Duffus W A, Levy-Mintz P, Klimjack M R, Kielian M
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Virol. 1995 Apr;69(4):2471-9. doi: 10.1128/JVI.69.4.2471-2479.1995.
The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28 degrees C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28 degrees C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.
Semliki森林病毒（SFV）的两个跨膜刺突蛋白亚基在糙面内质网中形成异二聚体复合物。然后该复合物被转运至质膜，在质膜处发生刺突-核衣壳结合和病毒出芽。通过使用感染性SFV克隆，我们已经表征了E1刺突亚基推定融合肽内的突变对刺突蛋白二聚化和病毒组装的影响。这些突变先前已被证明可阻断刺突蛋白膜融合活性（G91D）或导致融合pH阈值发生酸性偏移（G91A）。在37℃感染BHK细胞期间，含有任一突变的病毒刺突蛋白均能有效产生并转运至质膜，在质膜处它们与核衣壳结合。然而，突变刺突蛋白组装成成熟病毒粒子的过程严重受损，并且E1的一个裂解可溶性片段被释放到培养基中。相比之下，将感染突变体的细胞在较低温度（℃）下孵育可显著减少E1裂解，并允许形态正常的病毒粒子组装。脉冲标记研究表明，28℃孵育的关键时期是在病毒组装期间，而非刺突蛋白合成期间。因此，SFV推定融合肽中的突变赋予了强烈且热可逆的出芽缺陷。通过使用感染病毒突变体的细胞或在28℃组装的突变病毒粒子，分析了E1刺突蛋白亚基与E2的二聚化。G91D和G91A突变体的组装表型改变与E1-E2二聚体稳定性降低相关。
