Orozco B M, Kong L J, Batts L A, Elledge S, Hanley-Bowdoin L
Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
J Biol Chem. 2000 Mar 3;275(9):6114-22. doi: 10.1074/jbc.275.9.6114.
Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157-159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157-159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein.
番茄金色花叶病毒（TGMV）是双生病毒科的成员，编码一种必需的复制蛋白AL1，并从其植物宿主中招募其余的DNA复制装置。TGMV AL1是一种寡聚蛋白，可结合双链DNA并催化单链DNA的切割和连接。DNA结合所需的寡聚化结构域定位于与其他双生病毒Rep蛋白具有强序列和结构同源性的区域。为了评估保守残基的重要性，我们产生了一系列定点突变，并分析了它们在体外和体内对AL1功能的影响。双杂交实验表明，氨基酸157-159的突变抑制了AL1-AL1相互作用，而附近残基的突变降低了复合物的稳定性。157-159位的变化也破坏了昆虫细胞中全长突变蛋白与谷胱甘肽S-转移酶-AL1寡聚化结构域融合体之间的相互作用。当两种蛋白质都包含全长AL1序列时，这些突变对寡聚化没有可检测到的影响，表明AL1复合物可以通过寡聚化结构域之外的氨基酸来稳定。几乎所有的寡聚化结构域突变体在支持AL1导向的病毒DNA复制的能力上都受到抑制或严重减弱。相比之下，相同的突变体在AL1介导的转录抑制方面增强。复制缺陷型AL1突变体也干扰了编码野生型AL1的TGMV A DNA的复制。全长突变型AL1在干扰试验中比含有寡聚化结构域的截短蛋白更有效。总之，这些结果表明不同的AL1复合物介导病毒复制和转录调控，并且复制干扰涉及AL1蛋白的多个结构域。
