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Efficient Transduction of LEDGF/p75 Mutant Cells by Gain-of-Function HIV-1 Integrase Mutant Viruses.

Wang H, Shun MC, Li X, Di Nunzio F, Hare S, Cherepanov P, Engelman A - Mol Ther Methods Clin Dev (2014)

Bottom Line: Here we describe the selection and characterization of the K42E gain-of-function mutation in HIV-1 integrase, which greatly improves the efficiency of this system.Although the K42E mutation conferred functional gains to integrase mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant.We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge integrase mutant viral and LEDGF/p75 host proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Controlling the specificity of retroviral DNA integration could improve the safety of gene therapy vectors, and fusions of heterologous chromatin binding modules to the integrase-binding domain from the lentiviral integration host cofactor LEDGF/p75 are a promising retargeting strategy. We previously proposed the utility of integrase mutant lentiviral vectors that are selectively activated by complementary LEDGF/p75 variants, and our initial modifications in HIV-1 integrase and LEDGF/p75 supported about 13% of wild-type vector transduction activity. Here we describe the selection and characterization of the K42E gain-of-function mutation in HIV-1 integrase, which greatly improves the efficiency of this system. Both K42E and initial reverse-charge mutations in integrase negatively impacted reverse transcription and integration, yet when combined together boosted viral transduction efficiency to ~75% of the wild-type vector in a manner dependent on a complementary LEDGF/p75 variant. Although the K42E mutation conferred functional gains to integrase mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant. We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge integrase mutant viral and LEDGF/p75 host proteins.

No MeSH data available.


Related in: MedlinePlus

WT and IN mutant viral reverse transcription and integration activities. (a) Levels of WT, RR, K42E, and RRE HIV-Luc infectivity using the indicated T-cell line. Results were normalized to the level of WT HIV-Luc transduction in the presence of WT LEDGF/p75, which was set to 100%. (b) Levels of LRT product formation from the infections in panel a, normalized to the level of WT HIV-Luc DNA synthesis in cells expressing WT LEDGF/p75 (set at 100%). (c) Levels of 2-LTR circle formation from panel a infections, normalized to the WT virus in cells expressing WT LEDGF/p75. (d) Virus integration levels were normalized to the level of WT HIV-Luc virus in cells expressing WT LEDGF/p75, which was set at 100%. Values in panels a–d represent averages ± SD for two independent experiments. Panel a and d statistical comparisons evaluate RR versus RRE virus in EEE LEDGF/p75-expressing cells, whereas those in panels c and d compare the activities of the indicated viruses across cell lines. *P < 0.05; **P < 0.01. EEE, K401E/K402E/R405E; IN, integrase; LEDGF, lens epithelium–derived growth factor; NS, not significant; WT, wild type.
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fig6: WT and IN mutant viral reverse transcription and integration activities. (a) Levels of WT, RR, K42E, and RRE HIV-Luc infectivity using the indicated T-cell line. Results were normalized to the level of WT HIV-Luc transduction in the presence of WT LEDGF/p75, which was set to 100%. (b) Levels of LRT product formation from the infections in panel a, normalized to the level of WT HIV-Luc DNA synthesis in cells expressing WT LEDGF/p75 (set at 100%). (c) Levels of 2-LTR circle formation from panel a infections, normalized to the WT virus in cells expressing WT LEDGF/p75. (d) Virus integration levels were normalized to the level of WT HIV-Luc virus in cells expressing WT LEDGF/p75, which was set at 100%. Values in panels a–d represent averages ± SD for two independent experiments. Panel a and d statistical comparisons evaluate RR versus RRE virus in EEE LEDGF/p75-expressing cells, whereas those in panels c and d compare the activities of the indicated viruses across cell lines. *P < 0.05; **P < 0.01. EEE, K401E/K402E/R405E; IN, integrase; LEDGF, lens epithelium–derived growth factor; NS, not significant; WT, wild type.

Mentions: WT LEDGF/p75 expression in this series of experiments boosted HIV-Luc infection of TL3 cells ~27-fold, while EEE expression enhanced RR and RRE transduction ~14- and 9-fold over the levels supported by WT LEDGF/p75 (Figure 6a). As expected,10,27 WT HIV-1 DNA synthesis was independent of the level of LEDGF/p75 expression (Figure 6b). By contrast, the RR and K42E IN mutations caused approximately three- to fivefold reverse transcription defects. Because mutations in IN can affect HIV-1 replication at multiple steps, we previously established a classification system to delineate those mutations that specifically affect integration (class I) from those that additionally affect other steps in the viral life cycle (class II).34 The most common pleiotropic defect among class II HIV-1 IN mutant viruses is reverse transcription, and K42E and RR were accordingly typed as class II IN mutant viruses. Combining the K42E and RR mutations interestingly restored IN mutant reverse transcription to ~70% of the level of WT HIV-Luc (Figure 6b).


Efficient Transduction of LEDGF/p75 Mutant Cells by Gain-of-Function HIV-1 Integrase Mutant Viruses.

Wang H, Shun MC, Li X, Di Nunzio F, Hare S, Cherepanov P, Engelman A - Mol Ther Methods Clin Dev (2014)

WT and IN mutant viral reverse transcription and integration activities. (a) Levels of WT, RR, K42E, and RRE HIV-Luc infectivity using the indicated T-cell line. Results were normalized to the level of WT HIV-Luc transduction in the presence of WT LEDGF/p75, which was set to 100%. (b) Levels of LRT product formation from the infections in panel a, normalized to the level of WT HIV-Luc DNA synthesis in cells expressing WT LEDGF/p75 (set at 100%). (c) Levels of 2-LTR circle formation from panel a infections, normalized to the WT virus in cells expressing WT LEDGF/p75. (d) Virus integration levels were normalized to the level of WT HIV-Luc virus in cells expressing WT LEDGF/p75, which was set at 100%. Values in panels a–d represent averages ± SD for two independent experiments. Panel a and d statistical comparisons evaluate RR versus RRE virus in EEE LEDGF/p75-expressing cells, whereas those in panels c and d compare the activities of the indicated viruses across cell lines. *P < 0.05; **P < 0.01. EEE, K401E/K402E/R405E; IN, integrase; LEDGF, lens epithelium–derived growth factor; NS, not significant; WT, wild type.
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fig6: WT and IN mutant viral reverse transcription and integration activities. (a) Levels of WT, RR, K42E, and RRE HIV-Luc infectivity using the indicated T-cell line. Results were normalized to the level of WT HIV-Luc transduction in the presence of WT LEDGF/p75, which was set to 100%. (b) Levels of LRT product formation from the infections in panel a, normalized to the level of WT HIV-Luc DNA synthesis in cells expressing WT LEDGF/p75 (set at 100%). (c) Levels of 2-LTR circle formation from panel a infections, normalized to the WT virus in cells expressing WT LEDGF/p75. (d) Virus integration levels were normalized to the level of WT HIV-Luc virus in cells expressing WT LEDGF/p75, which was set at 100%. Values in panels a–d represent averages ± SD for two independent experiments. Panel a and d statistical comparisons evaluate RR versus RRE virus in EEE LEDGF/p75-expressing cells, whereas those in panels c and d compare the activities of the indicated viruses across cell lines. *P < 0.05; **P < 0.01. EEE, K401E/K402E/R405E; IN, integrase; LEDGF, lens epithelium–derived growth factor; NS, not significant; WT, wild type.
Mentions: WT LEDGF/p75 expression in this series of experiments boosted HIV-Luc infection of TL3 cells ~27-fold, while EEE expression enhanced RR and RRE transduction ~14- and 9-fold over the levels supported by WT LEDGF/p75 (Figure 6a). As expected,10,27 WT HIV-1 DNA synthesis was independent of the level of LEDGF/p75 expression (Figure 6b). By contrast, the RR and K42E IN mutations caused approximately three- to fivefold reverse transcription defects. Because mutations in IN can affect HIV-1 replication at multiple steps, we previously established a classification system to delineate those mutations that specifically affect integration (class I) from those that additionally affect other steps in the viral life cycle (class II).34 The most common pleiotropic defect among class II HIV-1 IN mutant viruses is reverse transcription, and K42E and RR were accordingly typed as class II IN mutant viruses. Combining the K42E and RR mutations interestingly restored IN mutant reverse transcription to ~70% of the level of WT HIV-Luc (Figure 6b).

Bottom Line: Here we describe the selection and characterization of the K42E gain-of-function mutation in HIV-1 integrase, which greatly improves the efficiency of this system.Although the K42E mutation conferred functional gains to integrase mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant.We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge integrase mutant viral and LEDGF/p75 host proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Controlling the specificity of retroviral DNA integration could improve the safety of gene therapy vectors, and fusions of heterologous chromatin binding modules to the integrase-binding domain from the lentiviral integration host cofactor LEDGF/p75 are a promising retargeting strategy. We previously proposed the utility of integrase mutant lentiviral vectors that are selectively activated by complementary LEDGF/p75 variants, and our initial modifications in HIV-1 integrase and LEDGF/p75 supported about 13% of wild-type vector transduction activity. Here we describe the selection and characterization of the K42E gain-of-function mutation in HIV-1 integrase, which greatly improves the efficiency of this system. Both K42E and initial reverse-charge mutations in integrase negatively impacted reverse transcription and integration, yet when combined together boosted viral transduction efficiency to ~75% of the wild-type vector in a manner dependent on a complementary LEDGF/p75 variant. Although the K42E mutation conferred functional gains to integrase mutant viral reverse transcription and integration, only the integration boost depended on the engineered LEDGF/p75 mutant. We conclude that the specificity of lentiviral retargeting strategies based on heterologous LEDGF/p75 fusion proteins will benefit from our optimized system that utilizes the unique complementation properties of reverse-charge integrase mutant viral and LEDGF/p75 host proteins.

No MeSH data available.


Related in: MedlinePlus