Sato Ryota, Okura Takashi, Kawahara Madoka, Takizawa Naoki, Momose Fumitaka, Morikawa Yuko
Graduate School for Infection Control, Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan.
Laboratory of Basic Biology, Institute of Microbial Chemistry, Tokyo, Japan.
Front Microbiol. 2019 Aug 13;10:1857. doi: 10.3389/fmicb.2019.01857. eCollection 2019.
The envelope proteins of influenza A virus, hemagglutinin (HA) and neuraminidase (NA), play critical roles in viral entry to host cells and release from the cells, respectively. After protein synthesis, they are transported from the -Golgi network (TGN) to the apical plasma membrane (PM) and assembled into virus particles. However, the post-TGN transport pathways of HA and NA have not been clarified. Temporal study by confocal microscopy revealed that HA and NA colocalized soon after their synthesis, and relocated together from the TGN to the upper side of the cell. Using the Rab family protein, we investigated the post-TGN transport pathways of HA and NA. HA partially colocalized with AcGFP-Rab15, Rab17, and Rab23, but rarely with AcGFP-Rab11. When analyzed in cells stably expressing AcGFP-Rab, HA/NA colocalized with Rab15 and Rab17, markers of apical sorting and recycling endosomes, and later colocalized with Rab23, which distributes to the apical PM and endocytic vesicles. Overexpression of the dominant-negative (DN) mutants of Rab15 and Rab17, but not Rab23, significantly delayed HA transport to the PM. However, Rab23DN impaired cell surface expression of HA. Live-cell imaging revealed that NA moved rapidly with Rab17 but not with Rab15. NA also moved with Rab23 in the cytoplasm, but this motion was confined at the upper side of the cell. A fraction of HA was localized to Rab17 and Rab23 double-positive vesicles in the cytoplasm. Coimmunoprecipitation indicated that HA was associated with Rab17 and Rab23 in lipid raft fractions. When cholesterol was depleted by methyl-β-cyclodextrin treatment, the motion of NA and Rab17 signals ceased. These results suggest that HA and NA are incorporated into lipid raft microdomains and are cotransported to the PM by Rab17-positive and followed by Rab23-positive vesicles.
甲型流感病毒的包膜蛋白血凝素（HA）和神经氨酸酶（NA）分别在病毒进入宿主细胞和从细胞中释放的过程中发挥关键作用。蛋白质合成后，它们从反式高尔基体网络（TGN）转运至顶端质膜（PM）并组装成病毒颗粒。然而，HA和NA在TGN后的转运途径尚未明确。共聚焦显微镜的时间研究表明，HA和NA在合成后不久共定位，并一起从TGN重新定位到细胞的上侧。利用Rab家族蛋白，我们研究了HA和NA在TGN后的转运途径。HA与AcGFP-Rab15、Rab17和Rab23部分共定位，但很少与AcGFP-Rab11共定位。在稳定表达AcGFP-Rab的细胞中分析时，HA/NA与顶端分选和再循环内体的标记物Rab15和Rab17共定位，随后与分布于顶端PM和内吞小泡的Rab23共定位。Rab15和Rab17的显性负性（DN）突变体而非Rab23的过表达显著延迟了HA向PM的转运。然而，Rab23DN损害了HA的细胞表面表达。活细胞成像显示，NA与Rab17一起快速移动，但与Rab15不一起移动。NA在细胞质中也与Rab23一起移动，但这种移动局限于细胞的上侧。一部分HA定位于细胞质中Rab17和Rab23双阳性小泡。免疫共沉淀表明，HA在脂筏组分中与Rab17和Rab23相关联。用甲基-β-环糊精处理耗尽胆固醇后，NA和Rab17信号的移动停止。这些结果表明，HA和NA被整合到脂筏微结构域中，并通过Rab17阳性囊泡随后是Rab23阳性囊泡共转运至PM。
