Tubulekas I, Liljeström P
Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden.
J Virol. 1998 Apr;72(4):2825-31. doi: 10.1128/JVI.72.4.2825-2831.1998.
The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.
塞姆利基森林病毒的E2刺突糖蛋白以p62前体蛋白的形式产生，该前体蛋白被宿主蛋白酶切割成其成熟形式E2。切割对于病毒粒子的形成或释放不是必需的，但对于感染性是必需的。先前的结果表明，切割缺陷型p62突变体的表型回复体产生了，并且我们在此表明这些回复体可能在切割位点附近含有第二位点抑制突变。这些热点位点发生突变以消除此类抑制突变的产生；然而，E2蛋白另一个遥远结构域中出现了二级突变，所有这些突变仍然导致切割缺陷型突变。此类突变体生长非常缓慢，并且在病毒进入和释放方面效率低下。突变位点定义了刺突蛋白的结构域，这些结构域可能相互作用以调节其结构和功能。由于其高度减毒的表型和较低的回复概率，靠近切割位点的新突变被用于制造新的辅助载体，用于将重组RNA包装成感染性粒子，从而进一步提高了基于塞姆利基森林病毒复制子的载体系统的生物安全性。
