Méndez Ernesto, Aguirre-Crespo Gabriela, Zavala Guadalupe, Arias Carlos F
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Colonia Miraval, Cuernavaca, Morelos 62250, Mexico.
J Virol. 2007 Oct;81(19):10649-58. doi: 10.1128/JVI.00785-07. Epub 2007 Jul 25.
VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on their intracellular distribution. VP90 was found in membrane-enriched fractions (mVP90), as well as in fractions enriched in cytosolic proteins (cVP90), while VP70 was found exclusively in the latter fractions. Upon trypsin activation, infectivity was detected in all VP90-containing fractions, confirming that both mVP90 and cVP90 are able to assemble into particles; however, the two forms of VP90 showed differential sensitivities to trypsin, especially at their carboxy termini, which in the case of mVP90 was shown to remain membrane associated after protease digestion. Structural protein oligomers were detected in purified VP70-containing viruses, as well as in membrane-enriched fractions, but they were less evident in cytosolic fractions. Ultrastructural studies of infected cells revealed different types of viral particles, some of which appeared to be associated with membranes. By immunoelectron microscopy, structural proteins were shown to form virus particles in clusters and to associate with the edges of vesicles induced during infection, which also appear to contain subviral particles inside. Nonstructural proteins and viral RNA colocalized with mVP90, but not with cVP90, suggesting that mVP90 might represent the form of the protein that is initially assembled into particles, at the sites where the virus genome is being replicated.
人星状病毒Yuc8的衣壳多聚蛋白前体VP90组装成病毒颗粒，其在羧基末端经细胞胱天蛋白酶加工产生VP70，这一过程与病毒的细胞释放相关。在此，我们表征了VP90 - VP70加工对这些蛋白质特性及其细胞内分布的影响。VP90存在于富含膜的组分（mVP90）以及富含胞质蛋白的组分（cVP90）中，而VP70仅存在于后者的组分中。经胰蛋白酶激活后，在所有含VP90的组分中均检测到感染性，证实mVP90和cVP90都能够组装成颗粒；然而，两种形式的VP90对胰蛋白酶表现出不同的敏感性，尤其是在其羧基末端，在蛋白酶消化后，mVP90的羧基末端仍与膜相关。在纯化的含VP70的病毒以及富含膜的组分中检测到结构蛋白寡聚体，但在胞质组分中不太明显。对感染细胞的超微结构研究揭示了不同类型的病毒颗粒，其中一些似乎与膜相关。通过免疫电子显微镜观察，结构蛋白显示在簇中形成病毒颗粒，并与感染期间诱导的囊泡边缘相关联，这些囊泡内部似乎也含有亚病毒颗粒。非结构蛋白和病毒RNA与mVP90共定位，但不与cVP90共定位，这表明mVP90可能代表最初在病毒基因组复制位点组装成颗粒的蛋白质形式。
