Barman Subrata, Adhikary Lopa, Kawaoka Yoshihiro, Nayak Debi P
Department of Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90095-1747, USA.
Virology. 2003 Jan 5;305(1):138-52. doi: 10.1006/viro.2002.1731.
Morphogenesis of influenza virus is a complex multistep process involving transport of all viral components as either individual or subviral components to the specified assembly site and interaction among the viral components in an ordered fashion to initiate the budding process. Envelope glycoprotein(s) is believed to be the major determinant in selecting the viral budding site since the majority of the viral glycoproteins are directed to the budding site independent of other viral components. Influenza viruses bud from the apical surface of polarized epithelial cells and all three envelope proteins, hemagglutinin (HA), neuraminidase (NA), and M2, are also targeted independently to the apical surface. Since HA is the major viral envelope protein, we decided to test whether basolaterally expressed HA can make the virus bud from the basolateral surface. Accordingly, we introduced the tyrosine-based basolateral-sorting signal to the cytoplasmic tail of HA by changing Cys561 --> Tyr561 and generated a transfectant virus by reverse genetics. Compared to the parent WSN virus, the mutant virus (HAtyr virus) contained less HA on its envelope. While the wild-type (wt) HA was >95% apical, the mutated HA (HAtyr) was approximately 60% basolateral in both transfected and virus-infected polarized MDCK cells. Also, HAtyr protein exhibited a much higher rate of endocytosis than the wt HA, in both apical and basolateral surface of transfected as well as virus-infected cells. However, the HAtyr virus, similar to wt WSN virus, was seen to bud almost exclusively (>99%) from the apical side of polarized MDCK cells. This finding was confirmed by using neuraminidase to facilitate virus release, by treating the collected virus particles with trypsin to cleave HA0 --> HA1 and HA2, by protein analysis of released virus particles, and finally, by electron microscopy. Therefore HA, the major glycoprotein alone, does not determine the budding site, and other factor(s), possibly both viral and host, is responsible for selecting the budding site of influenza virus.
流感病毒的形态发生是一个复杂的多步骤过程，涉及所有病毒成分作为单个或亚病毒成分运输到特定的组装位点，并使病毒成分以有序方式相互作用以启动出芽过程。包膜糖蛋白被认为是选择病毒出芽位点的主要决定因素，因为大多数病毒糖蛋白独立于其他病毒成分被导向出芽位点。流感病毒从极化上皮细胞的顶端表面出芽，并且所有三种包膜蛋白，血凝素（HA）、神经氨酸酶（NA）和M2，也独立地靶向顶端表面。由于HA是主要的病毒包膜蛋白，我们决定测试基底外侧表达的HA是否能使病毒从基底外侧表面出芽。因此，我们通过将Cys561变为Tyr561，将基于酪氨酸的基底外侧分选信号引入HA的细胞质尾部，并通过反向遗传学产生了一种转染病毒。与亲本WSN病毒相比，突变病毒（HAtyr病毒）包膜上的HA含量较少。在转染和病毒感染的极化MDCK细胞中，野生型（wt）HA>95%位于顶端，而突变的HA（HAtyr）约60%位于基底外侧。此外，在转染和病毒感染细胞的顶端和基底外侧表面，HAtyr蛋白的内吞率都比wt HA高得多。然而，与wt WSN病毒类似，HAtyr病毒几乎完全（>99%）从极化MDCK细胞的顶端侧出芽。通过使用神经氨酸酶促进病毒释放、用胰蛋白酶处理收集的病毒颗粒以将HA0切割为HA1和HA2、对释放的病毒颗粒进行蛋白质分析以及最后通过电子显微镜观察，这一发现得到了证实。因此，仅主要糖蛋白HA并不能决定出芽位点，其他因素，可能是病毒和宿主因素共同作用，负责选择流感病毒的出芽位点。
