Melanson Vanessa R, Iorio Ronald M
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0122, USA.
J Virol. 2006 Jan;80(2):623-33. doi: 10.1128/JVI.80.2.623-633.2006.
Most paramyxovirus fusion (F) proteins require the coexpression of the homologous attachment (HN) protein to promote membrane fusion, consistent with the existence of a virus-specific interaction between the two proteins. Analysis of the fusion activities of chimeric HN proteins indicates that the stalk region of the HN spike determines its F protein specificity, and analysis of a panel of site-directed mutants indicates that the F-interactive site resides in this region. Here, we use the addition of oligosaccharides to further explore the role of the HN stalk in the interaction with F. N-glycans were individually added at several positions in the stalk to determine their effects on the activities of HN, as well as its structure. N-glycan addition at positions 69 and 77 in the stalk specifically blocks fusion and the HN-F interaction without affecting either HN structure or its other activities. N-glycans added at other positions in the stalk modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as indicated by sucrose gradient sedimentation analyses. Finally, N-glycan addition in another region of HN (residues 124 to 152), predicted by a peptide-based analysis to mediate the interaction with F, does not significantly reduce the level of fusion, arguing strongly against this site being part of the F-interactive domain in HN. Our data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.
大多数副粘病毒融合（F）蛋白需要同源附着（HN）蛋白共同表达以促进膜融合，这与两种蛋白之间存在病毒特异性相互作用相一致。对嵌合HN蛋白融合活性的分析表明，HN刺突的柄部区域决定了其F蛋白特异性，而对一组定点突变体的分析表明，F相互作用位点位于该区域。在此，我们通过添加寡糖来进一步探究HN柄部在与F相互作用中的作用。在柄部的几个位置分别添加N-聚糖，以确定它们对HN活性及其结构的影响。在柄部69和77位添加N-聚糖可特异性阻断融合及HN-F相互作用，而不影响HN结构或其其他活性。在柄部其他位置添加的N-聚糖可调节位于HN球状头部的活性。这与蛋白质四聚体结构的改变相关，蔗糖梯度沉降分析表明了这一点。最后，在HN的另一个区域（124至152位残基）添加N-聚糖，基于肽的分析预测该区域介导与F的相互作用，但并未显著降低融合水平，这有力地反驳了该位点是HN中F相互作用结构域一部分的观点。我们的数据支持HN上的F相互作用位点由该蛋白的柄部区域定义这一观点。
