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

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 Dsequence-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.The adeno-associated virus 2 (AAV), a single-stranded DNAcontaining parvovirus, so far has not been shown to be associated with any pathology in humans (1, 2). The wild-type (wt) AAV genome has been shown to integrate in a sitespecific manner into human chromosome 19q13.3-qter (3-5). Thus, recombinant AAV vectors have emerged as useful alternatives to the more commonly used retroviral and adenoviral vectors for human gene therapy (6-10). However, recent studies from two independent laboratories have suggested that after infection, the leading strand viral DNA synthesis is a rate-limiting step in the efficient transduction by AAV vectors (11,12). AAV inverted terminal repeats (ITRs) contain 145 nt each, the terminal 125 nt of which are palindromic and form T-shaped hairpin (HP) structures. The 3Ј-HP structure serves as a primer for AAV DNA replication. AAV ITRs also contain an additional domain, designated the D sequence, which is a stretch of 20 nt that is not involved in HP formation (1, 2, 13). Our previous studies have indicated that the D sequence plays a crucial role in the efficient rescue, selective replication, and encapsidation of the AAV genome (14, 15). We hypothesized that a cellular protein(s) might interact with the D sequence and prevent the second-strand viral DNA synthesis. Indeed, using electrophoretic mobility-shift assays (EMSAs), we have recently provided evidence for the existence of a hitherto unknown host-c...

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

“…Regardless of the mechanism involved, the extent of viral second-strand DNA synthesis was consistent with the observed transcriptional activity of the vector genomes. [4][5][6][7][8][9][10][11] These data strongly argue that rather than strand-annealing, TC-PTP-mediated tyrosine dephosphorylation of FKBP52, or the absence of FKBP52, leads to efficient AAV second-strand DNA synthesis resulting in an increase in AAV-mediated transgene expression in hepatocytes in vivo. Although our data might appear at odds with the model proposed by Nakai et al, 2 which favors DNA strand-annealing as the mechanism of hepatocyte transduction, the simplest explanation is that viral secondstrand DNA synthesis is the rate-limiting step in B95% of hepatocytes, whereas DNA strand-annealing might be operative in B5% of hepatocytes.…”

“…2,3 Others and we, on the other hand, have suggested that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression. [4][5][6][7][8][9][10][11] We previously observed that following intravenous (i.v.) administration into the tail-vein, AAV vectors selectively target the liver in mice, but less than 5% of hepatocytes express the transgene.…”

“…Phosphorylated FKBP52 inhibits the viral second-strand DNA synthesis leading to inefficient transgene expression. [6][7][8][9][10][11] We have also documented that FKBP52 is dephosphorylated at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), 22,23 which leads to efficient viral …”

“…Phosphorylated FKBP52 inhibits the viral second-strand DNA synthesis leading to inefficient transgene expression. [6][7][8][9][10][11] We have also documented that FKBP52 is dephosphorylated at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), 22,23 which leads to efficient viral second-strand DNA synthesis and a significant increase in AAV-mediated transduction efficiency in established human cell lines as well as in primary hematopoietic stem cells from TC-PTP-transgenic mice. 9 In our current studies, we have examined the efficiency of AAV-mediated transgene expression in murine hepatocytes following i.v.…”

Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo

“…3,4 Thus, failure to undergo viral second-strand synthesis remains a predominant rate-limiting step in the observed low efficiency of singlestranded AAV (ssAAV) vector-mediated transgene expression both in vitro and in vivo. [5][6][7][8][9][10][11][12][13][14] The use of selfcomplementary AAV (scAAV) vectors that bypass the requirement for viral second-strand DNA synthesis can circumvent this problem. 13 However, their widespread use is limited by their limited packaging capacity (B3.3 kb), 15 which is significantly less than that of conventional ssAAV vectors (B6 kb).…”