A Single Amino Acid Change in nsP1 Attenuates Neurovirulence of the Sindbis-Group Alphavirus S.A.AR86

S.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c.) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence.

S.A.AR86属于α病毒（alphavirus）辛德毕斯病毒组，对成年小鼠具有神经毒力，其非结构蛋白1（nonstructural protein 1，nsP1）的538位氨基酸为独特的苏氨酸；该组其他非神经毒力的α病毒在此位置编码的则为异亮氨酸。研究人员将野生型S.A.AR86感染性克隆（infectious clone）ps55的538位氨基酸突变为异亮氨酸，获得突变克隆ps51；由该突变克隆拯救得到的病毒，与ps55来源的病毒相比，神经毒力显著减弱。颅内（intracranial, i.c.）感染ps55可导致89%的实验动物出现严重病症，包括后肢麻痹、结膜炎、体重下降乃至死亡。与之相比，ps51仅引发轻微临床症状，无动物死亡。不过，对突变株ps51与野生型ps55来源的S.A.AR86分子克隆病毒的比较分析显示，ps51在组织培养中的增殖能力与野生型ps55相当甚至更强；且颅内感染ps51后，小鼠脑组织内的病毒滴度（viral titers）与野生型ps55并无差异。通过原位杂交（in situ hybridization）分析脑内病毒复制情况发现，感染早期（12至72小时），两种病毒在脑内相似区域建立感染。但在感染后期，野生型ps55病毒已扩散至脑内大片区域，而ps51突变株的复制范围则受到限制。这提示ps51要么在感染后期无法在脑内广泛扩散，要么其清除速度快于ps55。后续实验进一步验证了nsP1第538位苏氨酸对S.A.AR86神经毒力的贡献：研究人员将对断乳小鼠（weanling mice）无神经毒力的辛德毕斯病毒TR339株的nsP1 538位突变为苏氨酸，获得重组病毒39ns1；该病毒展现出显著增强的神经毒力与发病症状，包括体重下降与后肢麻痹。上述结果证实了α病毒非结构蛋白基因在成年小鼠神经毒力中发挥的作用。