Ryan C, Ivanova L, Schlesinger M J
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Virology. 1998 Apr 10;243(2):380-7. doi: 10.1006/viro.1998.9074.
Assembly and budding of alphaviruses are postulated to occur by protein-protein interactions between sites on the cytoplasmic domain of the transmembranal envelope E2 glycoprotein and on the surface of the nucleocapsid protein subunits. Genetic data to support this model have been obtained by isolating revertants of two slow-growth mutants of Sindbis virus and analyzing the sequences of the genes encoding their structural proteins. The slow-growth phenotypes of the mutants were previously shown to result from site-directed mutations of 2 amino acids in the sequence corresponding to the 33 amino acids at the carboxyl terminus of E2, which are localized to the cytoplasmic face of the plasma membrane. Putative revertants of these two mutants with faster growth rates were isolated by sequential passaging of virus grown on insect cells or chicken embryo fibroblasts. Sequence analysis of plaque-purified viruses that grew significantly better than the original mutant revealed that the original E2 mutation was present and that there were additional amino acid changes in the virus capsid. Two of the latter were introduced separately into the wild-type virus cDNA and into the genomes of the original mutants. The new strains of virus that contained both capsid and E2 mutations produced many more extracellular particles than those with the E2 mutations alone, indicating substantial suppression of the original E2 mutation. Both capsid mutations appear to be localized near a hydrophobic pocket of the capsid, which is postulated to be the site for docking of hydrophobic amino acids of the E2 cytoplasmic domain. This genetic study provides strong support for the current models of alphavirus assembly.
甲病毒的装配和出芽被假定是通过跨膜包膜E2糖蛋白胞质结构域上的位点与核衣壳蛋白亚基表面之间的蛋白质-蛋白质相互作用而发生的。通过分离辛德毕斯病毒两个生长缓慢突变体的回复株并分析编码其结构蛋白的基因序列，已获得支持该模型的遗传数据。先前已表明，这些突变体的生长缓慢表型是由E2羧基末端33个氨基酸对应序列中2个氨基酸的定点突变导致的，这些氨基酸位于质膜的胞质面。通过在昆虫细胞或鸡胚成纤维细胞上连续传代培养病毒，分离出了这两个生长速度较快的突变体的假定回复株。对生长明显优于原始突变体的噬斑纯化病毒进行序列分析发现，原始E2突变仍然存在，并且病毒衣壳中还有其他氨基酸变化。将后两种变化分别引入野生型病毒cDNA和原始突变体的基因组中。同时含有衣壳和E2突变的新病毒株比仅含有E2突变的病毒株产生了更多的细胞外颗粒，这表明原始E2突变得到了显著抑制。两种衣壳突变似乎都位于衣壳的一个疏水口袋附近，该口袋被假定为E2胞质结构域疏水氨基酸对接的位点。这项遗传学研究为当前的甲病毒装配模型提供了有力支持。
