"Self-inactivating" rabies viruses are susceptible to loss of their intended attenuating modification

Abstract

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 development of a noncytotoxic version - a major advance - based on attenuating the rabies virus by 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 papers 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 three weeks postinjection. We conclude that the new approach is not robust but that it could become a viable technique given further optimization and validation. SIGNIFICANCE STATEMENTRabies 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.