Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides.
Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides.
复制标题
疏水性肽抑制仙台病毒与磷脂囊泡和人红细胞膜的融合。
DOI：
10.1016/0042-6822(91)90610-n
复制
发表时间：
1991
期刊：
影响因子：
3.7
通讯作者：
Yeagle,PL
中科院分区：
文献类型：
--
作者：
Kelsey,DR;Flanagan,TD;Young,JE;Yeagle,PL
能够抑制包膜病毒感染细胞的疏水二肽和三肽也能够抑制至少三种不同类型的膜融合事件。用挤压法制备了N-甲基二油酰磷脂酰乙醇胺(N-甲基涂料)的大单层囊泡(LUV)，该囊泡含有1-氨基-3，6，8-三磺酸(ANTS)和/或二甲苯双(溴化吡啶)(DPX)。然后用ANTS/DPX荧光分析法监测囊泡融合(内容物混合)和渗漏。用十八烷基罗丹明B氯化物(R18)标记的病毒荧光猝灭，测定了仙台病毒与脂泡的融合和仙台病毒与人红细胞膜的融合。本研究发现，羧基苯甲氧基-L-Phe-L-Phe(Z-L-Phe-L-Phe)、Z-L-Phe、Z-D-Phe和Z-GIY-L-Phe-L-Phe在抑制N-甲基兴奋剂LUV融合或病毒与N-甲基兴奋剂LUV融合方面的有效性也与已报道的它们阻断病毒感染性的能力相似。此外，Z-d-Phe-L-PheGly和Z-GIY-L-PheGly抑制仙台病毒与人红细胞膜的融合，其抑制作用与抑制囊泡-囊泡和病毒-囊泡融合的相对效力相同。证据表明，这些多肽通过包膜病毒发挥其抑制斑块形成的机制。这类抑制物显然是通过抑制病毒包膜与靶细胞膜的融合来发挥作用，从而防止病毒感染。研究了这些多肽抑制膜融合的物理途径。LUV中膜融合途径中的中间产物的31P核磁共振谱显示，有效的融合抑制剂Z-d-Phe-L-PheGly选择性地改变了假想的融合中间体的结构(或动力学)，而较差的抑制剂Z-GIY-L-Phe不能。对这些31P核磁共振结果的一种可能的解释是，当融合途径需要具有小曲率半径的膜缺陷时，抑制肽稳定了具有大曲率半径的膜结构。通过测定抑制性和非抑制性多肽对通过超声照射磷脂分散体形成小曲率半径的膜结构的影响，验证了这一假说。抑制肽能阻止小曲率半径的膜结构的形成，而非抑制肽不能阻止这种结构的形成。这些数据表明，抑制包膜病毒融合的多肽至少部分地可能通过干扰膜融合中间产物的形成(可能是由病毒引起的点状膜缺陷)来发挥作用，这些中间产物迫使脂质进入具有小曲率半径的结构。
Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) ofN-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/orp-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy-l-Phe-l-Phe (Z-l-Phe-l-Phe), Z-l-Phe, Z-D-Phe, and Z-GIy-l-Phe-l-Phe in inhibitingN-methyl DOPE LUV fusion or fusion of virus withN-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-d-Phe-l-PheGly and Z-GIy-l-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated.31P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-d-Phe-l-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-GIy-l-Phe did not. One possible interpretation of these31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures. These data taken collectively suggest that the peptides that inhibit the fusion of enveloped viruses could act, at least in part, by interfering with the formation of intermediates in membrane fusion (possibly punctate membrane defects induced by the virus) that force lipids into structures with small radii of curvature.