Qing K, Wang X S, Kube D M, Ponnazhagan S, Bajpai A, Srivastava A
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10879-84. doi: 10.1073/pnas.94.20.10879.
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.
腺相关病毒2型（AAV）是一种含单链DNA的非致病性人类细小病毒，作为一种潜在的有用的人类基因治疗载体已受到关注。然而，病毒基因组的单链性质显著影响转导效率，因为病毒第二链DNA合成是限速步骤。我们推测一种宿主细胞蛋白与AAV基因组反向末端重复结构内的单链D序列相互作用，并阻止病毒第二链DNA合成。实际上，已鉴定出一种细胞蛋白，它能特异性且优先地与AAV基因组3'端的D序列相互作用。这种蛋白被命名为单链D序列结合蛋白（ssD-BP），在酪氨酸残基处被磷酸化，并通过抑制前导链病毒DNA合成来阻断感染细胞中AAV介导的转基因表达。染料木黄酮对细胞蛋白酪氨酸激酶的抑制导致ssD-BP去磷酸化，不仅导致重组AAV转基因表达显著增强，还导致野生型AAV基因组的自主复制。ssD-BP的去磷酸化也与腺病毒感染或腺病毒E4orf6蛋白的表达相关，已知该蛋白可诱导AAV DNA复制和基因表达。因此，ssD-BP的磷酸化状态似乎在AAV的生命周期中起关键作用，并且可能被证明是在人类基因治疗中成功使用基于AAV的载体的一个重要决定因素。
