Studies on protein regulation of virus-mediated membrance fusion

1.在抗人副流感病毒2型（HPIV 2）诱导的细胞融合过程中，我们在抗HN单克隆抗体（MAb）的压力作用下，获得了4株逃避突变株。本研究进一步证明该单克隆抗体识别HN蛋白分子上的83个Asn和91个Lys。2.将3种副粘病毒（HPIV 2、HPIV 4和SV 41）的HN蛋白和F蛋白分别与编码各蛋白基因的重组质粒连接，在细胞中表达。HPIV 2 HN与HPIV 2 F蛋白的共表达导致诱导显著的细胞融合。当HPIV 2 HN与SV 41 F或HPIV 4 F蛋白共表达时，也能促进细胞融合，但程度较低。在此基础上，构建并表达了32个HPIV 2 HN和SV 41 HN的嵌合蛋白，并进行了进一步的分析。结果表明，HPIV 2 HN蛋白的37 Ser ~ 91 Ser（A区）和148 Gly ~ 20 Ala（B区）两个区域与F蛋白的功能性分子相互作用是细胞融合诱导的前提。另一方面，对嵌合F蛋白的类似分析表明，HPIV 2 F蛋白上的两个区域，范围从128 Ala到227 Ala，（区域Y）和430 Tur至551 Ser（Z区）参与与HN蛋白的功能相互作用。4.这些结果表明HN和F蛋白之间的分子相互作用对于细胞融合是不可或缺的，每个蛋白质都有两个参与相互作用的区域。值得注意的是，每种蛋白质的两个区域之一包括可以非常接近融合位点的跨膜结构域。

1.Previosly, we had obtained four escape mutants under pressure of an anti-HN monoclonal antibody (MAb), which inhibited cell fusion induced by human parainfluenza type 2 virus (HPIV2). In the present study, further analyzes have proved that the MAb recognizes 83Asn and 91Lys on the HN molecule.2.The HN and F proteins of three paramyxoviruses (HPIV2, HPIV4, and SV41) were expressed in cells by transfecting them with recombinant plasmid encoding each protein gene. Coexpression of HPIV2 HN with HPIV2 F protein resulted in induction of prominent cell fusion. HPIV2 HN could also promote cell fusion when coexpressed with SV41 F or HPIV4 F proteins, although at lower extents. SV41 HN or HPIV4 HN did not induce cell fusion by coexpressing with HPIV2 F protein.3.Based on the above observation, futher analyzes were performed by constructing and expressing 32 chimeric proteins of HPIV2 HN and SV41 HN proteins. It was revealed that two regions on the HPIV2 HN protein ranging from 37Ser to 91Ser (region A) and ranging from 148Gly to 20Ala (region B) are involved in functional molecular interaction with the F protein, which is a prerequiste for cell fusion induction. On the other hand, similar analyzes of chimeric F proteins have indicated that two regions on HPIV2 F protein ranging from 128Ala to 227Ala (region Y) and from 430Tur to 551Ser (region Z) are involved in the functional interaction with the HN protein.4.These results suggest that molecular interaction between HN and F proteins are indispensable for cell fusion, and each protein has two regions which are involed in the interaction. It is notewothy that one of the two regions of each protein includes the transmembrane domains which may be in close proximity to the fusion site.