Leser G P, Ector K J, Lamb R A
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Mol Biol Cell. 1996 Jan;7(1):155-72. doi: 10.1091/mbc.7.1.155.
The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.
副粘病毒猴病毒5（SV5）的血凝素神经氨酸酶（HN）和融合（F）糖蛋白在病毒感染细胞的表面表达。虽然发现F蛋白稳定表达，但HN蛋白从质膜内化。HN蛋白在其胞质结构域中缺乏许多通过网格蛋白包被小窝内化的整合膜蛋白所共有的已知内化信号。因此，对HN蛋白内化的细胞途径进行了研究。生化分析表明，HN从细胞表面消失的半衰期约为45 - 50分钟，周转半衰期约为2小时。免疫荧光分析显示，内化的SV5 HN呈囊泡样结构，位于近核区域，与卵清蛋白的定位一致。相比之下，高度相关的副流感病毒3（hPIV - 3）的SV5 F糖蛋白和HN糖蛋白仅在细胞表面被发现。对SV5感染的CV - 1细胞表面的HN进行免疫金染色并使用电子显微镜检查，结果显示表面标记较重，且随时间逐渐减少。同时，在内体系统中检测到金颗粒，随着时间的增加，观察到具有溶酶体形态的金标记结构。在质膜上，约5%的金标记HN存在于包被小窝中。通过胞质酸化抑制包被小窝从质膜上脱离，显著减少了HN的内化。内化的HN与早期内体区室的标志物金偶联转铁蛋白以及晚期内体区室的标志物金偶联牛血清白蛋白共定位。综上所述，这些数据强烈表明HN糖蛋白通过网格蛋白包被小窝内化并进入内吞途径。
