登革熱病毒感染細胞複製複合物之純化與活性分析
登革熱病毒隸屬於黃熱病毒科，為一種正股 RNA 病毒，其全長約有
10,800 個核苷酸。病毒 RNA 經轉譯可合成一約 3,390 個胺基酸之蛋 白。此病毒蛋白會被切割為三個參與病毒顆粒形成的結構蛋白，與七 個參與病毒複製的非結構蛋白。當病毒感染細胞後，細胞質內的高基 氏體、內質網與核膜間會形成特殊的膜狀結構，而參與複製的非結構 蛋白會在該結構中聚集成複製複合物並參與病毒基因體的複製。本實 驗擬自登革熱病毒感染的細胞中萃取複製複合物進行活體外的實驗，
分析病毒感染細胞萃取物能否以病毒基因體及外加病毒 RNA 片段為 模板進行複製。結果得知，以外加病毒 RNA 片段進行 RNA 合成實驗
，並未偵測到 RNA dependent RNA polymerase 的活性，但卻發現細胞 萃取物中具有端點轉移酶的活性，可藉由模板 RNA 的 3’-OH ，接入 32P 標定的核苷酸。另外、若以病毒感染細胞萃取物不外加 RNA 進 行活體外 RNA 合成實驗，發現有 32P 標定的核苷酸可接入至少為九 千個核苷酸的 RNA 中，且該 RNA 並非以端點轉移酶的活性生成，推 測該產物是以病毒基因體為 RNA 模板合成的產物。
The isolation and the analysis of replication complexes from Dengue virus infected cells
The genome of the mosquito-borne dengue virus, a member of positive stra nd RNA virus of the Flaviviridae family, consists of 10,800 nucleotides tha t encodes a 3,390 amino acid polyprotein. The polyprotein is processed int o three structural proteins that participate in virion formation, and seven no nstructural proteins that are involved in viral RNA replication. Flavivirus i nfection causes reorganization of host intracellular membranes, including Golgi apparatus, endoplasmic reticulum, and nuclear envelope, to form spe cialized structures, which is believed to harbor flaviviral replication compl exes (RC). In this study, I isolated the intracellular membrane from dengue virus infected cells and analyzed its RNA dependent RNA polymerase acti vity utilizing the virus genomic RNA or synthetic viral genome subfragme nt as the template. With the exogenous synthetic RNA templates, only the t erminal transferase activity that incoperated α-32P labeled NTP to 3’-OH was detected. Without the exogenous RNA template, α-32P labeled NTP w as incorporated into an RNA longer than 9 kb. The labeled RNA was not d erived from the terminal transferase, it might be synthesized from the viral RNA of the cell extract
