Adeno-associated virus type 2-mediated gene transfer: Correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein transgene expression in human cells in vitro and murine tissues in vivo

Keyun Qing, Benjawan Khuntirat, Cathryn Mah, Dagmar M. Kube, Xu Shan Wang, Selvarangan Ponnazhagan, Shangzhen Zhou, Varavani J. Dwarki, Mervin Yoder, Arun Srivastava

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Abstract

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single- stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3' end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34+ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin- primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

Original languageEnglish
Pages (from-to)1593-1599
Number of pages7
JournalJournal of Virology
Volume72
Issue number2
StatePublished - Feb 1998

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Dependovirus
Transgenes
gene transfer
transgenes
Tyrosine
tyrosine
binding proteins
phosphorylation
Carrier Proteins
Phosphorylation
mice
Genes
cells
dephosphorylation
Hematopoietic Stem Cells
gene therapy
Genetic Therapy
stem cells
tissues
In Vitro Techniques

ASJC Scopus subject areas

  • Immunology

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Adeno-associated virus type 2-mediated gene transfer : Correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein transgene expression in human cells in vitro and murine tissues in vivo. / Qing, Keyun; Khuntirat, Benjawan; Mah, Cathryn; Kube, Dagmar M.; Wang, Xu Shan; Ponnazhagan, Selvarangan; Zhou, Shangzhen; Dwarki, Varavani J.; Yoder, Mervin; Srivastava, Arun.

In: Journal of Virology, Vol. 72, No. 2, 02.1998, p. 1593-1599.

Research output: Contribution to journalArticle

Qing, Keyun ; Khuntirat, Benjawan ; Mah, Cathryn ; Kube, Dagmar M. ; Wang, Xu Shan ; Ponnazhagan, Selvarangan ; Zhou, Shangzhen ; Dwarki, Varavani J. ; Yoder, Mervin ; Srivastava, Arun. / Adeno-associated virus type 2-mediated gene transfer : Correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein transgene expression in human cells in vitro and murine tissues in vivo. In: Journal of Virology. 1998 ; Vol. 72, No. 2. pp. 1593-1599.
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abstract = "Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single- stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3' end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34+ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin- primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.",
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AU - Qing, Keyun

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AU - Mah, Cathryn

AU - Kube, Dagmar M.

AU - Wang, Xu Shan

AU - Ponnazhagan, Selvarangan

AU - Zhou, Shangzhen

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