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An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses.

Schreiber CA, Sakuma T, Izumiya Y, Holditch SJ, Hickey RD, Bressin RK, Basu U, Koide K, Asokan A, Ikeda Y - PLoS Pathog. (2015)

Bottom Line: Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction.In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression.Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

ABSTRACT
Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

No MeSH data available.


Related in: MedlinePlus

PHF5A blocks AAV vector transduction after second strand synthesis.(A) HeLa cells pre-treated with control or PHF5A siRNAs for 24hr were transduced with AAV9, 2, 6 or 8 vectors (MOI 104) expressing luciferase under the control of the SFFV retroviral promoter with no splicing unit. Relative increase in luciferase expression was determined 48 hours p.i. Averages of three independent experiments were shown. Error bars represent standard error of the mean. (B) Melanoma A375 cells and primary human fibroblasts were pre-treated with siRNAs for 24 hours, followed by transduction with the AAV2 CMV-Luc vector (MOI 104) for 48 hours. (C) HeLa cells were pre-treated with siRNAs for 24 hours and infected by the AAV9 CMV-Luc vector (MOI 8 x 104). Total cytoplasmic and nuclear DNA were isolated and AAV luciferase vector genome copies were determined by quantitative real-time PCR at 2, 6 and 24 hours p.i. All samples were prepared in duplicate, and results represent the average of three separate experiments. (D) Same as C, but total DNA at 6 hours p.i. was used to determine total and DNase-resistant AAV genome copies and assess the percent DNase-resistant AAV genomes. Samples were in duplicate and results show the average of two independent experiments. (E) siRNA-treated HeLa cells were infected with AAV9 CMV-Luc vector (MOI 8 x 104) for 1, 3 or 6 hours. Total nuclear DNA samples were used to detect the vector-derived single-stranded and double stranded monomers by Southern blotting. (F) HeLa cells were transfected with siRNAs for 24 hours, followed by infection with a GFP-expressing self-complementary (sc) AAV9 vector (MOI 2 x 104) for 48 hours. Flow cytometry analysis was performed to quantify GFP-positive cell populations. The graph represents percentage of GFP-positive cells from the R2-gated population. (G) HeLa cells were pre-treated or untreated with siRNAs for 24 hours, followed by transduction with the AAV9 CMV-Luc vector (MOI 4 x 105) for 36 hours. Nuclear and cytoplasmic RNA samples were subject to the Northern blotting analysis for detection of the luciferase transcripts. (H) HeLa cells were treated with no siRNA, control or PHF5A siRNAs for 24 hours and then transduced by AAV9 CMV-Luc (MOI 2 x 105). Thirty-six hours p.i., cells were harvested and levels of luciferase transcripts were determined by RT-qPCR. (I) HeLa cells were pre-treated with control or PHF5A siRNAs for 24 hours, followed by transfection with purified AAV CMV-Luc genomic DNA (0.1 μg/well). Luciferase activities were determined at 48 hours p.i. *p<0.05.
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ppat.1005082.g002: PHF5A blocks AAV vector transduction after second strand synthesis.(A) HeLa cells pre-treated with control or PHF5A siRNAs for 24hr were transduced with AAV9, 2, 6 or 8 vectors (MOI 104) expressing luciferase under the control of the SFFV retroviral promoter with no splicing unit. Relative increase in luciferase expression was determined 48 hours p.i. Averages of three independent experiments were shown. Error bars represent standard error of the mean. (B) Melanoma A375 cells and primary human fibroblasts were pre-treated with siRNAs for 24 hours, followed by transduction with the AAV2 CMV-Luc vector (MOI 104) for 48 hours. (C) HeLa cells were pre-treated with siRNAs for 24 hours and infected by the AAV9 CMV-Luc vector (MOI 8 x 104). Total cytoplasmic and nuclear DNA were isolated and AAV luciferase vector genome copies were determined by quantitative real-time PCR at 2, 6 and 24 hours p.i. All samples were prepared in duplicate, and results represent the average of three separate experiments. (D) Same as C, but total DNA at 6 hours p.i. was used to determine total and DNase-resistant AAV genome copies and assess the percent DNase-resistant AAV genomes. Samples were in duplicate and results show the average of two independent experiments. (E) siRNA-treated HeLa cells were infected with AAV9 CMV-Luc vector (MOI 8 x 104) for 1, 3 or 6 hours. Total nuclear DNA samples were used to detect the vector-derived single-stranded and double stranded monomers by Southern blotting. (F) HeLa cells were transfected with siRNAs for 24 hours, followed by infection with a GFP-expressing self-complementary (sc) AAV9 vector (MOI 2 x 104) for 48 hours. Flow cytometry analysis was performed to quantify GFP-positive cell populations. The graph represents percentage of GFP-positive cells from the R2-gated population. (G) HeLa cells were pre-treated or untreated with siRNAs for 24 hours, followed by transduction with the AAV9 CMV-Luc vector (MOI 4 x 105) for 36 hours. Nuclear and cytoplasmic RNA samples were subject to the Northern blotting analysis for detection of the luciferase transcripts. (H) HeLa cells were treated with no siRNA, control or PHF5A siRNAs for 24 hours and then transduced by AAV9 CMV-Luc (MOI 2 x 105). Thirty-six hours p.i., cells were harvested and levels of luciferase transcripts were determined by RT-qPCR. (I) HeLa cells were pre-treated with control or PHF5A siRNAs for 24 hours, followed by transfection with purified AAV CMV-Luc genomic DNA (0.1 μg/well). Luciferase activities were determined at 48 hours p.i. *p<0.05.

Mentions: The AAV CMV-Luc vector construct used in the library screening contained a human beta globin intron. To rule out the possibility of PHF5A modulating the CMV promoter activity or the intronic unit, we first replaced the CMV promoter and intron sequence in the AAV vector genome with an intron-less retroviral SFFV promoter. Disruption of PHF5A increased transduction by multiple AAV serotypes (Fig 2A), indicating that the PHF5A-mediated restriction was independent from internal promoters or receptors used by AAV vectors. Likewise, knocking down PHF5A was effective at increasing AAV vector transduction in other cell types, including A375 melanoma cells and primary cardiac fibroblasts (Fig 2B).


An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses.

Schreiber CA, Sakuma T, Izumiya Y, Holditch SJ, Hickey RD, Bressin RK, Basu U, Koide K, Asokan A, Ikeda Y - PLoS Pathog. (2015)

PHF5A blocks AAV vector transduction after second strand synthesis.(A) HeLa cells pre-treated with control or PHF5A siRNAs for 24hr were transduced with AAV9, 2, 6 or 8 vectors (MOI 104) expressing luciferase under the control of the SFFV retroviral promoter with no splicing unit. Relative increase in luciferase expression was determined 48 hours p.i. Averages of three independent experiments were shown. Error bars represent standard error of the mean. (B) Melanoma A375 cells and primary human fibroblasts were pre-treated with siRNAs for 24 hours, followed by transduction with the AAV2 CMV-Luc vector (MOI 104) for 48 hours. (C) HeLa cells were pre-treated with siRNAs for 24 hours and infected by the AAV9 CMV-Luc vector (MOI 8 x 104). Total cytoplasmic and nuclear DNA were isolated and AAV luciferase vector genome copies were determined by quantitative real-time PCR at 2, 6 and 24 hours p.i. All samples were prepared in duplicate, and results represent the average of three separate experiments. (D) Same as C, but total DNA at 6 hours p.i. was used to determine total and DNase-resistant AAV genome copies and assess the percent DNase-resistant AAV genomes. Samples were in duplicate and results show the average of two independent experiments. (E) siRNA-treated HeLa cells were infected with AAV9 CMV-Luc vector (MOI 8 x 104) for 1, 3 or 6 hours. Total nuclear DNA samples were used to detect the vector-derived single-stranded and double stranded monomers by Southern blotting. (F) HeLa cells were transfected with siRNAs for 24 hours, followed by infection with a GFP-expressing self-complementary (sc) AAV9 vector (MOI 2 x 104) for 48 hours. Flow cytometry analysis was performed to quantify GFP-positive cell populations. The graph represents percentage of GFP-positive cells from the R2-gated population. (G) HeLa cells were pre-treated or untreated with siRNAs for 24 hours, followed by transduction with the AAV9 CMV-Luc vector (MOI 4 x 105) for 36 hours. Nuclear and cytoplasmic RNA samples were subject to the Northern blotting analysis for detection of the luciferase transcripts. (H) HeLa cells were treated with no siRNA, control or PHF5A siRNAs for 24 hours and then transduced by AAV9 CMV-Luc (MOI 2 x 105). Thirty-six hours p.i., cells were harvested and levels of luciferase transcripts were determined by RT-qPCR. (I) HeLa cells were pre-treated with control or PHF5A siRNAs for 24 hours, followed by transfection with purified AAV CMV-Luc genomic DNA (0.1 μg/well). Luciferase activities were determined at 48 hours p.i. *p<0.05.
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Related In: Results  -  Collection

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ppat.1005082.g002: PHF5A blocks AAV vector transduction after second strand synthesis.(A) HeLa cells pre-treated with control or PHF5A siRNAs for 24hr were transduced with AAV9, 2, 6 or 8 vectors (MOI 104) expressing luciferase under the control of the SFFV retroviral promoter with no splicing unit. Relative increase in luciferase expression was determined 48 hours p.i. Averages of three independent experiments were shown. Error bars represent standard error of the mean. (B) Melanoma A375 cells and primary human fibroblasts were pre-treated with siRNAs for 24 hours, followed by transduction with the AAV2 CMV-Luc vector (MOI 104) for 48 hours. (C) HeLa cells were pre-treated with siRNAs for 24 hours and infected by the AAV9 CMV-Luc vector (MOI 8 x 104). Total cytoplasmic and nuclear DNA were isolated and AAV luciferase vector genome copies were determined by quantitative real-time PCR at 2, 6 and 24 hours p.i. All samples were prepared in duplicate, and results represent the average of three separate experiments. (D) Same as C, but total DNA at 6 hours p.i. was used to determine total and DNase-resistant AAV genome copies and assess the percent DNase-resistant AAV genomes. Samples were in duplicate and results show the average of two independent experiments. (E) siRNA-treated HeLa cells were infected with AAV9 CMV-Luc vector (MOI 8 x 104) for 1, 3 or 6 hours. Total nuclear DNA samples were used to detect the vector-derived single-stranded and double stranded monomers by Southern blotting. (F) HeLa cells were transfected with siRNAs for 24 hours, followed by infection with a GFP-expressing self-complementary (sc) AAV9 vector (MOI 2 x 104) for 48 hours. Flow cytometry analysis was performed to quantify GFP-positive cell populations. The graph represents percentage of GFP-positive cells from the R2-gated population. (G) HeLa cells were pre-treated or untreated with siRNAs for 24 hours, followed by transduction with the AAV9 CMV-Luc vector (MOI 4 x 105) for 36 hours. Nuclear and cytoplasmic RNA samples were subject to the Northern blotting analysis for detection of the luciferase transcripts. (H) HeLa cells were treated with no siRNA, control or PHF5A siRNAs for 24 hours and then transduced by AAV9 CMV-Luc (MOI 2 x 105). Thirty-six hours p.i., cells were harvested and levels of luciferase transcripts were determined by RT-qPCR. (I) HeLa cells were pre-treated with control or PHF5A siRNAs for 24 hours, followed by transfection with purified AAV CMV-Luc genomic DNA (0.1 μg/well). Luciferase activities were determined at 48 hours p.i. *p<0.05.
Mentions: The AAV CMV-Luc vector construct used in the library screening contained a human beta globin intron. To rule out the possibility of PHF5A modulating the CMV promoter activity or the intronic unit, we first replaced the CMV promoter and intron sequence in the AAV vector genome with an intron-less retroviral SFFV promoter. Disruption of PHF5A increased transduction by multiple AAV serotypes (Fig 2A), indicating that the PHF5A-mediated restriction was independent from internal promoters or receptors used by AAV vectors. Likewise, knocking down PHF5A was effective at increasing AAV vector transduction in other cell types, including A375 melanoma cells and primary cardiac fibroblasts (Fig 2B).

Bottom Line: Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction.In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression.Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America.

ABSTRACT
Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

No MeSH data available.


Related in: MedlinePlus