Mirza A M, Deng R, Iorio R M
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
J Virol. 1994 Aug;68(8):5093-9. doi: 10.1128/JVI.68.8.5093-5099.1994.
The sequence NRKSCS constitutes the longest linear stretch in the amino acid sequence of the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxoviruses that is completely conserved among all viruses in the group. We have used site-directed mutagenesis and expression of the mutated HN protein of one member of the group, Newcastle disease virus, to explore the role of this highly conserved sequence in the structure and function of the protein. Any substitution introduced for each of four residues in the sequence, N-234, R-235, K-236, or S-237, results in a drastic decrease in neuraminidase activity relative to that of the wild-type protein. Only substitutions for the terminal serine residue in the sequence had comparatively little effect on this activity. These findings are consistent with prior computer-based predictions of protein secondary structure which had suggested that this domain corresponds to one in the beta-sheet propeller structure of the neuraminidase protein of influenza virus closest to the center of the sialic acid binding site and forms part of the enzyme active site. Four of the substitutions, N-234-->Y and K-236-->E, -->Q, and -->S, apparently cause a local alteration in the antigenic structure of the protein. This is evidenced by (i) the diminished recognition of the protein only by monoclonal antibodies thought to bind at the neuraminidase active site, among an extensive panel of conformation-specific antibodies, and (ii) the slower rate of migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all except the K-236-->Q mutation. One of the mutations, K-236-->S, completely abolishes the ability of the protein to promote cellular fusion when coexpressed with the fusion protein. The latter cannot be explained by a decrease in the relative hemadsorption activity of the protein and suggests that the globular head of the protein may contribute to this process beyond providing receptor recognition.
序列NRKSCS构成了副粘病毒血凝素 - 神经氨酸酶（HN）糖蛋白氨基酸序列中最长的线性延伸部分，该序列在该病毒组的所有病毒中完全保守。我们利用定点诱变技术并表达了该病毒组中新城疫病毒一个成员的突变型HN蛋白，以探究这一高度保守序列在该蛋白结构和功能中的作用。对序列中的四个残基N - 234、R - 235、K - 236或S - 237中的任何一个进行替换，相对于野生型蛋白，神经氨酸酶活性都会急剧下降。只有对序列中末端丝氨酸残基的替换对该活性的影响相对较小。这些发现与先前基于计算机的蛋白质二级结构预测结果一致，该预测表明该结构域对应于流感病毒神经氨酸酶蛋白β - 折叠螺旋结构中最接近唾液酸结合位点中心的一个结构域，并构成酶活性位点的一部分。其中四个替换，N - 234→Y和K - 236→E、→Q以及→S，显然导致了该蛋白抗原结构的局部改变。这一点可通过以下两点得到证明：（i）在大量构象特异性抗体中，只有被认为在神经氨酸酶活性位点结合的单克隆抗体对该蛋白的识别能力减弱；（ii）除K - 236→Q突变外，所有突变体在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中的迁移速率都较慢。其中一个突变，K - 236→S，当与融合蛋白共表达时，完全消除了该蛋白促进细胞融合的能力。后者不能用该蛋白相对血细胞吸附活性的降低来解释，这表明该蛋白的球状头部可能在这一过程中发挥作用，而不仅仅是提供受体识别功能。
