登革熱病毒非結構蛋白NS3之腺苷三磷酸水解酶之研究-NS3蛋白和腺苷三磷酸及核醣核酸三者間之交互關係

The studies of the ATPase activity of Dengue virus nonstructural protein NS3-The interaction among NS3 protein, ATP and RNA.

陳建村(Chien-Tsun Chen)

指導教授 ： 吳惠南

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2005年)

摘要

登革熱病毒非結構蛋白3 (NS3) C端含有RNA解旋酶的保守性序列，具有NTPase、RNA triphosphatase及RNA解旋酶活性。本論文的目的是分析由大腸桿菌表現及純化的登革熱病毒NS3 C端的蛋白 (簡稱NS3H)的ATPase活性，並藉定點突變蛋白活性的變化，探討NS3H蛋白水解ATP的作用機制以及NS3H蛋白、ATP和RNA的關係。實驗結果發現NS3H蛋白水解ATP反應需要二價陽離子的參與，如Mg2+、Mn2+及Ca2+才可啟動，其中以Mg2+效果最佳。Mg2+的效應會被Mn2+抑制，顯示反應中MnATP複合物對NS3H蛋白的親和力不弱於MgATP。NS3H蛋白的ATPase活性可分被rU35和NaCl激活，兩因子激活NS3H ATPase反應對ATP的Km改變不大 (92-120 μM)，僅會提增ATP的水解速率。顯示了NaCl和rU35主要影響ATP進入ATPase活性區後的水解效能。再者rU35的激活效果會被高濃度 (10mM以上)的NaCl抑制，代表著高濃度的NaCl會降低NS3H和核酸間的離子作用力，使得NS3H不易與核酸結合，進而影響後續ATPase活性激活的效果。NS3H蛋白RNA解旋酶保守性區的motif I之K199A T200A置換突變和motif II 的D284A置換，均致使NS3H蛋白失去水解ATP的能力。而motif II的D284E置換突變蛋白，仍保有水解ATP的能力和受核酸激活的特性。因此motifs I和II與ATP的結合和水解有關連。NS3H蛋白RNA解旋酶保守區gatekeepers的M429和W488分別被置換時，ATPase活性均會受到影響。M429A突變蛋白ATPase活性仍可被核酸激活，此突變蛋白與ATP的Km值無論有否受rU35的激活，其Km值與NS3H相較差異不大。W488A的ATPase活性極低而且會受到核酸的抑制，且此受核酸的抑制效應和M429A受核酸的激活效應皆可被高濃度的NaCl所遮蔽，這表示M429A和W488A皆可與核酸結合，且結合後的酵素活性會因此而產生變化。至此我們認為NS3H蛋白與核酸結合後可能會誘導蛋白結構產生改變，在不影響ATP的Km情況下，此結構的改變會提增ATP在活性區內的水解速率，而gatekeeper的氨基酸會影響蛋白與核酸結合後的結構改變，進而影響後續ATPase的水解效果。

關鍵字

登革熱 ； 腺苷三磷酸水解酶

並列摘要

The C terminal domain of dengue virus nonstructural protein 3 (NS3) contains the conserved motifs present in several RNA helicases and has NTPase, RNA triphosphotase, and RNA helicase activities. In this research, we expressed and purified a His-tag fusion protein containing the C terminal domain of dengue virus NS3 (named as NS3H). In addition, we constructed and prepared the substitution mutants of NS3H. We studied the ATPase activity of the wild type and the mutant proteins, and examined the relationship among NS3H, ATP, and RNA. The ATPase activity required divalent cation to function. Mg2+ was more effective than Mn2+ and Ca2+ in catalyzing ATP hydrolysis, while Mn2+ strongly inhibited the stimulatory effect of Mg2+. Thus, MnATP appears to have a higher affinity to NS3H than that of MgATP. Both NaCl and rU35 (U homopolymer of 35 nt long) accelerated the rate of ATP hydrolysis, but they did not alter the Km to ATP (90-120 µM). The increase of NaCl concentration from 10 mM to 30 mM eliminated the stimulatory effect of rU35 on ATP hydrolysis, suggesting the interaction between rU35 and NS3H was mainly electrostatic. The K199A, T200A substitutions in the motif I as well as the D284A substitution in the motif II abolished the ATPase activity of NS3H, while the D284E substitution mutant possessed the RNA stimulated ATPase activity. These results reveal that the motifs I and II participate in ATP binding and ATP hydrolysis. M429 and W488 were the “gatekeepers” of the conserved RNA helicase domain. The M429A substitution mutant interacted with ATP and rU35 in a manner similar to the wild type protein, while it hydrolyzed ATP less efficiently than that of NS3H. The W488A substitution mutant had a very low ATPase activity. Instead of being stimulated by rU35, the ATPase activity of this mutant was inhibited by rU35, and the inhibitory effect of rU35 was attenuated when the NaCl concentration increased. Thus, the substitution at each gatekeeper may not affect the RNA binding activity of NS3H. In summary, the results of this study suggest that the binding of RNA to NS3H may induce the conformation rearrangement of NS3H that increases the ATP hydrolysis rate but does not alter the Km to ATP. The gatekeeper mutants may have defect in the conformation rearrangement process, as a result the ATPase activity is attenuated.

並列關鍵字

Dengue ； NS3 ； ATPase

參考文獻

Bazan, J. F. and Fletterick, R. J. (1989) Detection of a trypsin-like serine protease domain in flaviviruses and pestviruses. Virology 171: 637-639.

Baltelma, G. and Padmanabhan, R. (2002) Expression, purification, and characterization of the RNA 5’-triphosphatase activity of Dengue virus type 2 nonstructural protein 3. Virology 299: 122-132.

Brinton, M. A. (2002) The molecular biology of West Nile virus: a new invader of the western hemisphere. Annu. Rev. Microbiol. 56: 371-402.

Caruthers, J. M., Johnson, E. R., and Mckay, D. B. (2000) Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase. Proc. Natl. Acad. Sci. USA 97: 13080-13085.

Chambers, T. J., Hahn, C. S., Galler, R., and Rice, C.M. (1990) Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 44: 649-688.