intron to target a gene in mammalian cells, the LtrA protein must be modified to contain a nuclear localization signal to be directed to the nucleus and also must be codon-optimized for expression in mammalian cells. To facilitate splicing of the intron and formation of the ribonucleoprotein complex, it is important to co-localize the modified LtrA protein with the intron RNA. Furthermore, to avoid an immune response against this protein, it may be preferable to use liposomes to deliver the protein transiently or to use vectors allowing transient expression of this protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Guo, H., Karberg, M., Long, M., Jones, J. P., III, Sullenger, B. & Lambowitz, A. M. (2000). Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science 289, 452–457.
Jones, J. P., III, Kierlin, M. N., Coon, R. G., Perutka, J., Lambowitz, A. M. & Sullenger, B. A. (2005). Retargeting mobile group II introns to repair mutant genes. Mol Ther 11, 687–694.[CrossRef][Medline]
Joshi, S., Van Brunschot, A., Robson, I. & Bernstein, A. (1990). Efficient replication, integration, and packaging of retroviral vectors with modified LTRs containing the packaging signal. Nucleic Acids Res 18, 4223–4226.
Liem, S. E., Ramezani, A., Li, X. & Joshi, S. (1993). The development and testing of retroviral vectors expressing trans-dominant mutants of HIV-1 proteins to confer anti-HIV-1 resistance. Hum Gene Ther 4, 625–634.[Medline]
Magli, M. C., Dick, J. E., Huszar, D., Bernstein, A. & Phillips, R. A. (1987). Modulation of gene expression in multiple hematopoietic cell lineages following retroviral vector gene transfer. Proc Natl Acad Sci U S A 84, 789–793.
Menendez-Arias, L. (2002). Molecular basis of fidelity of DNA synthesis and nucleotide specificity of retroviral reverse transcriptases. Prog Nucleic Acid Res Mol Biol 71, 91–147.[Medline]
Received 15 May 2008;
accepted 17 June 2008.
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