紅龍果X病毒鞘蛋白次基因體啟動子之分析及應用

Abstract

次基因體RNA (subgenomic RNA, sgRNA)的合成為單鏈正意股RNA病毒表現基因之常用策略之一，其啟動子(subgenomic promoter, SGP)在合成sgRNA的過程中扮演不可或缺的角色。紅龍果X病毒(Pitaya virus X, PiVX)為Potexvirus屬之新種病毒，本研究藉由PiVX鞘蛋白基因SGP之分析，以增加對該病毒的了解。為了確認PiVX鞘蛋白SGP的位置，我們先以5’-RACE配合選殖與定序，定位出鞘蛋白sgRNA之轉錄起始點，其位於病毒全長第5865個核苷酸。以此位置為基準，建構一系列PiVX刪除突變株(deletion mutants)，藉由農桿菌注射法在菸草(Nicotiana benthamiana)上進行分析，以找到鞘蛋白SGP的位置。結果顯示，若要維持PiVX鞘蛋白sgRNA正常表現，以轉錄起始點作為+1，需保留-43至+23之序列，此即為PiVX的鞘蛋白SGP。接著，我們以複製鞘蛋白SGP之方式建構PiVX病毒載體，並嘗試表現外源基因，經實驗證實PiVX病毒載體可於白藜及菸草上複製，並且成功表現綠色螢光蛋白。另外，在進行序列比對時發現，大部分Potexvirus屬之病毒在鞘蛋白基因轉譯起始點上游處，有一保守性序列GUUAACUU，但在以仙人掌科為寄主之potexviruses中，此保守序列第一核苷酸皆為C，為了要了解此核苷酸在PiVX感染過程中是否具有特殊意義，故建構了pGR-PiVX5C5843G。確定PiVX5C5843G在菸草原生質體中之複製及轉錄和野生型PiVX5沒有差別後，將質體DNA接種至三週大之白藜上，發現接種葉的病徵減弱、鞘蛋白累積量降低，且病毒無法移動至系統葉。初步推測可能是因為將C改變為G，進而改變RNA-鞘蛋白之間的交互作用，導致病毒之移動受到影響。而在確認上述推論前，我們想先確定鞘蛋白是否參與在PiVX之細胞間移動，故將鞘蛋白基因之轉譯起始碼突變為終止碼，建構了pGR-PiVX5-CP*，並接種在菸草原生質體及白藜植株上。結果顯示，當PiVX鞘蛋白不被轉譯時，PiVX之基因體RNA在菸草原生質體中之累積量皆明顯降低；而於白藜接種葉上此差異更加明顯，推測鞘蛋白位置之轉譯作用的進行或是鞘蛋白之存在，可能有助於病毒之累積，但此實驗無法確定PiVX鞘蛋白是否參與在病毒細胞間之移動。本研究不僅讓我們對PiVX有更多認識，也能以此為基礎，發展各式各樣的病毒載體以供利用。

Transcription of subgenomic RNA (sgRNA) is one of the strategies used by eukaryotic positive sense RNA viruses for gene expression and its promoter plays an important role in sgRNA synthesis. Pitaya virus X (PiVX), a recently identified potexvirus, was investigated for its subgenomic promoter (SGP) in this study. In order to find out the SGP of PiVX capsid protein (CP) gene, the transcription start site of CP sgRNA was first studied and mapped to nucleotide (nt) 5865 by 5’-RACE analysis. Based on this result, a series of PiVX deletion mutants were constructed and used to define the functional promoter region of CP sgRNA by Agrobacteium-mediated inoculation on Nicotiana benthamiana. The results showed that the region between nt -43 and +23 relative to the CP transcription start site (+1) of PiVX supported the full promoter activity to generate CP sgRNA. Subsequently, we constructed a PiVX-based vector that contains duplicated full-length CP SGP, which could replicate and successfully express enhanced green fluorescent protein (EGFP) on Chenopodium quinoa and N. benthamiana. One interesting finding from the multiple sequence alignment indicted that sequences GUUAACUU upstream of CP gene translation start site are conserved in most members of the genus Potexvirus, but the first nt is C instead of G in potexviruses infecting Cactaceae plants. To study the significance of this nucleotide for PiVX, we performed the site-directed mutagenesis to create a mutant PiVXG5843C, its C5843 replaced by G5843. The replication and transcription had no significant difference between PiVXG5843C and wild type PiVX in tobacco protoplasts. After inoculation of plasmid DNA on 3-week-old C. quinoa mechanically, the results revealed that the symptoms caused by PiVXG5843C were weaker, and the CP levels were lower in the inoculated leaves compared to wild type. Moreover, the mutated virus PiVXG5843C could not be detected in the systemic leaves. Thus, these data suggested that G5843 in PiVX may change the interaction of RNA and CP, and then affect the virus movement in C. quinoa plant. In order to confirm if PiVX CP participates in cell-to-cell movement, we constructed pGR-PiVX5-CP* whose start codon of CP was mutated to stop codon and inoculated to N. benthamiana protoplasts and C. quinoa plants. The results showed that the accumulation of PiVX gRNA reduced in tobacco protoplasts and even became worse in the inoculated leaves of C. quinoa. It is suggested that translation of CP gene or CP protein itself may be important for the accumulation of PiVX gRNA. By this study, we can not only understand the characteristics of PiVX CP SGP but also obtain a benefit for further viral vector development.