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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

The U2snRNP complex plays the key role in restricting AAV vector transduction.(A) HeLa cells were transfected with control siRNA, or siRNAs targeting PHF5A, histone 4, U2AF1, SF3B1, SF3B2 and SF3B3 for 24 hours, followed by the AAV9 CMV-Luc vector transduction (MOI 104). Relative luciferase expression was determined 48 hours p.i. (B) Same as A, but luciferase-expressing adenoviral vector was used to transduce siRNA-treated cells (MOI of 3 x 102). (C) Same as A, except that the siRNA-treated cells were transfected with the vector genome plasmid, pAAV CMV-Luc (0.2 μg/well)for 48 hours. Note that this plasmid was used to generate the infectious AAV9 CMV-Luc vector used in (A). (D) HeLa cells were treated with increasing concentrations of U2 snRNP inhibitor, meayamycin B, followed by transduction with the AAV9 CMV-Luc vector (MOI 104). Relative luciferase expression was determined 48 hours p.i. (E) Same as D, except that a prespliceosome/A complex inhibitor, Isoginkgetin, was used. (F) HeLa cells were treated with indicated spliceosome inhibitors for 8 and 24 hours and levels of unspliced and spliced cellular MAPT (microtubule associated protein tau) transcripts were determined by RT-PCR. (G) HeLa cells were treated with 20 nM meayamycin B at various time points before or after AAV9 CMV-Luc vector infection (MOI 104). Relative luciferase expression was determined 48 hours p.i. (H) HeLa cells were infected by AAV9 CMV-GFP (MOI 103) or scAAV9 CMV-GFP vectors (MOI 6 x 103), followed by treatment with 20 nM meayamycin B at 8 hours p.i. Flow cytometry analysis was performed to see GFP-positive cell populations at 48 hours p.i. (I) Co-treatment of HeLa cells with SF3B1 siRNA and Meayamycin B. HeLa cells were treated with the siRNAs for 48 hr, followed by infection with AAV9 CMV-Luc (MOI 104). At 9 hours p.i. Meayamycin B (5nM) was added, and cells were harvested for the luciferase assay 48 hours p.i. (J) Co-treatment of HeLa cells with MG-132 and Meayamycin B. 30 min prior to AAV infection cells were treated with MG-132. 9 hours after infection with AAV9 CMV-Luc (MOI 104), cells were treated with meayamycin B, and harvested for luciferase assay 20 hours later. Due to notable toxicity of MG-132, we needed to harvest cells at this early time point. (K) Influence of dual treatment with human adenovirus 5 infection and SF3B1 disruption on AAV vector infection. HeLa cells were treated with control or SF3B1 siRNAs for 24 hours, followed by infection with AAV2 CMV-Luc (MOI 104) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 104) for 48 hours. (L) Influence of adenovirus 5 infection on subcellular localization of SF3B1 in HeLa cells. HeLa cells were infected with human adenovirus 5 (MOI 104) for 24 hours, and SF3B1 in control and infected HeLa cells was visualized by anti-SF3B1 antibody (red). Nuclei were counter-stained by DAPI (blue). (A-E, G, I, J and K) Samples were run in triplicate and results are the average of two independent experiments. *p<0.05.
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ppat.1005082.g003: The U2snRNP complex plays the key role in restricting AAV vector transduction.(A) HeLa cells were transfected with control siRNA, or siRNAs targeting PHF5A, histone 4, U2AF1, SF3B1, SF3B2 and SF3B3 for 24 hours, followed by the AAV9 CMV-Luc vector transduction (MOI 104). Relative luciferase expression was determined 48 hours p.i. (B) Same as A, but luciferase-expressing adenoviral vector was used to transduce siRNA-treated cells (MOI of 3 x 102). (C) Same as A, except that the siRNA-treated cells were transfected with the vector genome plasmid, pAAV CMV-Luc (0.2 μg/well)for 48 hours. Note that this plasmid was used to generate the infectious AAV9 CMV-Luc vector used in (A). (D) HeLa cells were treated with increasing concentrations of U2 snRNP inhibitor, meayamycin B, followed by transduction with the AAV9 CMV-Luc vector (MOI 104). Relative luciferase expression was determined 48 hours p.i. (E) Same as D, except that a prespliceosome/A complex inhibitor, Isoginkgetin, was used. (F) HeLa cells were treated with indicated spliceosome inhibitors for 8 and 24 hours and levels of unspliced and spliced cellular MAPT (microtubule associated protein tau) transcripts were determined by RT-PCR. (G) HeLa cells were treated with 20 nM meayamycin B at various time points before or after AAV9 CMV-Luc vector infection (MOI 104). Relative luciferase expression was determined 48 hours p.i. (H) HeLa cells were infected by AAV9 CMV-GFP (MOI 103) or scAAV9 CMV-GFP vectors (MOI 6 x 103), followed by treatment with 20 nM meayamycin B at 8 hours p.i. Flow cytometry analysis was performed to see GFP-positive cell populations at 48 hours p.i. (I) Co-treatment of HeLa cells with SF3B1 siRNA and Meayamycin B. HeLa cells were treated with the siRNAs for 48 hr, followed by infection with AAV9 CMV-Luc (MOI 104). At 9 hours p.i. Meayamycin B (5nM) was added, and cells were harvested for the luciferase assay 48 hours p.i. (J) Co-treatment of HeLa cells with MG-132 and Meayamycin B. 30 min prior to AAV infection cells were treated with MG-132. 9 hours after infection with AAV9 CMV-Luc (MOI 104), cells were treated with meayamycin B, and harvested for luciferase assay 20 hours later. Due to notable toxicity of MG-132, we needed to harvest cells at this early time point. (K) Influence of dual treatment with human adenovirus 5 infection and SF3B1 disruption on AAV vector infection. HeLa cells were treated with control or SF3B1 siRNAs for 24 hours, followed by infection with AAV2 CMV-Luc (MOI 104) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 104) for 48 hours. (L) Influence of adenovirus 5 infection on subcellular localization of SF3B1 in HeLa cells. HeLa cells were infected with human adenovirus 5 (MOI 104) for 24 hours, and SF3B1 in control and infected HeLa cells was visualized by anti-SF3B1 antibody (red). Nuclei were counter-stained by DAPI (blue). (A-E, G, I, J and K) Samples were run in triplicate and results are the average of two independent experiments. *p<0.05.

Mentions: PHF5A has been reported to interact with various proteins, including the U2 snRNP proteins, SF3B1, SF3B2, SF3B3 [34,35,36], U2AF1, ATP-dependent helicases EP400 and DDX1, and arginine-serine-rich domains of splicing factor SFRS5 [36]. Additionally, through co-immuno-precipitation of HA-tagged PHF5A, we identified potential PHF5A-interacting proteins, including FUS, EEF1, EEF2 and HIST1H4B. To further understand the underlying mechanism, we assessed the effects of disrupting those proteins on expression from AAV vectors. After verification of reduction in corresponding transcripts upon transfection of specific siRNAs (S4A Fig), siRNA-treated cells were infected with AAV9 CMV-Luc vectors at 24 hours post transfection, with luciferase activity assayed 48 hours p.i. Ablation of U2 snRNP components and U2 snRNP-associated factor (U2AF1) resulted in a substantial increase in luciferase activity relative to HeLa cells pre-treated with a control siRNA (Figs 3A and S4B). Disruption of HIST1H4B, one of histone H4 genes, also showed a modest increase, while ablation of other factors showed no notable effect. Of note, disruption of spliceosome proteins involved in other splicing steps, including SNRNP200 and PRPF31, essential factors for U4/U6-U5 formation and function, did not increase the AAV vector transduction (S4A and S4B Fig). These results suggest that PHF5A blocks AAV vector transduction through an interaction with U2 snRNP proteins and associated U2AF1, independently of cellular RNA spliceosome function. Similar to the effects of PHF5A knockdown, disruption of U2 snRNP components or U2AF1 did not enhance the luciferase expression from an adenoviral vector or a transfected AAV vector plasmid, pAAV CMV-Luc (Fig 3B and 3C). Taken together, we conclude that infectious AAV particles and all steps in intracellular trafficking pathway are essential for the restriction of transduction by U2 snRNP and associated proteins.


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)

The U2snRNP complex plays the key role in restricting AAV vector transduction.(A) HeLa cells were transfected with control siRNA, or siRNAs targeting PHF5A, histone 4, U2AF1, SF3B1, SF3B2 and SF3B3 for 24 hours, followed by the AAV9 CMV-Luc vector transduction (MOI 104). Relative luciferase expression was determined 48 hours p.i. (B) Same as A, but luciferase-expressing adenoviral vector was used to transduce siRNA-treated cells (MOI of 3 x 102). (C) Same as A, except that the siRNA-treated cells were transfected with the vector genome plasmid, pAAV CMV-Luc (0.2 μg/well)for 48 hours. Note that this plasmid was used to generate the infectious AAV9 CMV-Luc vector used in (A). (D) HeLa cells were treated with increasing concentrations of U2 snRNP inhibitor, meayamycin B, followed by transduction with the AAV9 CMV-Luc vector (MOI 104). Relative luciferase expression was determined 48 hours p.i. (E) Same as D, except that a prespliceosome/A complex inhibitor, Isoginkgetin, was used. (F) HeLa cells were treated with indicated spliceosome inhibitors for 8 and 24 hours and levels of unspliced and spliced cellular MAPT (microtubule associated protein tau) transcripts were determined by RT-PCR. (G) HeLa cells were treated with 20 nM meayamycin B at various time points before or after AAV9 CMV-Luc vector infection (MOI 104). Relative luciferase expression was determined 48 hours p.i. (H) HeLa cells were infected by AAV9 CMV-GFP (MOI 103) or scAAV9 CMV-GFP vectors (MOI 6 x 103), followed by treatment with 20 nM meayamycin B at 8 hours p.i. Flow cytometry analysis was performed to see GFP-positive cell populations at 48 hours p.i. (I) Co-treatment of HeLa cells with SF3B1 siRNA and Meayamycin B. HeLa cells were treated with the siRNAs for 48 hr, followed by infection with AAV9 CMV-Luc (MOI 104). At 9 hours p.i. Meayamycin B (5nM) was added, and cells were harvested for the luciferase assay 48 hours p.i. (J) Co-treatment of HeLa cells with MG-132 and Meayamycin B. 30 min prior to AAV infection cells were treated with MG-132. 9 hours after infection with AAV9 CMV-Luc (MOI 104), cells were treated with meayamycin B, and harvested for luciferase assay 20 hours later. Due to notable toxicity of MG-132, we needed to harvest cells at this early time point. (K) Influence of dual treatment with human adenovirus 5 infection and SF3B1 disruption on AAV vector infection. HeLa cells were treated with control or SF3B1 siRNAs for 24 hours, followed by infection with AAV2 CMV-Luc (MOI 104) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 104) for 48 hours. (L) Influence of adenovirus 5 infection on subcellular localization of SF3B1 in HeLa cells. HeLa cells were infected with human adenovirus 5 (MOI 104) for 24 hours, and SF3B1 in control and infected HeLa cells was visualized by anti-SF3B1 antibody (red). Nuclei were counter-stained by DAPI (blue). (A-E, G, I, J and K) Samples were run in triplicate and results are the average of two independent experiments. *p<0.05.
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ppat.1005082.g003: The U2snRNP complex plays the key role in restricting AAV vector transduction.(A) HeLa cells were transfected with control siRNA, or siRNAs targeting PHF5A, histone 4, U2AF1, SF3B1, SF3B2 and SF3B3 for 24 hours, followed by the AAV9 CMV-Luc vector transduction (MOI 104). Relative luciferase expression was determined 48 hours p.i. (B) Same as A, but luciferase-expressing adenoviral vector was used to transduce siRNA-treated cells (MOI of 3 x 102). (C) Same as A, except that the siRNA-treated cells were transfected with the vector genome plasmid, pAAV CMV-Luc (0.2 μg/well)for 48 hours. Note that this plasmid was used to generate the infectious AAV9 CMV-Luc vector used in (A). (D) HeLa cells were treated with increasing concentrations of U2 snRNP inhibitor, meayamycin B, followed by transduction with the AAV9 CMV-Luc vector (MOI 104). Relative luciferase expression was determined 48 hours p.i. (E) Same as D, except that a prespliceosome/A complex inhibitor, Isoginkgetin, was used. (F) HeLa cells were treated with indicated spliceosome inhibitors for 8 and 24 hours and levels of unspliced and spliced cellular MAPT (microtubule associated protein tau) transcripts were determined by RT-PCR. (G) HeLa cells were treated with 20 nM meayamycin B at various time points before or after AAV9 CMV-Luc vector infection (MOI 104). Relative luciferase expression was determined 48 hours p.i. (H) HeLa cells were infected by AAV9 CMV-GFP (MOI 103) or scAAV9 CMV-GFP vectors (MOI 6 x 103), followed by treatment with 20 nM meayamycin B at 8 hours p.i. Flow cytometry analysis was performed to see GFP-positive cell populations at 48 hours p.i. (I) Co-treatment of HeLa cells with SF3B1 siRNA and Meayamycin B. HeLa cells were treated with the siRNAs for 48 hr, followed by infection with AAV9 CMV-Luc (MOI 104). At 9 hours p.i. Meayamycin B (5nM) was added, and cells were harvested for the luciferase assay 48 hours p.i. (J) Co-treatment of HeLa cells with MG-132 and Meayamycin B. 30 min prior to AAV infection cells were treated with MG-132. 9 hours after infection with AAV9 CMV-Luc (MOI 104), cells were treated with meayamycin B, and harvested for luciferase assay 20 hours later. Due to notable toxicity of MG-132, we needed to harvest cells at this early time point. (K) Influence of dual treatment with human adenovirus 5 infection and SF3B1 disruption on AAV vector infection. HeLa cells were treated with control or SF3B1 siRNAs for 24 hours, followed by infection with AAV2 CMV-Luc (MOI 104) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 104) for 48 hours. (L) Influence of adenovirus 5 infection on subcellular localization of SF3B1 in HeLa cells. HeLa cells were infected with human adenovirus 5 (MOI 104) for 24 hours, and SF3B1 in control and infected HeLa cells was visualized by anti-SF3B1 antibody (red). Nuclei were counter-stained by DAPI (blue). (A-E, G, I, J and K) Samples were run in triplicate and results are the average of two independent experiments. *p<0.05.
Mentions: PHF5A has been reported to interact with various proteins, including the U2 snRNP proteins, SF3B1, SF3B2, SF3B3 [34,35,36], U2AF1, ATP-dependent helicases EP400 and DDX1, and arginine-serine-rich domains of splicing factor SFRS5 [36]. Additionally, through co-immuno-precipitation of HA-tagged PHF5A, we identified potential PHF5A-interacting proteins, including FUS, EEF1, EEF2 and HIST1H4B. To further understand the underlying mechanism, we assessed the effects of disrupting those proteins on expression from AAV vectors. After verification of reduction in corresponding transcripts upon transfection of specific siRNAs (S4A Fig), siRNA-treated cells were infected with AAV9 CMV-Luc vectors at 24 hours post transfection, with luciferase activity assayed 48 hours p.i. Ablation of U2 snRNP components and U2 snRNP-associated factor (U2AF1) resulted in a substantial increase in luciferase activity relative to HeLa cells pre-treated with a control siRNA (Figs 3A and S4B). Disruption of HIST1H4B, one of histone H4 genes, also showed a modest increase, while ablation of other factors showed no notable effect. Of note, disruption of spliceosome proteins involved in other splicing steps, including SNRNP200 and PRPF31, essential factors for U4/U6-U5 formation and function, did not increase the AAV vector transduction (S4A and S4B Fig). These results suggest that PHF5A blocks AAV vector transduction through an interaction with U2 snRNP proteins and associated U2AF1, independently of cellular RNA spliceosome function. Similar to the effects of PHF5A knockdown, disruption of U2 snRNP components or U2AF1 did not enhance the luciferase expression from an adenoviral vector or a transfected AAV vector plasmid, pAAV CMV-Luc (Fig 3B and 3C). Taken together, we conclude that infectious AAV particles and all steps in intracellular trafficking pathway are essential for the restriction of transduction by U2 snRNP and associated proteins.

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