Thammawat Sutthiwan, Sadlon Tania A, Hallsworth Peter G, Gordon David L
Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
J Virol. 2008 Dec;82(23):11767-74. doi: 10.1128/JVI.01208-08. Epub 2008 Sep 10.
Human metapneumovirus (hMPV) is an important cause of lower respiratory tract disease, particularly in infants and young children. hMPV has two major glycoproteins, G and F, which are responsible for virus attachment and membrane fusion, respectively. We investigated the role of cellular glycosaminoglycans (GAGs) and G protein in hMPV infection. The pretreatment of hMPV with soluble heparin markedly inhibited the infection of HEp-2 cells. Recombinant G protein, comprising the extracellular domain of G, bound to heparin-agarose columns and also to HEp-2 cells. hMPV infection and G protein binding to HEp-2 cells was inhibited by other soluble GAGs, including chondroitin sulfates, by the enzymatic removal of cell surface GAGs with GAG lyases or by the pretreatment of cells with basic fibroblast growth factor. The role of cellular GAGs was confirmed by the binding of G protein to wild-type CHO cells but not to GAG-deficient CHO-pgsA745 cells. An analysis of the G protein sequence revealed two adjacent clusters of positively charged amino acids ((149)EKKKTRA(155) and (159)QRRGKGKE(166)). Truncated G fragments were expressed, and only the fragment containing these putative heparin binding domains retained heparin binding. The alanine mutagenesis of charged residues in either of these regions resulted in the loss of binding to heparin and to HEp-2 cells, suggesting that both sites are likely to be required for hMPV attachment. These results, taken together with the inhibition of hMPV infection by soluble G protein, indicate an important role for G protein and cellular GAGs in hMPV infection.
人偏肺病毒（hMPV）是下呼吸道疾病的重要病因，尤其是在婴幼儿中。hMPV有两种主要糖蛋白，即G蛋白和F蛋白，分别负责病毒的附着和膜融合。我们研究了细胞糖胺聚糖（GAGs）和G蛋白在hMPV感染中的作用。用可溶性肝素预处理hMPV可显著抑制HEp-2细胞的感染。包含G蛋白胞外域的重组G蛋白能与肝素琼脂糖柱结合，也能与HEp-2细胞结合。其他可溶性GAGs（包括硫酸软骨素）、用GAG裂解酶酶解去除细胞表面GAGs或用碱性成纤维细胞生长因子预处理细胞，均可抑制hMPV感染及G蛋白与HEp-2细胞的结合。G蛋白与野生型CHO细胞结合，但不与缺乏GAG的CHO-pgsA745细胞结合，从而证实了细胞GAGs的作用。对G蛋白序列的分析揭示了两个相邻的带正电荷氨基酸簇（（149）EKKKTRA（155）和（159）QRRGKGKE（166））。表达了截短的G片段，只有包含这些假定肝素结合域的片段保留了肝素结合能力。对这两个区域中任何一个区域的带电荷残基进行丙氨酸诱变，都会导致与肝素和HEp-2细胞的结合丧失，这表明两个位点可能都是hMPV附着所必需的。这些结果与可溶性G蛋白对hMPV感染的抑制作用相结合，表明G蛋白和细胞GAGs在hMPV感染中起重要作用。
