A型流感病毒PB1-F2蛋白質於人類細胞株之穩定性與蛋白酶體降解效率之研究

Abstract

已知A型流感病毒的PB1-F2蛋白質會調控病毒PB1聚合酶活性，亦會進入宿主細胞粒線體內外膜之間，促使膜電位下降，引發單核球細胞凋亡、調控細胞先天性免疫反應。此外PB1-F2也會抑制干擾素的生成，並增加二次細菌性肺炎感染的風險。然而，來自不同病毒株的PB1-F2在不同宿主細胞中的定位、表現量和功能都有不少差異，因此研究PB1-F2的穩定性、功能及調控機制，也許可以進一步了解不同流感病毒株致病力有所差異的原因。本研究將四株不同亞型的病毒株A/Puerto Rico/8/1934 (H1N1)、A/Udorn/307/1972 (H3N2)、A/Hong Kong/156/1997 (H5N1)、A/Taiwan/01/2013 (H7N9)的PB1-F2基因於HEK293細胞中表現時，發現其表現量有極大的差異，且皆會因加入蛋白酶體抑制劑MG132而有不同程度的上升，表示PB1-F2會受到蛋白酶體降解系統的調控。將四株不同亞型病毒株的PB1-F2進行部分胺基酸序列互換時，發現2、10、11、14和68-71會影響H1N1、H3N2和H7N9 PB1-F2在HEK293細胞中的穩定性，然而對於H5N1則沒有顯著影響。此外，細胞影像的結果顯示PB1-F2 68-71的胺基酸序列會影響PB1-F2在細胞中的分佈位置，其中ILVF有粒線體標的功能，會使PB1-F2位於粒線體。為了探究PB1-F2的穩定性是否受到泛素化的調控，將四株病毒株之PB1-F2上的所有離胺酸突變為精胺酸後，發現H1N1、H3N2、H7N9之PB1-F2皆因泛素化位點的突變而提升的在細胞中的穩定性，而H5N1 PB1-F2的穩定性則沒有因突變而有顯著提升，因此可知PB1-F2可經由泛素依賴型 (dependent) 或不依賴型 (independent) 路徑而被蛋白酶體降解。此外，分別將PA28α、PA28β或PA28γ與PB1-F2共轉染至HEK293細胞中，四株病毒株的PB1-F2的表現量皆有非常明顯的下降，顯示PA28可以促進PB1-F2的降解。

Influenza A virus (IAV) protein Polymerase basic 1-frame 2 (PB1-F2) regulates viral polymerase activity, induces apoptosis in host immune cells, interferes the host innate immune response and enhances the pathogenesis of secondary bacterial pneumonia. Moreover, PB1-F2 derived from different virus strains may perform different functions, expression levels and cellular localization, leading to their various strain-specific virulence in host cells. Therefore, studying the principles which determine the stability, functions and regulation mechanisms of PB1-F2 might help us know further about the pathogenesis of various influenza A virus strains. In this study, HEK293 cells expressed PB1-F2 from four IAV strains (A/Puerto Rico/8/1934 (H1N1), A/Udorn/307/1972 (H3N2), A/Hong Kong/156/1997 (H5N1), A/Taiwan/01/2013 (H7N9)) in extremely various expression levels. Moreover, the expression levels of PB1-F2 were increased upon MG132 treatment and showed different sensitivity to MG132, indicating that PB1-F2 may undergo proteasome-mediated degradation. Swapping of equivalent amino acid residues 2, 10, 11, 14, and 68-71 of PB1-F2 among these four IAV strains may alter their protein stability. Moreover, the cell images showed that amino acid residues 68-71 might modulate the localization of PB1-F2. In addition, PB1-F2 residues I68, L69, V70, and F71 are mitochondrial targeting sequence. In order to clarify whether PB1-F2 stability is ubiquitination-mediated, all of the lysine residues of PB1-F2 were mutated to arginine residues to inhibit the possible ubiquitination of PB1-F2. It is found that the expression levels of H1N1, H3N2 and H7N9 PB1-F2 were greatly increased, but H5N1 PB1-F2 did not increase significantly, indicating that the ubiquitin-dependent and ubiquitin-independent degradation pathway are all involved in regulation of PB1-F2 stability. Furthermore, the expression levels of PB1-F2 were markedly decreased while the proteasome activator PA28α/PSEM1, PA28β/PSME2, or PA28γ/PSME3 was co-transfected with PB1-F2, suggested that PA28 can promote degradation of PB1-F2.