Bouchard M J, Lam D H, Racaniello V R
Department of Microbiology, Columbia University College of Physicians & Surgeons, New York, New York 10032, USA.
J Virol. 1995 Aug;69(8):4972-8. doi: 10.1128/JVI.69.8.4972-4978.1995.
To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.
为了确定脊髓灰质炎病毒1型Sabin疫苗株减毒的决定因素，利用强毒株1型脊髓灰质炎病毒P1/Mahoney和减毒疫苗株1型P1/Sabin的感染性cDNA克隆构建了一系列重组病毒。将这些病毒脑内接种到表达人脊髓灰质炎病毒受体的转基因小鼠中，确定了基因组中赋予神经毒力降低的区域。通过交换较小的限制性片段和定点诱变来确定导致减毒的核苷酸变化。VP4的935位核苷酸、VP3的2438位核苷酸以及VP1的2795和2879位核苷酸处的P1/Sabin突变均被证明是减毒的决定因素。重组病毒和定点突变体还用于确定与P1/Sabin温度敏感性有关的核苷酸变化。在HeLa细胞中，这种表型的决定因素定位于VP4的935位核苷酸、VP3的2438位核苷酸和VP1的2741位核苷酸处的变化。P1/Sabin的3Dpol基因与其亲本P1/Mahoney相比有三个氨基酸差异，这也导致了P1/Sabin的温度敏感性；然而，含有单个氨基酸变化的突变体在高温下的生长情况与P1/Mahoney相同，这表明温度敏感性可能需要某些组合或所有三个变化。此外，P1/Sabin的3'非编码区增强了由3Dpol赋予的温度敏感表型。尽管P1/Sabin的2741位核苷酸、3Dpol基因和3'非编码区导致了P1/Sabin的温度敏感性，但它们对表达脊髓灰质炎病毒受体的转基因小鼠的减毒没有作用，这表明减毒和温度敏感性的决定因素在遗传上是可以分开的。
