"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification.
"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification.
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DOI：
10.1073/pnas.2023481120
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发表时间：
2023-02-14
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
--
作者：
狂犬病病毒在突触连接的神经元之间传播，一直是神经科学家用来揭示大脑组织的主要工具之一。最近有报道称，狂犬病病毒的一种新的修改允许映射连接的神经元，而不会对细胞的健康产生不利影响，这与早期版本不同。在这里，我们表明该研究的结论可能是不正确的，并且基于使用了由于突变而失去预期修饰的病毒。我们还表明，保留预期修饰的狂犬病病毒在先前报道的条件下不会在神经元之间有效传播;然而，它确实在不同条件下在神经元之间传播，这表明如果进一步改进，该方法可能是成功的。狂犬病病毒单突触示踪是神经科学中的一项重要技术，它可以在突触前直接标记神经元。2017年的一篇文章报道了一种无细胞毒性版本的开发-一个主要的进步-基于通过在病毒蛋白的C末端添加不稳定结构域来减弱狂犬病病毒。然而，这种修饰似乎并没有阻碍病毒在神经元之间传播的能力。我们分析了作者提供的两种病毒，并在这里表明，这两种病毒都是失去了预期修饰的突变体，解释了论文的矛盾结果。然后，我们制造了一种病毒，它实际上至少在大多数病毒体中具有预期的修饰，并发现它在原始论文中描述的条件下没有有效地传播，即没有表达外源蛋白酶以去除去稳定化结构域。我们发现，当提供蛋白酶时，它确实扩散，尽管这似乎也导致大多数源细胞在注射后3周死亡。我们的结论是，新的方法是不稳健的，但它可能成为一个可行的技术，进一步优化和验证。
Rabies virus, which spreads between synaptically connected neurons, has been one of the primary tools used by neuroscientists to reveal the organization of the brain. A new modification to rabies virus was recently reported to allow the mapping of connected neurons without adverse effects on the cells' health, unlike earlier versions. Here, we show that the conclusions of that study were probably incorrect and based on having used viruses that had lost the intended modification because of mutations. We also show that a rabies virus that does retain the intended modification does not spread efficiently between neurons under the conditions reported previously; however, it does spread between neurons under different conditions, suggesting that the approach may be successful if refined further. Monosynaptic tracing using rabies virus is an important technique in neuroscience, allowing brain-wide labeling of neurons directly presynaptic to a targeted neuronal population. A 2017 article reported the development of a noncytotoxic version—a major advance—based on attenuating the rabies virus by the addition of a destabilization domain to the C terminus of a viral protein. However, this modification did not appear to hinder the ability of the virus to spread between neurons. We analyzed two viruses provided by the authors and show here that both were mutants that had lost the intended modification, explaining the paper's paradoxical results. We then made a virus that actually did have the intended modification in at least the majority of virions and found that it did not spread efficiently under the conditions described in the original paper, namely, without an exogenous protease being expressed in order to remove the destabilization domain. We found that it did spread when the protease was supplied, although this also appeared to result in the deaths of most source cells by 3 wk postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation.
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