Biochemical activities of minute virus of mice nonstructural protein NS1 are modulated In vitro by the phosphorylation state of the polypeptide.
Biochemical activities of minute virus of mice nonstructural protein NS1 are modulated In vitro by the phosphorylation state of the polypeptide.
复制标题
小鼠非结构蛋白NS1微小病毒的生化活性在体外通过多肽的磷酸化状态进行调节。
DOI：
10.1128/jvi.72.10.8002-8012.1998
复制
发表时间：
1998
影响因子：
5.4
通讯作者：
Rommelaere,J
中科院分区：
文献类型：
--
作者：
Nüesch,JP;Corbau,R;Tattersall,P;Rommelaere,J
NS 1是小鼠微小病毒（MVM）的83-kDa主要非结构蛋白，是一种多功能核磷蛋白，其在子代病毒产生期间的各种步骤中所需，在感染期间的早期和晚期。NS 1是病毒DNA复制的起始蛋白。它特异性结合靶DNA基序;具有位点特异性单链切口酶、内在ATP酶和解旋酶活性;反式调节病毒和细胞启动子;并对宿主细胞施加细胞毒性应激。为了研究NS 1的这些多重活性是否依赖于翻译后修饰，特别是磷酸化，我们通过使用重组牛痘病毒在HeLa细胞中表达His标记的NS 1，用小牛肠碱性磷酸酶在丝氨酸和苏氨酸残基处使其去磷酸化，并比较纯化的磷酸化（NS 1 O）和天然（NS 1 P）多肽的生物化学活性。复制功能的生化分析表明，NS 1 O的内在解旋酶活性严重降低，ATP酶和切口酶活性降低，但程度较轻，而其对靶DNA序列[ACCA]2- 3的亲和力比NS 1 P增强。在复制提取物中发现的内源性蛋白激酶存在下，NS 10显示出3′二聚体桥的分解和复制所需的所有功能，表明NS 10通过再磷酸化而重新激活。当NS 10与蛋白激酶C孵育时，也发现解旋酶活性的部分再活化。
NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities;transregulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1Orevealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1Oshowed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1Oby rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1Owas incubated with protein kinase C.