Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression - PubMed (original) (raw)

Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression

K Qing et al. Proc Natl Acad Sci U S A. 1997.

Abstract

The adeno-associated virus 2 (AAV), a single-stranded DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the single-stranded nature of the viral genome significantly impacts upon the transduction efficiency, because the second-strand viral DNA synthesis is the rate-limiting step. We hypothesized that a host-cell protein interacts with the single-stranded D sequence within the inverted terminal repeat structure of the AAV genome and prevents the viral second-strand DNA synthesis. Indeed, a cellular protein has been identified that interacts specifically and preferentially with the D sequence at the 3' end of the AAV genome. This protein, designated the single-stranded D-sequence-binding protein (ssD-BP), is phosphorylated at tyrosine residues and blocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis. Inhibition of cellular protein tyrosine kinases by genistein results in dephosphorylation of the ssD-BP, leading not only to significant augmentation of transgene expression from recombinant AAV but also to autonomous replication of the wild-type AAV genome. Dephosphorylation of the ssD-BP also correlates with adenovirus infection, or expression of the adenovirus E4orf6 protein, which is known to induce AAV DNA replication and gene expression. Thus, phosphorylation state of the ssD-BP appears to play a crucial role in the life cycle of AAV and may prove to be an important determinant in the successful use of AAV-based vectors in human gene therapy.

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Figures

Figure 1

Differential interaction of the D-BP with the D(−) and D(+) sequences in the AAV ITRs. EMSAs were carried out with D(−) or D(+) 20-nt synthetic oligonucleotides (A) as previously described (16). Various indicated unlabeled single- or double-stranded oligonucleotides representing the D sequence, a non-AAV substitute (S) sequence, and AAV capsid (cap) gene sequences were used in competition experiments (B). Comparative analysis of D-BP interaction with the single-stranded D(−) sequence versus the double-stranded D(±) sequence (C). The arrow indicates the single-stranded D-BP, and the arrowhead indicates the double-stranded D-BP.

Figure 2

The ssD-BP undergoes modification following growth-arrest of HeLa cells. EMSAs were carried out with WCEs prepared under different conditions as described in the text. D(−) or D(±) probes were used under identical conditions as described in the legend to Fig. 1.

Figure 3

Ad2 infection or the AdE4orf6 protein expression leads to ssD-BP modification. Cells were either mock-infected or infected with the wt AAV or Ad2, or were coinfected with AAV+Ad2 at the indicated moi, and WCEs prepared from these cells were used in EMSAs with the D(−) probe (A). EMSAs were also carried out with WCEs prepared from cells transfected with the indicated plasmids (B).

Figure 4

The ssD-BP is phosphorylated at tyrosine residues. The ssD-BP was immunoprecipitated from equivalent amounts of WCEs prepared from cells following mock-infection, infection with AAV or Ad2, or treatment with HU, with the anti-phosphotyrosine antibody (αP-Tyr Ab), followed by precipitation with protein A agarose beads as previously described (–23). Supernatants were collected, and immunoprecipitates were washed with PBS and dissolved in EMSA reaction buffer. Ten microliters of each of the supernatants (Sup.) and resuspended pellet solution (Pellet) were used for EMSA with the D(−) oligonucleotide probe.

Figure 5

Inhibition of protein tyrosine kinases leads to increased transduction efficiency of recombinant AAV. HeLa cells were infected with 20 moi of vCMVp-lacZ following either no treatment (A) or treatment with 10 mM HU for 24 hr (B), 150 μM genistein for 2 hr (C), or 1 mM NaOV for 2 hr (D). Forty-eight hours postinfection, cells were fixed and the recombinant AAV-mediated transgene expression was detected as previously described (26, 27).

Figure 6

Inhibition of cellular protein tyrosine kinases leads to autonomous replication of the wt AAV genome. Cells were infected with the wt AAV at an moi of 2 following either no treatment or treatment with HU, genistein, or NaOV, and low-_M_r DNA samples isolated at various indicated times postinfection were analyzed on Southern blots using an AAV-specific DNA probe. d, m, and ss denote the dimeric and monomeric replicative DNA intermediates and single-stranded progeny AAV genomes, respectively.

Figure 7

A possible model for the role of the tyrosine-phosphorylated ssD-BP in the viral second-strand DNA synthesis and AAV-mediated transgene expression. The phosphorylated form of ssD-BP preferentially complexes with the D(−) sequence in the AAV ITR and blocks initiation of DNA replication from the 3′ OH end. Various indicated treatments cause dephosphorylation of the ssD-BP, leading to some type of conformational change, which results in accessibility of the 3′ OH end as a primer for the second-strand DNA synthesis followed by gene expression.

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Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression - PubMed (original) (raw)