Limits...
Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection.

Kimpel J, Braun SE, Qiu G, Wong FE, Conolle M, Schmitz JE, Brendel C, Humeau LM, Dropulic B, Rossi JJ, Berger A, von Laer D, Johnson RP - PLoS ONE (2010)

Bottom Line: However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells.These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo.Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

View Article: PubMed Central - PubMed

Affiliation: Angewandte Virologie und Gentherapie, Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Germany.

ABSTRACT
Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Show MeSH

Related in: MedlinePlus

Schematic diagrams of the viral transfer plasmids.The lentiviral vectors HIV-shI-GFP, VRX494, M589, HJ57, and M420, are based on HIV-1NL4-3 or HIV-1HXB2. The viral inhibitors encoded by each of the experimental vectors are noted in parentheses. Where indicated, vectors use a heterologous CMV promoter to initiate transcription of the genomic RNA with a self-inactivating 3′ LTR. The VRX494 vector uses a functional HIV-1 LTR, which is upregulated after viral infection [36]. All vectors contain cis-acting regulatory domains (the ψ packaging signal, the central polypurine tract [cppt], the Rev response element [RRE], and, in some cases, the Woodchuck post-transcriptional regulatory element [wPRE]), and eGFP. The vector HIV-shI-GFP contains the U6 promoter regulating a shRNA targeting exon 1 of HIV-1 tat and rev (shI) [10]. The lentiviral vector VRX494 contains 937 bp of antisense (AS) HIV-1 envelope, and eGFP transcriptionally regulated by the HIV-1 LTR [32]. The vector M589 contains an internal SFFV promoter regulating expression of the C46 heptad repeat-anchored with a linker and transmembrane domain:GFP fusion protein (maC46:GFP). The control vectors HJ57 and M420 do not contain an inhibitor cassette.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2925957&req=5

pone-0012357-g001: Schematic diagrams of the viral transfer plasmids.The lentiviral vectors HIV-shI-GFP, VRX494, M589, HJ57, and M420, are based on HIV-1NL4-3 or HIV-1HXB2. The viral inhibitors encoded by each of the experimental vectors are noted in parentheses. Where indicated, vectors use a heterologous CMV promoter to initiate transcription of the genomic RNA with a self-inactivating 3′ LTR. The VRX494 vector uses a functional HIV-1 LTR, which is upregulated after viral infection [36]. All vectors contain cis-acting regulatory domains (the ψ packaging signal, the central polypurine tract [cppt], the Rev response element [RRE], and, in some cases, the Woodchuck post-transcriptional regulatory element [wPRE]), and eGFP. The vector HIV-shI-GFP contains the U6 promoter regulating a shRNA targeting exon 1 of HIV-1 tat and rev (shI) [10]. The lentiviral vector VRX494 contains 937 bp of antisense (AS) HIV-1 envelope, and eGFP transcriptionally regulated by the HIV-1 LTR [32]. The vector M589 contains an internal SFFV promoter regulating expression of the C46 heptad repeat-anchored with a linker and transmembrane domain:GFP fusion protein (maC46:GFP). The control vectors HJ57 and M420 do not contain an inhibitor cassette.

Mentions: We directly compared the potency of viral inhibition and the selective advantage of several lentiviral vectors expressing genetic inhibitors of HIV-1 replication: 1. HIV-shI-GFP, which contains the U6 promoter expressing a shRNA targeting exon 1 of HIV-1 tat and rev, as well as GFP expressed by an internal CMV promoter [11]; 2. M589, which contains an internal SFFV promoter expressing a membrane-anchored fusion protein (maC46) consisting of the N-terminal C46 heptad repeat from HIV-1 gp41 anchored with a linker and a transmembrane domain fused to GFP at the intracellular C-terminus [9], [23]; and 3) VRX494, which contains 937 bp of an RNA antisense HIV-1 envelope sequence and GFP transcriptionally regulated by the HIV-1 LTR [12] (Figure 1). The lentiviral vectors HJ57 and M420, which do not contain inhibitor genes but do express GFP, were included as controls. These genetic inhibitors were selected based on previous reports demonstrating their efficacy in inhibiting HIV-1 replication [9], [11], [12], their ability to target distinct sites in the retroviral life cycle, including viral entry (maC46) and as well as post-integration events (shI, VRX494), and their use, either alone [14], [18] or in combination with other genetic inhibitors [24] in human clinical trials.


Survival of the fittest: positive selection of CD4+ T cells expressing a membrane-bound fusion inhibitor following HIV-1 infection.

Kimpel J, Braun SE, Qiu G, Wong FE, Conolle M, Schmitz JE, Brendel C, Humeau LM, Dropulic B, Rossi JJ, Berger A, von Laer D, Johnson RP - PLoS ONE (2010)

Schematic diagrams of the viral transfer plasmids.The lentiviral vectors HIV-shI-GFP, VRX494, M589, HJ57, and M420, are based on HIV-1NL4-3 or HIV-1HXB2. The viral inhibitors encoded by each of the experimental vectors are noted in parentheses. Where indicated, vectors use a heterologous CMV promoter to initiate transcription of the genomic RNA with a self-inactivating 3′ LTR. The VRX494 vector uses a functional HIV-1 LTR, which is upregulated after viral infection [36]. All vectors contain cis-acting regulatory domains (the ψ packaging signal, the central polypurine tract [cppt], the Rev response element [RRE], and, in some cases, the Woodchuck post-transcriptional regulatory element [wPRE]), and eGFP. The vector HIV-shI-GFP contains the U6 promoter regulating a shRNA targeting exon 1 of HIV-1 tat and rev (shI) [10]. The lentiviral vector VRX494 contains 937 bp of antisense (AS) HIV-1 envelope, and eGFP transcriptionally regulated by the HIV-1 LTR [32]. The vector M589 contains an internal SFFV promoter regulating expression of the C46 heptad repeat-anchored with a linker and transmembrane domain:GFP fusion protein (maC46:GFP). The control vectors HJ57 and M420 do not contain an inhibitor cassette.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2925957&req=5

pone-0012357-g001: Schematic diagrams of the viral transfer plasmids.The lentiviral vectors HIV-shI-GFP, VRX494, M589, HJ57, and M420, are based on HIV-1NL4-3 or HIV-1HXB2. The viral inhibitors encoded by each of the experimental vectors are noted in parentheses. Where indicated, vectors use a heterologous CMV promoter to initiate transcription of the genomic RNA with a self-inactivating 3′ LTR. The VRX494 vector uses a functional HIV-1 LTR, which is upregulated after viral infection [36]. All vectors contain cis-acting regulatory domains (the ψ packaging signal, the central polypurine tract [cppt], the Rev response element [RRE], and, in some cases, the Woodchuck post-transcriptional regulatory element [wPRE]), and eGFP. The vector HIV-shI-GFP contains the U6 promoter regulating a shRNA targeting exon 1 of HIV-1 tat and rev (shI) [10]. The lentiviral vector VRX494 contains 937 bp of antisense (AS) HIV-1 envelope, and eGFP transcriptionally regulated by the HIV-1 LTR [32]. The vector M589 contains an internal SFFV promoter regulating expression of the C46 heptad repeat-anchored with a linker and transmembrane domain:GFP fusion protein (maC46:GFP). The control vectors HJ57 and M420 do not contain an inhibitor cassette.
Mentions: We directly compared the potency of viral inhibition and the selective advantage of several lentiviral vectors expressing genetic inhibitors of HIV-1 replication: 1. HIV-shI-GFP, which contains the U6 promoter expressing a shRNA targeting exon 1 of HIV-1 tat and rev, as well as GFP expressed by an internal CMV promoter [11]; 2. M589, which contains an internal SFFV promoter expressing a membrane-anchored fusion protein (maC46) consisting of the N-terminal C46 heptad repeat from HIV-1 gp41 anchored with a linker and a transmembrane domain fused to GFP at the intracellular C-terminus [9], [23]; and 3) VRX494, which contains 937 bp of an RNA antisense HIV-1 envelope sequence and GFP transcriptionally regulated by the HIV-1 LTR [12] (Figure 1). The lentiviral vectors HJ57 and M420, which do not contain inhibitor genes but do express GFP, were included as controls. These genetic inhibitors were selected based on previous reports demonstrating their efficacy in inhibiting HIV-1 replication [9], [11], [12], their ability to target distinct sites in the retroviral life cycle, including viral entry (maC46) and as well as post-integration events (shI, VRX494), and their use, either alone [14], [18] or in combination with other genetic inhibitors [24] in human clinical trials.

Bottom Line: However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells.These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo.Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

View Article: PubMed Central - PubMed

Affiliation: Angewandte Virologie und Gentherapie, Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt, Germany.

ABSTRACT
Although a variety of genetic strategies have been developed to inhibit HIV replication, few direct comparisons of the efficacy of these inhibitors have been carried out. Moreover, most studies have not examined whether genetic inhibitors are able to induce a survival advantage that results in an expansion of genetically-modified cells following HIV infection. We evaluated the efficacy of three leading genetic strategies to inhibit HIV replication: 1) an HIV-1 tat/rev-specific small hairpin (sh) RNA; 2) an RNA antisense gene specific for the HIV-1 envelope; and 3) a viral entry inhibitor, maC46. In stably transduced cell lines selected such that >95% of cells expressed the genetic inhibitor, the RNA antisense envelope and viral entry inhibitor maC46 provided the strongest inhibition of HIV-1 replication. However, when mixed populations of transduced and untransduced cells were challenged with HIV-1, the maC46 fusion inhibitor resulted in highly efficient positive selection of transduced cells, an effect that was evident even in mixed populations containing as few as 1% maC46-expressing cells. The selective advantage of the maC46 fusion inhibitor was also observed in HIV-1-infected cultures of primary T lymphocytes as well as in HIV-1-infected humanized mice. These results demonstrate robust inhibition of HIV replication with the fusion inhibitor maC46 and the antisense Env inhibitor, and importantly, a survival advantage of cells expressing the maC46 fusion inhibitor both in vitro and in vivo. Evaluation of the ability of genetic inhibitors of HIV-1 replication to confer a survival advantage on genetically-modified cells provides unique information not provided by standard techniques that may be important in the in vivo efficacy of these genes.

Show MeSH
Related in: MedlinePlus