Jackson Cheryl A, Messinger Jeff, Palmer Matthew T, Peduzzi Jean D, Morrow Casey D
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Virology. 2003 Sep 15;314(1):45-61. doi: 10.1016/s0042-6822(03)00385-4.
The spread of intramuscularly inoculated poliovirus to the central nervous system (CNS) has been documented in humans, monkeys, and mice transgenic for the human poliovirus receptor. Poliovirus spread is thought to be due to infection of the peripheral nerve and retrograde transport of poliovirus through the axon to the neuron cell body, where final virus uncoating occurs and translation/replication ensues. In previous studies, we have shown that polio-based vectors (replicons) can be used for gene delivery to motor neurons of the CNS. Using a replicon that encodes green fluorescent protein (GFP), we found that following intrathecal inoculation, GFP expression was confined to motorneurons of the spinal cord. To further characterize the gene expression of poliovirus in the periphery and CNS, we have intramuscularly inoculated transgenic mice with poliovirus replicons encoding GFP. Expression of GFP was demonstrated in the muscle, sciatic nerve, dorsal root ganglion, and the ventral horn motorneurons following intramuscular inoculation. There was no evidence of paralysis or behavioral abnormalities in the mice following intramuscular inoculation of the replicon encoding GFP. Injection of replicon RNA alone (naked RNA) into the muscle of transgenic mice or rats, which do not express the poliovirus receptor, also resulted in expression of GFP in the muscle, sciatic nerve, dorsal root ganglion, and ventral horn motorneurons, indicating that transport of the replicon RNA from the periphery to CNS had occurred. GFP expression was found in the muscles and sciatic nerve as early as 6 h after injection of replicons or replicon RNA, even after sciatic nerve section. Analysis at longer times postinjection revealed GFP expression similar to 6 h levels in the cut sciatic nerves and robust expression in the nerves of uncut animals. The infection and expression of GFP in the CNS following intramuscular inoculation of encapsidated replicons encoding GFP occurred in juvenile or adult animals. The expression of GFP in the CNS of juvenile animals was more intense and lasted for up to 5 weeks, in contrast to the duration of expression of approximately 96 h for adult animals. The results of these studies establish that poliovirus replicon RNA is expressed locally within the sciatic nerve and transported from the periphery to the CNS via axonal transport and support the potential of replicons for gene delivery to the CNS.
脊髓灰质炎病毒经肌肉注射后向中枢神经系统（CNS）扩散的情况已在人类、猴子以及转人脊髓灰质炎病毒受体基因的小鼠中得到证实。脊髓灰质炎病毒的扩散被认为是由于其感染外周神经，并通过轴突逆向运输至神经元细胞体，在那里病毒最终脱壳并随后进行翻译/复制。在先前的研究中，我们已经表明基于脊髓灰质炎病毒的载体（复制子）可用于向中枢神经系统的运动神经元进行基因传递。使用编码绿色荧光蛋白（GFP）的复制子，我们发现经鞘内注射后，GFP表达局限于脊髓的运动神经元。为了进一步表征脊髓灰质炎病毒在外周和中枢神经系统中的基因表达，我们给转脊髓灰质炎病毒受体基因的小鼠肌肉注射了编码GFP的脊髓灰质炎病毒复制子。肌肉注射后，在肌肉、坐骨神经、背根神经节和腹角运动神经元中均证实了GFP的表达。肌肉注射编码GFP的复制子后，小鼠没有出现麻痹或行为异常的迹象。将单独的复制子RNA（裸RNA）注射到不表达脊髓灰质炎病毒受体的转基因小鼠或大鼠的肌肉中，也导致GFP在肌肉、坐骨神经、背根神经节和腹角运动神经元中表达，这表明复制子RNA已从外周运输至中枢神经系统。早在注射复制子或复制子RNA后六小时，甚至在坐骨神经切断后，在肌肉和坐骨神经中就发现了GFP表达。注射后更长时间的分析显示，切断的坐骨神经中GFP表达水平与六小时时相似，而未切断神经的动物神经中GFP表达强烈。在幼年或成年动物中，肌肉注射封装的编码GFP的复制子后，中枢神经系统中出现了GFP的感染和表达。与成年动物约96小时的表达持续时间相比，幼年动物中枢神经系统中GFP的表达更强烈，持续长达5周。这些研究结果表明，脊髓灰质炎病毒复制子RNA在坐骨神经内局部表达，并通过轴突运输从外周运输至中枢神经系统，支持了复制子向中枢神经系统进行基因传递的潜力。
