Genevaux Pierre, Lang Florence, Schwager Françoise, Vartikar Jai V, Rundell Kathleen, Pipas James M, Georgopoulos Costa, Kelley William L
Département de Biochimie Médicale, Centre Médical Universitaire, Université de Genève, CH-1211 Genève 14, Switzerland.
J Virol. 2003 Oct;77(19):10706-13. doi: 10.1128/jvi.77.19.10706-10713.2003.
The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.
DNA肿瘤病毒T抗原的N端外显子代表一个J结构域，它可介导与宿主编码的热休克蛋白70（Hsp70）伴侣蛋白的相互作用。我们利用针对大肠杆菌的快速热休克蛋白40（Hsp40）共伴侣蛋白检测方法，评估了猿猴病毒40（SV40）编码的J结构域的功能丧失情况。我们发现，大肠杆菌中共伴侣蛋白功能的丧失与SV40生长缺陷之间存在很强的相关性，这表明DNA肿瘤病毒中J结构域的主要作用是提供共伴侣蛋白功能。我们还报告了天然SV40病毒T抗原在大肠杆菌中的表达情况。我们的结果表明，在有限的生长条件下，小t抗原而非大T抗原（LT）或LT截短体TN125或TN136能够在体内功能性替代DnaJ（Hsp40）的功能。此外，纯化的小t抗原在体外能够有效刺激大肠杆菌DnaK（Hsp70）的ATP酶活性，因此表现得像一个真正的共伴侣蛋白。此外，与病毒J结构域相邻的小t抗原的83至174位氨基酸，即使与它们的DnaJ对应序列在氨基酸上没有显著相似性，也能够替代紧邻J结构域序列的大肠杆菌DnaJ J结构域富含甘氨酸 - 苯丙氨酸的结构域。综上所述，我们的研究表明，来自哺乳动物病毒系统的功能相关的Hsp40蛋白能够在细菌中得到快速研究，并可用于探究普遍保守的Hsp70伴侣蛋白机制。
