Polo J M, Davis N L, Rice C M, Huang H V, Johnston R E
Department of Microbiology, North Carolina State University, Raleigh 27695-7615.
J Virol. 1988 Jun;62(6):2124-33. doi: 10.1128/JVI.62.6.2124-2133.1988.
Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.
通过使用病毒的全长cDNA克隆（Toto1101），研究了影响新生小鼠中辛德毕斯病毒发病机制的基因位点。全长cDNA与噬菌体SP6启动子相连，以促进体外感染性RNA转录本的合成。源自Toto1101的病毒在新生小鼠中显示出毒力降低（减毒）。用源自强毒株AR339（SB）的克隆E1和6K基因替换Toto1101的E1糖蛋白和6K基因，产生了一种新的构建体TR2000，其产生了强毒病毒。对TR2000、Toto1101以及相关强毒株和减毒株的整个替换区域进行序列测定，确定了Toto1101和TR2000之间E1的三个编码差异。这些差异单独或组合起来，可能是导致减毒表型的原因。本实验室先前的研究在E2糖蛋白的第114位氨基酸处鉴定出另一个减毒突变（N.L. 戴维斯、F.J. 富勒、W.G. 多尔蒂、R.A. 奥尔姆斯特德和R.E. 约翰斯顿，《美国国家科学院院刊》83:6771 - 6775，1986年）。突变体SB - RL中第114位的精氨酸替代SB中的第114位丝氨酸似乎赋予了三种显著的表型：在新生小鼠中减毒、对特定E2单克隆抗体的敏感性增加以及BHK细胞的加速穿透。用SB或SB - RL的cDNA克隆的相应片段替换包含E2第114位氨基酸密码子的TR2000序列，产生了两种辛德毕斯病毒株（TR2100和TR2200），它们除了E2第114位密码子（分别为丝氨酸和精氨酸）外是同基因的。这三种诊断表型根据E2第114位氨基酸密码子的来源共分离，证实了这一单氨基酸替换对这三种表型的显著影响。
