登革熱病毒感染細胞複製複合物之純化與活性分析
The isolation and the analysis of replication complexes from Dengue virus infected cells
中文摘要
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。
英文摘要
The genome of the mosquito-borne dengue virus, a member of positive strand RNA virus of the Flaviviridae family, consists of 10,800 nucleotides that encodes a 3,390 amino acid polyprotein. The polyprotein is processed into three structural proteins that participate in virion formation, and seven nonstructural proteins that are involved in viral RNA replication. Flavivirus infection causes reorganization of host intracellular membranes, including Golgi apparatus, endoplasmic reticulum, and nuclear envelope, to form specialized structures, which is believed to harbor flaviviral replication complexes (RC). In this study, I isolated the intracellular membrane from dengue virus infected cells and analyzed its RNA dependent RNA polymerase activity utilizing the virus genomic RNA or synthetic viral genome subfragment as the template. With the exogenous synthetic RNA templates, only the terminal transferase activity that incoperated α-32P labeled NTP to 3’-OH was detected. Without the exogenous RNA template, α-32P labeled NTP was incorporated into an RNA longer than 9 kb. The labeled RNA was not derived from the terminal transferase, it might be synthesized from the viral RNA of the cell extract
