“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 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.

Long-term labeling and imaging of synaptically connected neuronal networks in vivo using double-deletion-mutant rabies viruses

“…In previous work, we used longitudinal two-photon microscopy to show that neurons labeled with a second-generation (∆GL) RV stayed alive for as long as we imaged them and also that it did not appear to perturb their visual response properties 4 . Because we had only used the Cre-expressing versions of ∆G and ∆GL viruses for the longitudinal two-photon imaging work from our previous publications 4 , 11 , we first conducted a similar set of experiments using the Flpo-expressing RV∆GL and confirmed that it was similarly nontoxic (Supplementary Fig. 1 ).…”

“…More recently, it was reported that adding a destabilization domain to the C terminus of the viral nucleoprotein rendered the virus nontoxic, allowing monosynaptic tracing ‘with no adverse effects on neural physiology, circuit function and circuit-dependent computations’ 10 . We have since shown that those results were probably obtained using reversion mutants that had lost the intended C-terminal addition 11 , although we also showed that the technique may be salvageable 11 , and the authors of the original study have persevered with their approach 12 . From our own laboratory, we have introduced second-generation (∆GL) rabies viral vectors, which have the viral polymerase gene L (for large protein; NCBI gene symbol RABVgp5) deleted along with G, and showed that they do not appear to perturb the structure or function of labeled neurons 4 .…”

“…EnvA-enveloped ∆G RVs were made as described previously 11 , 65 , 66 , using genome plasmids pRV∆G-4Flpo (Addgene, 122050) and pRV∆G-4Cre (Addgene, 98034).…”

“…Recombinase-expressing RVs were titered primarily on the reporter cell lines 293T-FLEX-BC, 293T-F14F15S-BC, 293T-FLEX-BC-TVA and 293T-F14F15S-BC-TVA as described 11 but using threefold serial dilutions, instead of tenfold dilutions, for higher precision. For the corticostriatal experiment, to directly compare viral titers in a way that did not depend on the efficacy of the encoded recombinases, we made the BHK-B19L-TVA950 cell line (described above), which expresses both TVA, to allow infection by EnvA-enveloped viruses, and L, to allow intracellular replication of ∆GL viruses so that infected cells could be clearly labeled using immunostaining against the RV nucleoprotein.…”

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA andin situsequencing

“…We injected AAV2-retro-synP-mCre (Jin et al 2023) into the dorsal striatum, and two helper viruses, AAV1-synP-FLEX-splitTVA-EGFP-tTA and AAV1-TREtight-mTagBFP2-B19G (Liu et al 2017), into the somatosensory cortex. This combination allows expression of TVA and the rabies glycoprotein in corticostriatal neurons (Jin et al 2023). After seven days, we injected a barcoded EnvA-pseudotyped ΔG rabies virus library into the somatosensory cortex.…”