Knight Ronald L, Schultz Kimberly L W, Kent Rebekah J, Venkatesan Meera, Griffin Diane E
Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Medicine, Baltimore, Maryland 21205, USA.
J Virol. 2009 Jun;83(11):5640-7. doi: 10.1128/JVI.02427-08. Epub 2009 Mar 18.
Each Sindbis virus (SINV) surface glycoprotein has two sites for N-linked glycosylation (E1 positions 139 and 245 [E1-139 and E1-245] and E2 positions 196 and 318 [E2-196 and E2-318]). Studies of SINV strain TE12 mutants with each site eliminated identified the locations of carbohydrates by cryo-electron microscopy (S. V. Pletnev et al., Cell 105:127-136, 2001). In the current study, the effects of altered glycosylation on virion infectivity, growth in cells of vertebrates and invertebrates, heparin binding, virulence in mice, and replication in mosquitoes were assessed. Particle-to-PFU ratios for E1-139 and E2-196 mutant strains were similar to that for TE12, but this ratio for the E1-245 mutant was 100-fold lower than that for TE12. Elimination of either E2 glycosylation site increased virus binding to heparin and increased replication in BHK cells. Elimination of either E1 glycosylation site had no effect on heparin binding but resulted in an approximately 10-fold decrease in virus yield from BHK cells compared to the TE12 amount. No differences in pE2 processing were detected. E2-196 and E2-318 mutants were more virulent in mice after intracerebral inoculation, while E1-139 and E1-245 mutants were less virulent. The E1-245 mutant showed impaired replication in C7/10 mosquito cells and in Culex quinquefasciatus after intrathoracic inoculation. We conclude that the increased replication and virulence of E2-196 and E2-318 mutants are primarily due to increased efficiency of binding to heparan sulfate on mammalian cells. Lack of glycosylation at E1-139 or E1-245 impairs replication in vertebrate cells, while E1-245 also severely affects replication in invertebrate cells.
每个辛德毕斯病毒（SINV）表面糖蛋白都有两个N - 连接糖基化位点（E1的139位和245位[E1 - 139和E1 - 245]以及E2的196位和318位[E2 - 196和E2 - 318]）。对每个位点被消除的SINV TE12株突变体的研究通过冷冻电子显微镜确定了碳水化合物的位置（S. V. Pletnev等人，《细胞》105:127 - 136，2001年）。在当前研究中，评估了糖基化改变对病毒粒子感染性、在脊椎动物和无脊椎动物细胞中的生长、肝素结合、对小鼠的毒力以及在蚊子中的复制的影响。E1 - 139和E2 - 196突变株的粒子与空斑形成单位（PFU）的比率与TE12相似，但E1 - 245突变株的这一比率比TE12低100倍。消除任何一个E2糖基化位点都会增加病毒与肝素的结合，并增加在BHK细胞中的复制。消除任何一个E1糖基化位点对肝素结合没有影响，但与TE12相比，BHK细胞中的病毒产量降低了约10倍。未检测到pE2加工的差异。E2 - 196和E2 - 318突变体在脑内接种后对小鼠的毒力更强，而E1 - 139和E1 - 245突变体的毒力较弱。E1 - 245突变体在胸内接种后在C7/10蚊子细胞和致倦库蚊中显示出复制受损。我们得出结论，E2 - 196和E2 - 318突变体复制和毒力增加主要是由于与哺乳动物细胞上硫酸乙酰肝素结合效率提高。E1 - 139或E1 - 245缺乏糖基化会损害在脊椎动物细胞中的复制，而E1 - 245也严重影响在无脊椎动物细胞中的复制。
