Adeno-associated virus Rep78 protein interacts with protein kinase a and its homolog PRKX and inhibits CREB-dependent transcriptional activation
Adeno-associated virus Rep78 protein interacts with protein kinase a and its homolog PRKX and inhibits CREB-dependent transcriptional activation
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DOI：
10.1128/jvi.72.10.7916-7925.1998
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发表时间：
1998-10-01
影响因子：
5.4
通讯作者：
Stacey, SN
中科院分区：
文献类型：
--
作者：
Di Pasquale, G;Stacey, SN
腺相关病毒（AAV）是一种依赖病毒属的人细小病毒。AAV的复制主要局限于与辅助病毒共感染的细胞。在没有辅助病毒的情况下，AAV基因组可以整合到特定的染色体位点，在那里它保持潜伏状态，直到宿主细胞被适当的辅助病毒重复感染而重新激活。AAV的复制功能已经定位到Rep68和Rep78基因产物上。Rep蛋白具有DNA结合、核酸内切酶和解旋酶活性，并参与AAV和异源启动子的转录调节。在一系列病毒性和非病毒性肿瘤中，AAV与抑制致癌性有关。在这项研究中，我们试图鉴定和研究AAV Rep的细胞蛋白靶点，以便更好地了解Rep的各种活性。我们使用酵母双杂交系统鉴定了与AAV 2型Rep78相互作用的HeLa细胞蛋白，我们分离了几个强相互作用的克隆，随后鉴定为PRIM（以前命名为PKX1），这是最近描述的蛋白激酶a （PKA）催化亚基（PKA,）的同源物。相互作用通过pMal-Rep下拉试验在体外得到证实。Rep78的相互作用区域被定位到c端锌指状结构域；缺乏该结构域的Rep68不与PRIM相互作用，PRIM表现出对组蛋白H1和PKA寡肽靶点的自磷酸化和激酶活性。与Rep78的相互作用抑制了自磷酸化。在转染实验中，一个PRIM表达载体被证明能够激活creb依赖性转录。这种激活被Rep78抑制，而不被Rep68抑制。由于PRIM是PKA的同源基因，我们研究了Rep78是否可以直接与PKA相互作用。发现pMal-Rep78与纯化的PKA结合，抑制其激酶活性。共转染实验表明，Rep78可以阻断PKA表达载体对CREB的激活。这些实验表明，AAV可能干扰感染细胞中正常的循环AMP反应途径。
Adeno-associated virus (AAV) is a human parvovirus of the genus Dependovirus. AAV replication is largely restricted to cells which are coinfected with a helper virus. In the absence of a helper virus, the AAV genome can integrate into a specific chromosomal site where it remains latent until reactivated by superinfection of the host cell with an appropriate helper virus. Replication functions of AAV have been mapped to the Rep68 and Rep78 gene products. Rep proteins demonstrate DNA binding, endonuclease, and helicase activities and are involved in regulation of transcription from both AAV and heterologous promoters. AAV has been associated with suppression of oncogenicity in a range of viral and nonviral tumors. In this study we sought to identify and study cellular protein targets of AAV Rep, in order to develop a better understanding of the various activities of Rep. We used the yeast two-hybrid system to identify HeLa cell proteins that interact with AAV type 2 Rep78, We isolated several strongly interacting clones which were subsequently identified as PRIM (previously named PKX1), a recently described homolog of the protein kinase A (PKA) catalytic subunit (PKA,). The interaction was confirmed in vitro by using pMal-Rep pull-down assays. The region of Rep78 which interacts was mapped to a C-terminal zinc finger-like domain; Rep68, which lacks this domain, did not interact with PRIM. PRIM demonstrated autophosphorylation and kinase activity towards histone H1 and a PKA oligopeptide target. Autophosphorylation was inhibited by interaction with Rep78. In transfection assays, a PRIM expression vector was shown to be capable of activating CREB-dependent transcription. This activation was suppressed by Rep78 but not by Rep68. Since PRIM is a close homolog of PKA,, we investigated whether Rep78 could interact directly with PKA,. pMal-Rep78 was found to associate with purified PKA, and inhibited its kinase activity. Cotransfection experiments demonstrated that Rep78 could block the activation of CREB by a PKA, expression vector. These experiments suggest that AAV may perturb normal cyclic AMP response pathways in infected cells.