禽流感病毒血凝素HA的可变性解析

以重组的蒙古鸭H5N2禽流感病毒A/Duck/Mongolia/54/01的血凝素HA蛋白的cDNA为模板,进行PCR随机突变,表达只有单个氨基酸突变的H5HA基因共计38个.根据红细胞吸附反应,分析这些突变HA的功能,仍然具有红细胞吸附活性的单个氨基酸突变的HA约占89%,说明H5HA单个氨基酸突变的容许率是相当高的.HA1区突变数目大约是HA2区的两倍.对失去红细胞吸附功能和某些仍然拥有红细胞吸附功能的HA及单个氨基酸突变的位置与结构的关系进行探讨.有两个位点氨基酸突变了两次,但都不影响红细胞吸附功能,对红细胞吸附功能的影响,似乎主要由位置决定,而不是取决于取代的氨基酸的种类.位点179位和122位的突变是不允许的;位点179位于H5N1的受体结合区域RBD内,122位位于A抗原决定簇区附近,推测在H5HA三维结构上,这两个位点位于HA分子的内部,维持着H5HA的结构.HA1Cys位点4和HA2Cys位点148的突变是不允许的.这两个Cys正好形成HA1和HA2连接的桥梁,对维持H5HA结构也是相当重要的.本实验中HA先后失去了三个糖基化位点,但并不影响吸附红细胞的功能.总之,通过实验分析以研究某些氨基酸改变的效果,寻找关键位点是否突变,可以作为评估H5N1野毒株大流行潜力的分子标志.

Analysis of the Amino Acid Changes of the Hemagglutinin of H5 Avian Influenza Virus

We introduced 38 single-point amino acid changes into the hemagglutinin(HA) protein of the reassortmented A/Duck/Mongolia/54/01(H5N2) strain by a PCR random mutation method.The percentage of amino acid changes on the HA domain that did not abrogate hemadsorption activity was calculated to be 89%.Changes in the amino acids of the HA2 domain were observed to be about half of those in the HA1 domain of these mutants.We assumed that amino acid changes in the HA1 domain afforded more flexibility in maintaining the functions of the HA protein than did those in the HA2 domain.Changes at two positions allowed the mutants to have same characteristics with respect to HA function despite the difference in the substituted amino acid.The results suggested that the effect on hemadsorption activity of an amino acid change on the HA protein primarily depends on the position rather than the species of substituted amino acid.An amino acid change at residue 122 from Trp to Arg and 179 from His to Arg resulted in the loss of hemadsorption activity of the HA protein.Site 122 is near the antibody binding site A,and site 179 is in the receptor binding domain(RBD) of H5HA.So that we suggest residue position 179 or 122 is very important to maintain the structure of RBD or antigenic site of H5HA.Position 4 in HA1 changed from Cys to Arg and position 148 in HA2 changed from Cys to Tyr also resulted in the loss of hemadsorption activity of the HA protein.Cys plays an important role in maintaining the structure of HA protein by means of S-S bonds.3 potential glycosylation sites(Asn-X-Ser/Thr) were lost in our experiment that did not lose the hemadsorption activity of HA.Some interesting positions need to be analyzed more finely.Some amino acid changes identified in vitro experiment may serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.