ALTERATION OF THE PH-DEPENDENCE OF CORONAVIRUS-INDUCED CELL-FUSION - EFFECT OF MUTATIONS IN THE SPIKE GLYCOPROTEIN
ALTERATION OF THE PH-DEPENDENCE OF CORONAVIRUS-INDUCED CELL-FUSION - EFFECT OF MUTATIONS IN THE SPIKE GLYCOPROTEIN
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DOI：
10.1128/jvi.65.4.1916-1928.1991
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发表时间：
1991-04-01
影响因子：
5.4
通讯作者：
BUCHMEIER, MJ
中科院分区：
文献类型：
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作者：
GALLAGHER, TM;ESCARMIS, C;BUCHMEIER, MJ
感染易感鼠细胞的冠状病毒小鼠肝炎病毒4型（MHV4）的感染导致pHS的广泛细胞融合从5.5到8.5。内体性弱碱氯喹和氯化铵不能预防MHV4感染。与形成鲜明对比的是，我们从持续感染MHV4的神经细胞系中选择了变体，这些变体完全依赖于酸pH来融合宿主细胞，并受到内体性弱碱基的强烈抑制。在融合活性表面（S）糖蛋白基因的水平上比较了野生型和变体病毒。每个4,131碱基开放阅读框的克隆和测序预测总共八个氨基酸差异，分为三个不同的簇。每种S糖蛋白从cDNA表达时，都以等效量合成，并将相似的比例转运到细胞表面。野生型在中性pH下诱导的细胞 - 细胞融合，而变体需要长时间暴露于酸性pH，以诱导融合。通过在野生型和变体cDNA之间交换限制片段制备的杂交基因的表达表明，消除中性pH融合仅取决于位置1067（q至h），1094（q至H）的氨基酸改变，而1114 （L到R）。这些变化在于S（S2）的跨膜裂解片段的预测七个区域。这些发现表明，冠状病毒融合的pH依赖性高度可变，并且该变异性可以由几个氨基酸残基确定。
Infection of susceptible murine cells with the coronavirus mouse hepatitis virus type 4 (MHV4) results in extensive cell-cell fusion at pHs from 5.5 to 8.5. The endosomotropic weak bases chloroquine and ammonium chloride do not prevent MHV4 infection. In marked contrast, we have selected variants from a neural cell line persistently infected with MHV4 which are entirely dependent on acid pH to fuse host cells and are strongly inhibited by endosomotropic weak bases. Wild-type and variant viruses were compared at the level of the fusion-active surface (S) glycoprotein gene. Cloning and sequencing of each 4,131-base open reading frame predicted a total of eight amino acid differences which fell into three distinct clusters. Each S glycoprotein, when expressed from cDNA, was synthesized in equivalent amounts, and similar proportions were transported to the cell surface. Wild-type S induced cell-cell fusion at neutral pH, whereas variant S required prolonged exposure to acidic pH to induce fusion. Expression of hybrid S genes prepared by exchange of restriction fragments between wild-type and variant cDNAs revealed that elimination of neutral pH fusion was solely dependent on amino acid alterations at positions 1067 (Q to H), 1094 (Q to H), and 1114 (L to R). These changes lie within a predicted heptad region of the transmembrane cleavage fragment of S (S2). These findings demonstrate that the pH dependence of coronavirus fusion is highly variable and that this variability can be determined by a few as three amino acid residues.