Limits...
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

Meayamycin B increases AAV vector transduction of clinically relevant cell types.(A) Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. (B) Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. (C) Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. (D) Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 104 was used.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526370&req=5

ppat.1005082.g005: Meayamycin B increases AAV vector transduction of clinically relevant cell types.(A) Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. (B) Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. (C) Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. (D) Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 104 was used.

Mentions: Finally, we tested the ability of meayamycin B to boost AAV transduction in various cell types, relevant to gene therapy applications. When primary pancreatic islets were transduced with AAV8 CMV-GFP and treated with 2 nM meayamycin B 3 hours p.i., there were increased numbers of GFP expressing cells in drug treated mouse islets (Fig 5A). When primary human pancreatic islets were infected with AAV2 or AAV9 CMV-Luc vectors and treated with 0, 2, 5, or 20 nM meayamycin B at 7 hours p.i., we found dose-dependent increases in luciferase expression in AAV2 and AAV9 infected cells (Fig 5B). Likewise, meayamycin B treatment increased AAV2 and AAV9 transduction of primary neonatal rat cardiomyocytes as well as porcine hepatocytes (Fig 5C and 5D). These results demonstrate that meayamycin B enhances AAV vector transduction of a variety of cell types from different host species. Although we typically observed no notable toxicity in primary cells treated with 5 nM meayamycin B, prolonged treatment with over 10 nM meayamycin B showed anti-proliferative effects as we reported previously [42]. Since meayamycin B is rapidly cleared from circulation by unknown mechanism(s) [42], we were unable to evaluate drug doses high enough to test the impact on AAV vector transduction in vivo.


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)

Meayamycin B increases AAV vector transduction of clinically relevant cell types.(A) Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. (B) Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. (C) Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. (D) Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 104 was used.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526370&req=5

ppat.1005082.g005: Meayamycin B increases AAV vector transduction of clinically relevant cell types.(A) Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. (B) Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. (C) Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. (D) Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 104 was used.
Mentions: Finally, we tested the ability of meayamycin B to boost AAV transduction in various cell types, relevant to gene therapy applications. When primary pancreatic islets were transduced with AAV8 CMV-GFP and treated with 2 nM meayamycin B 3 hours p.i., there were increased numbers of GFP expressing cells in drug treated mouse islets (Fig 5A). When primary human pancreatic islets were infected with AAV2 or AAV9 CMV-Luc vectors and treated with 0, 2, 5, or 20 nM meayamycin B at 7 hours p.i., we found dose-dependent increases in luciferase expression in AAV2 and AAV9 infected cells (Fig 5B). Likewise, meayamycin B treatment increased AAV2 and AAV9 transduction of primary neonatal rat cardiomyocytes as well as porcine hepatocytes (Fig 5C and 5D). These results demonstrate that meayamycin B enhances AAV vector transduction of a variety of cell types from different host species. Although we typically observed no notable toxicity in primary cells treated with 5 nM meayamycin B, prolonged treatment with over 10 nM meayamycin B showed anti-proliferative effects as we reported previously [42]. Since meayamycin B is rapidly cleared from circulation by unknown mechanism(s) [42], we were unable to evaluate drug doses high enough to test the impact on AAV vector transduction in vivo.

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