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Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.


Deletion of aptamer cassette in RNA transcripts from the Phoenix(amphotropic) packaging cell lines. The Phoenix (amphotropic) packaging cell lines were transfected with the different aptamer vectors. Total RNA was isolated and subjected to amplification by RT-PCR with (+RT) or without (-RT) reverse transcriptase using the 3'GFP-for and 3' UTR-rev primers. Amplification of the MMP 70.15 plasmid was included as control. The DNA ladder is the 100 bp DNA ladder from Invitrogen.
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Figure 6: Deletion of aptamer cassette in RNA transcripts from the Phoenix(amphotropic) packaging cell lines. The Phoenix (amphotropic) packaging cell lines were transfected with the different aptamer vectors. Total RNA was isolated and subjected to amplification by RT-PCR with (+RT) or without (-RT) reverse transcriptase using the 3'GFP-for and 3' UTR-rev primers. Amplification of the MMP 70.15 plasmid was included as control. The DNA ladder is the 100 bp DNA ladder from Invitrogen.

Mentions: To determine whether the truncated aptamer cassette originated during post-transcriptional splicing of the mRNA or after later steps in the retroviral life cycle, we isolated total RNA from the Phoenix (amphotropic) packaging cell lines after transfection with each of the aptamer vectors. Under these conditions, the inhibitor cassette will have been transcribed and the RNA processed, but will not have progressed to reverse transcription and/or integration. The total RNA from the packaging cell line was amplified by RT-PCR with or without reverse transcriptase using the same primer combination (Figure 1a) as was used previously to amplify the aptamer in Figure 3. In all four transfected Phoenix (amphotropic) populations, both the full-length and the truncated product were detected (Figure 6), while amplification of the MMP-70.15 plasmid generated only the full-length product and amplification of the RNA samples without RT were negative (Figure 6). The ratio of full-length to truncated product during one round of transcription and mRNA processing favors the full-length product (Figure 6). Because the RT-PCR reaction was not designed or evaluated for quantitative analysis, the efficiency of RNA processing cannot be determined. These results demonstrate that the deletion in the U6 promoter when transduced in the antisense orientation within a retroviral vector is caused by splicing of the retroviral genomic RNA at cryptic splice donor/splice acceptor sequences.


Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Deletion of aptamer cassette in RNA transcripts from the Phoenix(amphotropic) packaging cell lines. The Phoenix (amphotropic) packaging cell lines were transfected with the different aptamer vectors. Total RNA was isolated and subjected to amplification by RT-PCR with (+RT) or without (-RT) reverse transcriptase using the 3'GFP-for and 3' UTR-rev primers. Amplification of the MMP 70.15 plasmid was included as control. The DNA ladder is the 100 bp DNA ladder from Invitrogen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211285&req=5

Figure 6: Deletion of aptamer cassette in RNA transcripts from the Phoenix(amphotropic) packaging cell lines. The Phoenix (amphotropic) packaging cell lines were transfected with the different aptamer vectors. Total RNA was isolated and subjected to amplification by RT-PCR with (+RT) or without (-RT) reverse transcriptase using the 3'GFP-for and 3' UTR-rev primers. Amplification of the MMP 70.15 plasmid was included as control. The DNA ladder is the 100 bp DNA ladder from Invitrogen.
Mentions: To determine whether the truncated aptamer cassette originated during post-transcriptional splicing of the mRNA or after later steps in the retroviral life cycle, we isolated total RNA from the Phoenix (amphotropic) packaging cell lines after transfection with each of the aptamer vectors. Under these conditions, the inhibitor cassette will have been transcribed and the RNA processed, but will not have progressed to reverse transcription and/or integration. The total RNA from the packaging cell line was amplified by RT-PCR with or without reverse transcriptase using the same primer combination (Figure 1a) as was used previously to amplify the aptamer in Figure 3. In all four transfected Phoenix (amphotropic) populations, both the full-length and the truncated product were detected (Figure 6), while amplification of the MMP-70.15 plasmid generated only the full-length product and amplification of the RNA samples without RT were negative (Figure 6). The ratio of full-length to truncated product during one round of transcription and mRNA processing favors the full-length product (Figure 6). Because the RT-PCR reaction was not designed or evaluated for quantitative analysis, the efficiency of RNA processing cannot be determined. These results demonstrate that the deletion in the U6 promoter when transduced in the antisense orientation within a retroviral vector is caused by splicing of the retroviral genomic RNA at cryptic splice donor/splice acceptor sequences.

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.