Viral factors responsible for the neurovirulence of SSPE virus

为了确定亚急性硬化性全脑炎（SSPE）病毒神经毒力的病毒因子，我们测定了大坂-1，-2和-3株SSPE病毒的核苷酸序列。比较P-M连接区的序列发现SSPE病毒之间存在变异。对于Osaka-1株，野生型P基因末端（3 ′-UUUUU-5 ′）通过单核苷酸缺失突变为3 ′-UUUUU-5 ′，或者对于Osaka-2株的OSA-2/Fr/B同胞病毒突变为3 ′-UUUCUU-5 ′。为了阐明这些突变对P基因转录终止的意义，我们构建了包含萤火虫和海肾荧光素酶基因的双顺反子微型基因组，通过在两个报告基因之间插入不同类型的突变P-M连接序列来分离。荧光素酶测定表明，这些突变是唯一负责通读mRNA合成。F基因序列分析表明，所有三个SSPE病毒的F蛋白的胞质结构域有显着的突变。通过用克隆的H cDNA转染的Vero细胞的红细胞吸附试验测定病毒与细胞受体CD 46的结合。SSPE株H基因转染细胞的负血吸附结果表明，在持续感染过程中，麻疹病毒祖先的H蛋白的原始性质是保守的。此外，一些特定的氨基酸取代负责H蛋白和CD 46之间的相互作用。从SSPE病毒克隆的F和H cDNA的共表达实验再现了类似于病毒感染的细胞病变效应。对F和H蛋白突变的进一步功能研究将揭示SSPE和麻疹毒株之间的差异。这些结果也将为SSPE病毒的受体提供新的见解。

To Identify viral factors responsible for the neurovirulence of subacute sclerosing panencephalitis(SSPE) virus, we have determined nucleotide sequences of the Osaka-1, -2, and -3 strains of SSPE virus. Comparison of the sequence of the P-M junction revealed a variation among SSPE viruses. The wild-type P gene end(3'-UUUUUU-5')was mutated to 3'-UUUUU-5' for the Osaka-1 strain by a single nucleotide deletion, or mutated to 3'-UUUCUU-5' for the OSA-2/Fr/B sibling virus of the Osaka-2 strain. To clarify the significance of these mutations for the transcriptional termination of the P gene, we have constructed dicistronic minigenomes which contained the Firefly and the Renilla luciferase genes, separated by insertion of different types of mutated P-M junction sequence between the two reporter genes. The luciferase assay demonstrated that these mutations were solely responsible for the read-through of mRNA synthesis. The F gene sequence analysis revealed that all the three SSPE viruses had significant mutations in the cytoplasmic domain of the F protein. The binding of the virus with the cellular receptor CD46 was determined by the hemadsorption assay of Vero cells transfected with the cloned H cDNAs. The results of the negative hemadsorption of cells transfected with H genes from SSPE strains revealed that the original property of the H protein of the progenitor measles virus was conserved during persistent infection. In addition, some specific amino acid substitutions were responsible for the interaction between the H protein and CD46. Co-expression experiments of the cloned F and H cDNAs from SSPE viruses reproduced the cytopathic effect similarly to the viral infection. Further functional studies of the mutations in the F and H protein will reveal the differences between SSPE and measles strains. These results will also provide a new insight into the receptor for SSPE viruses.