烯醇化酶與磷酸甘油酸變位酶在圓葉菸草中對竹嵌紋病毒累積量所扮演之角色

Abstract

病毒需要藉助其宿主的蛋白進行複製，然而目前所知參與病毒複製的宿主蛋白仍相當有限。本研究室利用strepto tag方法選取了一些與馬鈴薯X病毒屬的單股正極RNA病毒──竹嵌紋病毒(Bamboo mosaic virus, BaMV)──及其衛星核酸(satBaMV)之5’或是3’端非轉譯區結合的宿主候選蛋白。本研究測試作用於糖解作用的兩種蛋白質──烯醇化酶(enolase, ENL)與磷酸甘油酸變位酶(phosphoglycerate mutase, PGM)對BaMV複製的影響。將這兩種蛋白的部分內生基因以菸草脆裂病毒(Tobacco rattle virus)為載體系統分別靜默圓葉菸草(Nicotiana benthamiana)，發現BaMV在ENL靜默的植物中累積量下降至對照組之10%，而在PGM靜默的植物中卻與對照組沒有顯著差異。相反地，當ENL在菸草中過量表現，BaMV的累積量便會上升至150%，而在ENL靜默的菸草中過量表現ENL，則可回復BaMV的累積量。而BaMV在原生質體的累積量並不會受到ENL抑制劑影響。另外測試另一種馬鈴薯X病毒屬病毒──馬鈴薯病毒X (Potato virus X, PVX)──也有相同的結果，其累積量在ENL靜默的植物中下降至約30%。除此之外，ENL與PGM在菸草中的表現量並不會受到BaMV感染的影響。整體而言，ENL對於BaMV、satBaMV及PVX在圓葉菸草的累積量有正向調控的影響。

Viruses cannot replicate without recruitment of host proteins. However, the information of host proteins that are involved in virus replication is quite limited. Previously, some host factor candidates were found to be bound with the 5’ and 3’ untranslated regions of Bamboo mosaic virus (BaMV), a positive-sense RNA virus, and the satellite of BaMV (satBaMV) by strepto tag method. This study aims to examine the effects of enolase (ENL) and phosphoglycerate mutase (PGM), two enzymes from glycolysis pathway, on BaMV replication. We first knocked down these genes by the Tobacco rattle virus-based virus-induced gene silencing (VIGS) system and found that the accumulation of BaMV decreased to 10% of the mock-treated control when ENL was knocked down in Nicotiana benthamiana, while knocking down of PGM did not significantly change BaMV accumulation. In contrast, BaMV accumulation increased to 150% when ENL was overexpressed. The overexpression of ENL recovered the accumulation of BaMV in ENL knocked down N. benthamiana. These results suggest that ENL positively regulates the accumulation of BaMV in N. benthamiana but no difference in BaMV accumulation was observed when protoplasts of N. benthamiana were treated with ENL inhibitor, NaF. Similarly, the accumulation of satBaMV decreased to about 40% and accumulation of another Potexvirus, Potato virus X (PVX), decreased to about 30% in ENL knocked down N. benthamiana compared with that in mock-treated control. In addition, the expression level of ENL and PGM was not significantly changed during BaMV infection. In summary, ENL positively regulates the accumulation of BaMV, satBaMV and PVX in N. benthamiana.