ROLE OF TYROSINE PHOSPHORYLATION OF A CELLULAR PROTEIN IN ADENOASSOCIATED VIRUS 2-MEDIATED TRANSGENE EXPRESSION

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 nat ure of the viral genome significantly impacts upon the transduction ef ficiency, because the second-strand viral DNA synthesis is the rate-li miting step. We hypothesized that a host-cell protein interacts with t he single-stranded D sequence within the inverted terminal repeat stru cture of the AAV genome and prevents the viral second-strand DNA synth esis, Indeed, a cellular protein has been identified that interacts sp ecifically and preferentially with the D sequence at the 3' end of the AAV genome, This protein, designated the single-stranded D-sequence-b inding protein (ssD-BP), is phosphorylated at tyrosine residues and bl ocks AAV-mediated transgene expression in infected cells by inhibiting the leading strand viral DNA synthesis, Inhibition of cellular protei n tyrosine kinases by genistein results in dephosphorylation of the ss D-BP, leading not only to significant augmentation of transgene expres sion from recombinant AAV but also to autonomous replication of the wi ld-type AAV genome, Dephosphorylation of the ssD-BP also correlates wi th adenovirus infection, or expression of the adenovirus E40rf6 protei n, which is known to induce AAV DNA replication and gene expression, T hus, phosphorylation state of the ssD-BP appears to play a crucial rol e in the life cycle of AAV and may prove to be an important determinan t in the successful use of AAV-based vectors in human gene therapy.