探討TGB1蛋白於仙人掌X病毒與紅龍果X病毒之感染中所扮演之角色

紅龍果具高經濟價值，為國內重要熱帶果樹之一。根據實驗室前人調查，台灣田間的紅龍果普遍受到仙人掌X病毒 (Cactus virus X, CVX)、紅龍果X病毒 (Pitaya virus X, PiVX) 以及蟹爪蘭X病毒 (Zygocactus virus X, ZyVX) 三種 potexviruses 所感染，且複合感染的情形相當常見，其中尤以 CVX 與 PiVX 的感染率較高。在 potexvirus 的基因體中，由 ORF2 編碼的蛋白為 triple gene block 1 (TGB1)，其帶有典型的 NTPase/helicase domain，具 ATP酶、RNA 解旋酶的活性以及結合 RNA 的能力，被認為是一種多功能性蛋白。CVX 與 PiVX之 TGB1 蛋白已被證實具有 RNA 靜默抑制子之功能，並且透過圓葉菸草原生質體接種試驗，發現 TGB1 的存在有助於 CVX 與 PiVX 之間協力作用的發生。本研究目的為探討 CVX 與 PiVX 之 TGB1在病毒感染過程中的角色，聚焦於 TGB1 蛋白對於病毒複製、細胞間移動以及 CVX-PiVX 之間協力作用發生的重要性。除了利用野生型與 TGB1 缺失之病毒株進行分析比較之外，本研究亦對potexvirus 之 TGB1進行胺基酸序列排併比對，以及預測CVX 與 PiVX 之 TGB1蛋白三級結構，據此建構出兩病毒 TGB1 蛋白T115A、T190A 與 T211A 之三種單點胺基酸突變株。前人研究曾發現當兩病毒之 TGB1 基因短暫表現於圓葉菸草葉片，除可抑制由正股RNA所誘發的RNA 靜默外，亦會造成植物的細胞死亡。本研究結果顯示 CVX TGB1-T190A、CVX TGB1-T211A 與 PiVX TGB1-T211A 三種TGB1突變蛋白，在失去抑制 RNA 靜默能力的同時，亦變成不會誘發植物細胞死亡。其他三種保有 RNA 靜默抑制子能力的突變蛋白 CVX TGB1 T115A、PiVX TGB1-T115A 與 PiVX TGB1-T190A ，則與野生型 TGB1 的表現一致。藉由北方雜合法分析 TGB1 缺失或單點胺基酸突變之 CVX 與 PiVX 病毒株，結果顯示其在圓葉菸草或紅龍果原生質體中的 RNA 累積量與野生型病毒相近。為了瞭解 TGB1 對於 CVX-PiVX 協力作用的重要性，將病毒轉錄體以不同組合同時接種至紅龍果原生質體，經RT-qPCR分析發現 TGB1 的缺失或是單點突變，皆會顯著影響CVX 與 PiVX提升對方病毒RNA的累積量。最後，為瞭解 TGB1 對於 CVX 與 PiVX 在細胞間移動能力的影響，將帶有 eGFP 螢光蛋白的病毒株接種於白藜葉片，以螢光顯微鏡觀察，發現TGB1基因帶有 T115A 突變的病毒，其移動速率與野生型病毒相似；而 TGB1缺失及具T211A 突變的CVX 與 PiVX，皆喪失細胞間移動能力。而T190A 突變會使CVX 被侷限在單細胞中，但只會減緩PiVX的細胞間移動速度。綜合以上研究結果，TGB1 應該沒有參與CVX 與 PiVX的複製過程，但其會影響CVX 與 PiVX之間的協力作用；TGB1是病毒在白藜葉片細胞間移動的必要因子，此移動能力與其 RNA 靜默抑制能力或許具相關性。此外，TGB1 抑制 RNA 靜默的能力與其誘發圓葉菸草細胞死亡可能有關，而此推測需更進一步的研究來證實。

關鍵字

仙人掌X病毒；紅龍果X病毒； TGB1 ； RNA 靜默作用；細胞間移動；協力作用

並列摘要

Pitaya is one of the important tropical crops with high economic value in Taiwan. Based on previous surveys, pitayas co-infected by three potexviruses, Cactus virus X (CVX), Pitaya virus X (PiVX), and Zygocactus virus X (ZyVX), are common in field. Among them, CVX and PiVX have a higher infection rate. ORF2 in potexviral genome encodes triple gene block 1 (TGB1) protein. TGB1 is a multifunctional protein containing a canonical NTPase/helicase domain and has ATPase and RNA helicase activity as well as RNA binding ability. The TGB1 proteins of CVX and PiVX are confirmed as RNA silencing suppressors. Furthermore, the existence of these viral proteins is thought to be important to the synergistic interaction between CVX and PiVX in tobacco protoplasts. This study aims to further investigate the role of TGB1 of CVX and PiVX in the virus infection process, focusing on the importance of TGB1 for virus replication, cell-to-cell movement, and the synergistic interaction between CVX and PiVX. In addition to the wild type and TGB1 deletion mutant viruses, three TGB1 point mutants of CVX and PiVX were constructed based on amino acid sequence alignment and protein tertiary structure prediction. Each TGB1 point mutant has a single mutated amino acid (T115A, T190A, or T211A), which is predicted to be related to the ability of RNA silencing suppression. According to previous research, overexpression of the TGB1 proteins of CVX and PiVX can suppress RNA silencing and also cause cell death on Nicotiana benthamiana leaves. In this study, we found that within six TGB1 point mutants, CVX-TGB1-T190A, CVX-TGB1-T211A, and PiVX-TGB1-T211A, lost their ability to suppress RNA silencing and did not induce cell death. While CVX-TGB1-T115A, PiVX-TGB1-T115A, and PiVX-TGB1-T190A had the same behavior as wild-type TGB1. Through northern blot analyses of N. benthamiana and pitaya protoplast inoculation assays, the RNA accumulation of TGB1 mutants of CVX and PiVX were similar to wild type viruses. To investigate the importance of TGB1 to CVX-PiVX synergism, two different virus transcripts were co-transfected to pitaya protoplasts and analyzed by RT-qPCR. The results indicated that both the loss and point mutation of TGB1 affect the ability of CVX or PiVX to increase each other’s viral RNA accumulation. To confirm the effect of TGB1 on CVX and PiVX during cell-to-cell movement, transcripts of viruses tagged with an enhanced green fluorescent protein (eGFP) were inoculated on Chenopodium quinoa leaves. From the observation with a fluorescent microscope, viruses containing T115A mutation on TGB1 were found to have similar cell-to-cell movement ability to wild type viruses. However, CVX and PiVX with TGB1 deletion and T211A mutation lost the ability to move between cells. Interestingly, T190A mutation caused CVX to be restricted to single cells, but only slowed down the cell-to-cell movement of PiVX. In summary, TGB1 may not participate in the replication of CVX and PiVX, but influences the synergism between CVX and PiVX. TGB1 is a necessary factor for CVX and PiVX to move between cells of C. quinoa, and this movement ability may be related to its ability to suppress RNA silencing. Additionally, the ability of TGB1 of CVX and PiVX to suppress RNA silencing may also associate with the cell death of N. benthamiana induced by them, although this speculation needs further research to confirm.