Genomic stability of self-inactivating rabies
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The authors previously developed a tool with the goal of non-toxic trans-synaptic tracing using a modified rabies virus, an important goal for the neuroscience field. The tool has the propensity to accumulate mutations over time that promote toxicity, and the manuscript here describes techniques to avoid these mutations. It remains important to show that the non-mutated virus can serve as an effective trans-synaptic tracing tool.
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Abstract
Transsynaptic viral vectors provide means to gain genetic access to neurons based on synaptic connectivity and are essential tools for the dissection of neural circuit function. Among them, the retrograde monosynaptic ΔG-Rabies has been widely used in neuroscience research. A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits. However, the high mutational rate of the rabies virus poses a risk that mutations targeting the key genetic regulatory element in the SiR genome could emerge and revert it to a canonical ΔG-Rabies. Such revertant mutations have recently been identified in a SiR batch. To address the origin, incidence and relevance of these mutations, we investigated the genomic stability of SiR in vitro and in vivo. We found that “revertant” mutations are rare and accumulate only when SiR is extensively amplified in vitro, particularly in suboptimal production cell lines that have insufficient levels of TEV protease activity. Moreover, we confirmed that SiR-CRE, unlike canonical ΔG-Rab-CRE or revertant-SiR-CRE, is non-toxic and that revertant mutations do not emerge in vivo during long-term experiments.
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Author Response
Reviewer #1 (Public Review):
This manuscript describes conditions under which "Self-inactivating Rabies" (SiR) can be grown to limit mutations that would allow the virus to replicate in the absence of TEV protease. It is also shown that neurons directly infected with a non-mutated virus remain healthy and that the virus does not mutate in the brain in vivo. Remarkably there is nothing in the manuscript to address the obvious question that is raised by the observation that such mutations were occurring around the time of the initial description of circuit tracing with this virus. Can the transsynaptic tracing experiments in the absence of TEV expression (as described in their original Neuron paper) be replicated with SiR that is not mutated? This obvious omission suggests that the authors might have conducted such …
Author Response
Reviewer #1 (Public Review):
This manuscript describes conditions under which "Self-inactivating Rabies" (SiR) can be grown to limit mutations that would allow the virus to replicate in the absence of TEV protease. It is also shown that neurons directly infected with a non-mutated virus remain healthy and that the virus does not mutate in the brain in vivo. Remarkably there is nothing in the manuscript to address the obvious question that is raised by the observation that such mutations were occurring around the time of the initial description of circuit tracing with this virus. Can the transsynaptic tracing experiments in the absence of TEV expression (as described in their original Neuron paper) be replicated with SiR that is not mutated? This obvious omission suggests that the authors might have conducted such experiments and were unable to replicate their published results. It is imperative that the authors be forthcoming about whether they have conducted such experiments and what were the results. If they have not conducted such experiments, they should do them and include the results here. Regardless of the outcome, the results should be published. If they cannot replicate their results, then the reliability of the Neuron paper is in doubt.
How do the results presented here relate to the results published in the Neuron paper and why are they not definitive with respect to the utility of SiR? The original publication in Neuron presents results that do not appear to be plausible and are best explained by the possibility that some experiments described in that manuscript were conducted using mutated SiR. This became most apparent when shortly after the Neuron publication, the Tripodi lab shared SiR as well as TEV expressing cell lines for propagation with other labs. Several of those groups observed that when they progagated the SiR received from the Tripodi lab, there was a mutation that removed the linkage of the PEST targeting sequence to N. This would be expected to allow the virus to replicate and spread without the need for TEV protease to remove the PEST sequence - precisely the phenotype observed in the trans-synaptic tracing experiments described in the Neuron paper. In the Neuron paper, culture experiments showed that the N-PEST (SiR) rabies could not replicate in the absence of TEV. And additional experiments showed that the virus is not toxic to neurons directly infected. These are the same experiments that are replicated in this submission. But then (in the Neuron paper) comes the unlikely report that this virus can spread trans-synaptically in vivo, in the absence of TEV expression. An alternative explanation would be that the virus used for those experiments was mutated and that is why TEV expression was not needed. There are no experiments in the original Neuron paper that address this possibility. Specifically, the experiments in Neuron describing cell survival during trans-synaptic tracing are not adequate to rule this out. This is because the two timepoints during which neurons were counted correspond to an early time when labeled neurons would be expected to still be accumulating and a later time that might be past the peak and represent a time when many neurons have died. To quantify proportions of neurons that survive, it is necessary to follow the same neurons over time, as has been done to demonstrate that only about half of neurons infected with G-deleted rabies die (half survive). Until tests are conducted testing whether TEV expression is required to obtain trans-synaptic labeling with an SiR that is known to not be mutated, it is irrelevant whether mutations can be prevented under particular culture conditions. The utility of this virus depends on whether it can be used for trans-synaptic tracing without toxicity and this manuscript presents no experiments to address that. Further, the omission of such experiments is glaring, as it is difficult to imagine that they have not been attempted.
We thank the reviewer for giving us the opportunity to improve on this point. We have performed additional experiments to confirm the ability of revertant-free SiR virus to spread transsynaptically in vivo. Our data shows that non-mutated SiR spreads transsynpatically in the mouse brain when complemented with G. In addition, we also tested the effect of the addition of TEVp to the starter neuronal population and found that it can significantly improve spreading efficiency. These data confirm the transsynaptic spreading capabilities of unmutated SiR in line with our original report. Furthermore, the data show the enhancing effect on the spreading efficacy of supplementing TEVp to the starter cells, broadly in line with what was recently reported by Jin et al., 2023. We have discussed the implications of these findings and suggested future directions in the main text and discussion.
Additionally, for completeness, we also assessed the spread efficiency of the recently generated SiR-N2c (based on the CVS-N2c rabies strain) in presence and absence of TEVp. We found that SiR-N2c spreads significantly better in the BLA-> NAc circuit than the original SiR (based on the SAD-B19 strain), and that the same spreading efficiency is not achieved by complementing SiR-B19 with the G from CVS_N2c Rabies strain. Interestingly, we found only a very small effect of the addition TEVp to the starting cells on the number of transsynaptically labelled cells with SiR-N2c. We have discussed the implications of these findings in the main text and discussion.
Changes in the manuscript: We have updated Figure 1 with the addition of a 6-month time point and update the main text accordingly. The updated paragraph is provided here:
"Results, SiR transsynaptic spreading.
We then tested the ability of revertant-free SiR to trace neural circuits transsynaptically in the mouse brain. ΔG-Rabies vectors can be pseudotyped with the chimeric EnvA glycoprotein to selectively infect neurons expressing the TVA receptor, which is not endogenously expressed by mammalian cells (Wickersham et al., 2007b). We injected the nucleus accumbens (NAc) of CRE-dependent tdTomato reporter mice with an AAV expressing either TVA and the rabies G or TVA only. After 3 weeks, we re-injected the NAc with EnvA-pseudotyped revertant-free SiR-CRE or EnvA-pseudotyped SiR-G453X-CRE and assessed the CRE-dependent tdTomato expression presynaptically, in the basolateral amygdala (BLA). At 1 month post SiR injection, we detected no tdTomato+ cells in the BLA in TVA-only-injected animals, confirming the G-dependency for SiR transsynaptic spreading (Fig 5B-C). In contrast, as expected, transsynaptic spreading was apparent in the TVA+G condition. We observed similar numbers of presynaptically traced neurons in both SiR-CRE and SiR-G453X-CRE injected brains (169 ± 24 and 190 ± 36 tdTomato+ neurons, respectively; two-tailed t-test, P = 0.64; Fig 5B-C). However, tdTomato+ microglial cells were only detected in the SiR-G453X-CRE condition indicating the re-emergence of toxicity of the revertant mutants (Fig 5B). We also tested the effect of supplying TEV protease to the starting cells, as this has been suggested to be a necessary step to ensure transsynapitc spreading. While the previous experiments unambiguously show that TEVp is not necessary for the transsynaptic spreading of SiR, the injection of an AAV expressing TEVp in the NAc did lead to an increase in the number of transsynaptically labelled BLA neurons (366 ± 69 tdTomato+ neurons; two-tailed t-test, P = 0.04; Fig 5C), indicating that TEVp-dependent SiR reactivation in starter cells can improve its spreading (Jin et al., 2023).
We recently showed that a novel SiR-N2c vector, derived from the neurotropic CVS-N2c Rabies strain, displays enhanced transsynaptic spreading and improved peripheral neurotropism over the original SAD B19-derived SiR (Lee et al., 2023). Hence, for completeness, we compared the transynaptic spreading efficacty of EnvA-pseudotyped revertant-free SiR-N2c and the original SiR. SiR-N2c labelled a greater number of BLA neurons at 1 month p.i. than what was detected with SiR (1691 ± 112 tdTomato+ neurons traced by SiR-N2c; two-tailed t-test, P = 2x105; Fig 5D-E). Additionally, TEVp expression in the starter cells in SiR-N2c tracing experiments had a negligible effect on the overall transsynaptic spreading (1934 ± 135 tdTomato+ neurons traced by SiR-N2c in presence of TEVp; two-tailed t-test, P = 0.24; Fig 5D-E). Since the use of G from the CVS-N2c Rabies strain (G_N2c) has been shown to improve ΔG-Rabies (SAD-B19) retrograde tracing (Zhu et al., 2020), we tested if complementing EnvA-pseudotyped SiR with G_N2c in the NAc could increase its spreading. While we detected more BLA tdTomato+ neurons than in our previous experiments, complementing SiR with G_N2c still labelled less neurons than SiR-N2c, even when TEVp was provided to the starter cells (487 ± 164 and 844 ± 14 tdTomato+ neurons traced by SiR in absence or presence of TEVp, respectively; Fig 5D-E)."
Discussion
"ΔG-Rabies vectors are powerful tools for the dissection of neural circuit organization thanks to their ability to spread retrogradely to synpatically-connected neurons. Here, we show that EnvA-pseudotyped revertant-free SiR vectors effectively spread transsynpatically in the mouse brain. Importantly, the co-delivery of an AAV expressing TEVp in addition to G increase the number of traced neurons in presynaptic areas, likely due to the TEVp-dependent reactivation of SiR in vivo (Ciabatti et al., 2017), in line with recent results (Jin et al., 2023). This should be considered when planning transsynaptic tracing experiments using SiR. To improve SiR spreading efficiency, further studies should investigate the use of inducible TEVp, as we previously showed (Ciabatti et al., 2017), that could maximise spreading efficiency while minimising possible side effects of prolonged protease expression.
Interestingly, we found that the recently developed SiR-N2c vector, generated by applying the same proteasome-targeting modification to the genome of the CVS-N2c ΔG-Rabies strain (Lee et al., 2023), show a higher number of retrogradely labelled neurons compared to the original SiR (SAD-B19) (Fig 5). Additionally, the co-delivery of TEVp had a smaller effect on the number of neurons transsynaptically-traced by SiR-N2c. Interestingly, the gap in trassynaptic spreading efficacy between SiR (SAD-B19) and SiR-N2c could not be filled by complementing the SiR with the neurotropic G_N2c. This could be linked to a more efficient packaging of SiR-N2c by G_N2c (Reardon et al., 2016; Sumser et al., 2022) or by the particularly high speed of CVS-N2c strain propagation (~12hrs)(Callaway, 2008; Hoshi et al., 2005). These results point to SiR-N2c as the vector of choice for transsynaptic experiments."
Other comments:
"A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, represents the first tool for open-ended genetic manipulation of neural circuits." It is not clear what the authors intend to be claiming with respect to "open-ended genetic manipulation of neural circuits" but it is clear that this assertion is overblown. There are numerous tools that are available for genetic manipulation of neural circuits. This is not the first, won't be the last, and it is arguably not the best.
We have rephrased this sentence.
Changes in the manuscript: The updated paragraph and figure panel is provided here:
Abstract
"A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, allows the long term genetic manipulation of neural circuits."
"Interestingly, a fraction of tdTomato+ neurons survived in ΔG- Rab-CRE-injected brains, differing from what we observed when injecting ΔGRab-GFP, where no cells were detected at 3 weeks p.i. (Fig 3CD) (Ciabatti et al., 2017). " This is a known result (same as Chatterjee et al., 2018) with a known mechanism. GFP expression is not observed because the rabies virus transitions from transcription to replication resulting in the termination of GFP expression. But Cre-recombination of the genome permanently labels cells with TdTomato. This is how Chatterjee et al. demonstrated that half of the neurons infected with G-deleted rabies survive. They imaged cells and saw that the GFP disappeared but the cells marked by Cre-recombination and RFP expression remained healthy indefinitely. The consideration of this in the Introduction is strange. There is no reason to suppose that Cre expression would somehow protect cells from rabies infection and there is no need to propose any such mechanism to explain the observed results.
This consideration is a response to the suggestion, proposed in Matsuyama et al 2019, that the toxicity reduction observed in ΔG-Rab-CRE could be linked to the expression of Cre recombinase compared to a cytosolic protein.
"Here we show that revertant-free SiR-CRE efficiently traces neurons in vivo without toxicity in cortical and subcortical regions for several months p.i.."
This wording is disingenuous and appears to be intentionally misleading. "Trace" implies that circuits were traced by transynaptic labeling, which they were not.
To avoid any misunderstanding, we have now changed trace to infect.
Changes in the manuscript: The updated sentence is provided here:
Abstract
"Here we show that revertant-free SiR-CRE efficiently infect neurons in vivo without toxicity in cortical and subcortical regions for several months p.i.."
Reviewer #2 (Public Review):
The study by Ciabatti et al examined the mutation issue for self-inactivating rabies (SiR), which was found by other labs. The authors identified the mutations in the rabies genome and showed that this mutation occurred more frequently after multiple passage of production cell lines with suboptimal TEVp expressions. The authors further showed that such mutation did not accumulate in vivo and that SiR-labeled cells remained alive across longitudinal imaging in vivo.
In this study, the rabies genome is rigorously examined by sequencing many viral particles from independent preparations. The rabies with point mutation in the PEST domain is directly engineered for sequencing and infection test. Overall, the mutation issue is well addressed by the authors and the conclusions are well supported, but some more aspects of discussion and data analysis need to be extended for an easier production of SiR in a condition not that optimal.
- The authors stated that one should produce SiR from cDNA in order to avoid the potential mutation in SiR. From a practical point of view, it would be much better to amplify the rabies from a stock virus directly in the production cell lines. Any discussion or exploration on this direction would be appreciated in the field.
We thank the reviewer for giving us the opportunity to improve on this point. We have added in the discussion a paragraph suggesting the number of passages to be used during production for the packaging cells and viral stocks, referring to the equivalent passage in our experiments.
Changes in the manuscript: The updated paragraph is provided here:
Discussion
"Notably, we found that TEVp activity inevitably decreases after several passages of amplification of HEK-TTG, thus fresh low passage packaging cells should always be used to produce SiR preparations. Our results suggest that stock for packaging cells should be made within a couple of passage after selection is established, and then used freshly defrosted to produce SiR viruses (equivalent to P0 cells in Fig 2B-C). Similarly, SiR supernatant stocks should be made directly from cDNA transfection and amplified for a maximum of 2 passages (equivalent to SiR P0 in Fig 2E) before being used for large scale SiR productions."
- 6 passages of production cell lines are not that extensive. In Fig.2C, there was already some level of TEVp activity reduction at 2nd passage. It is not clear to me that how the TEVp activity reduction naturally happens. Is there some room to play around puromycin concentration to maintain high TEVp activity?
As mentioned in the previous point, we have added in the discussion a paragraph describing the recommended number of passages to be used during production of the packaging cells and viral stocks, referring to the equivalent passage in our experiments. We clarified that our starting P0 conditions for packaging cells and stock SiR viruses were equivalent to already amplified stocks ready for viral production, which would add only 1-2 passages.
Reviewer #3 (Public Review):
This paper is a response to the report by Lin et al., bioRxiv 2022 (DOI: https://doi.org/10.1101/550640) that mutations in the genome of SiR were identified, which could result in a canonical G-deleted Rabies virus.
Strengths:
First, the authors found that SiR production from cDNA leads to revertant-free viruses by analyzing a total of 400 individual viral particles obtained from 8 independent viral productions with Sanger sequencing. Next, they identified the molecular mechanisms of mutations in the SiR; they found that extensive amplification of packaging cells HEK-TGG leads to the selection of clones with suboptimal TEVp expression level, which leads to the accumulation of revertant mutants, where, as the authors discuss, the revertant mutants have a specific replication advantage. Based on these observations, the authors recommend producing SiR freshly from cDNA with low passage packaging cells. Lastly, the authors observed that SiR-infected hippocampal and cortical neurons can survive for longer periods of time than the neurons infected with revertant mutants or a canonical G-deleted Rabies virus by combining next-generation sequencing of RNAs isolated from infected tissue and 2-photon in vivo longitudinal imaging of infected cortical neurons. Together, these findings support the idea that the degradation of N by PEST-mediated cellular mechanism results in the self-inactivation of SiR as suggested in the original SiR manuscript (Ciabatti et al., Cell 2017). Thus, SiR remains a powerful viral tool for the chronic investigation of neuronal circuitry and function as long as the virus is prepared in a way the authors recommend.
Weaknesses:
While most of the findings are solid, some conclusions are not fully supported by the data presented. The authors need to address the following points: Reviewer #3
- In Figure 3B-D, the authors concluded that SiR-CRE -infected cells did not show cell death in contrast to Rab-CRE and SiR-G453X, but it cannot be fully supported only by this experiment. The authors should consider the potential variance in infection efficiency in each experimental animal and show evidence of suppressed cell death. In addition, it needs to be confirmed that SiR-Cre is diminished in infected cells at later times. The authors should explain and address these concerns by conducting additional experiments, for example, cleaved caspase-3 staining and quantification of virus RNA levels in each time point as performed in their previous study Ciabatti et al., Cell 2017 (DOI: 10.1016/j.cell.2017.06.014).
We thank the reviewer for the suggestion and give the opportunity to strengthen our work. We have added an analysis of the rabies transcripts over time in SiR-infected hippocampi (Fig S4). The drastic decrease of SiR RNA, along with the finding that the numbers of tdTomato-positive cells remain comparable at each time points support the reduction in mortality in SiR infected cells. We have added this data and clarified this point in the text..
Changes in the manuscript: The updated paragraph is provided here:
Results: Difference in cytotoxicity between ΔG-Rabies, PEST-mutant SiR and SiR
"We detected no decrease of tdTomato+ neurons in SiR-infected hippocampi (4109 ± 266 tdTomato+ neurons at 1 week p.i.; 4458 ± 739 tdTomato+ neurons at 2 months p.i.; one-way ANOVA, F = 0.08, p = 0.92, Fig 3C-D) while only 44% of tdTomato+ neurons were detected in Rabies-targeted and 60% in SiR-G453X-targeted hippocampi at 2 months p.i. (1422 ± 184 at 1 week versus 624 ± 114 at 2 months p.i. for ΔGRab; one-way ANOVA, F = 11.55, p = 0.003; 3052+508 at 1 week versus 1829+198 at 2 months p.i. for SiR-G453X; one-way ANOVA, F = 4.27, p = 0.05; Fig 3C-D). Additionally, we confirmed inactivation of revertant-free SiR by analysing the decrease of Rabies transcripts in the infected hippocampi over times (Fig S4). These results support the lack of toxicity of SiR on the infected neurons, in line with our previous findings (Ciabatti et al., 2017). Moreover, these data confirm the requirement for an intact PEST sequence to sustain the self-inactivating behaviour of SiR and suggest that PEST-targeting mutations do not occur in vivo."
- In Figure 3E-F, to ensure the long-term stability of SiR-Cre in the vivo mouse brain, authors conducted SMRT sequencing 1 week after the virus infection. To test the potential slow accumulation of mutations at 1-month and 2-month, the authors should perform the same experiment at these time points. Only when SiR-Cre was undetected at 1-month and 2-month, would it be reasonable to show only 1-week data, however, such data is not presented.
We thank the reviewer for the suggestion. We have added an analysis of the Rabies transcript in the infected Hippocampi showing a drastic decrease of SiR RNA over time. This result, along with the finding that similar numbers of tdTomato-positive cells are detected in the infected hippocampi over time, support our choice of an early time point to find emergence and accumulation of revertant mutations.
- In figure 4, the authors used only 2 mice for this experiment, although this is one of the most important experiments to ensure SiR-infected cells stay alive for the long term in vivo animals. It should be confirmed whether the conclusion remains the same by increasing the number of animals.
While we understand why the reviewer put forward this suggestion, we believe that our choice of number of animals is appropriate as the investment in time and resources to adding further animals would not strengthen our conclusion (which we have indirectly assessed previously (Ciabatti et al 2017) and here in Fig 3). For completeness, we have added a Fig4_S1with the images of all the ROI at every time points used in Fig 4.
- The legend in Table 3 doesn't match the contents.
We thank the reviewer for pointing this out, in response we have now updated Table 3.
-
eLife assessment
The authors previously developed a tool with the goal of non-toxic trans-synaptic tracing using a modified rabies virus, an important goal for the neuroscience field. The tool has the propensity to accumulate mutations over time that promote toxicity, and the manuscript here describes techniques to avoid these mutations. It remains important to show that the non-mutated virus can serve as an effective trans-synaptic tracing tool.
-
Reviewer #1 (Public Review):
This manuscript describes conditions under which "Self-inactivating Rabies" (SiR) can be grown to limit mutations that would allow the virus to replicate in the absence of TEV protease. It is also shown that neurons directly infected with a non-mutated virus remain healthy and that the virus does not mutate in the brain in vivo. Remarkably there is nothing in the manuscript to address the obvious question that is raised by the observation that such mutations were occurring around the time of the initial description of circuit tracing with this virus. Can the transsynaptic tracing experiments in the absence of TEV expression (as described in their original Neuron paper) be replicated with SiR that is not mutated? This obvious omission suggests that the authors might have conducted such experiments and were …
Reviewer #1 (Public Review):
This manuscript describes conditions under which "Self-inactivating Rabies" (SiR) can be grown to limit mutations that would allow the virus to replicate in the absence of TEV protease. It is also shown that neurons directly infected with a non-mutated virus remain healthy and that the virus does not mutate in the brain in vivo. Remarkably there is nothing in the manuscript to address the obvious question that is raised by the observation that such mutations were occurring around the time of the initial description of circuit tracing with this virus. Can the transsynaptic tracing experiments in the absence of TEV expression (as described in their original Neuron paper) be replicated with SiR that is not mutated? This obvious omission suggests that the authors might have conducted such experiments and were unable to replicate their published results. It is imperative that the authors be forthcoming about whether they have conducted such experiments and what were the results. If they have not conducted such experiments, they should do them and include the results here. If they cannot replicate their results, then the reliability of the Neuron paper is in doubt.
How do the results presented here relate to the results published in the Neuron paper and why are they not definitive with respect to the utility of SiR? The original publication in Neuron presents results that do not appear to be plausible and are best explained by the possibility that some experiments described in that manuscript were conducted using mutated SiR. This became most apparent when shortly after the Neuron publication, the Tripodi lab shared SiR as well as TEV expressing cell lines for propagation with other labs. Several of those groups observed that when they progagated the SiR received from the Tripodi lab, there was a mutation that removed the linkage of the PEST targeting sequence to N. This would be expected to allow the virus to replicate and spread without the need for TEV protease to remove the PEST sequence - precisely the phenotype observed in the trans-synaptic tracing experiments described in the Neuron paper. In the Neuron paper, culture experiments showed that the N-PEST (SiR) rabies could not replicate in the absence of TEV. And additional experiments showed that the virus is not toxic to neurons directly infected. These are the same experiments that are replicated in this submission. But then (in the Neuron paper) comes the unlikely report that this virus can spread trans-synaptically in vivo, in the absence of TEV expression. An alternative explanation would be that the virus used for those experiments was mutated and that is why TEV expression was not needed. There are no experiments in the original Neuron paper that address this possibility. Specifically, the experiments in Neuron describing cell survival during trans-synaptic tracing are not adequate to rule this out. This is because the two timepoints during which neurons were counted correspond to an early time when labeled neurons would be expected to still be accumulating and a later time that might be past the peak and represent a time when many neurons have died. To quantify proportions of neurons that survive, it is necessary to follow the same neurons over time, as has been done to demonstrate that only about half of neurons infected with G-deleted rabies die (half survive). Until tests are conducted testing whether TEV expression is required to obtain trans-synaptic labeling with an SiR that is known to not be mutated, it is irrelevant whether mutations can be prevented under particular culture conditions. The utility of this virus depends on whether it can be used for trans-synaptic tracing without toxicity and this manuscript presents no experiments to address that. Further, the omission of such experiments is glaring, as it is difficult to imagine that they have not been attempted.
Other comments:
"A recently developed engineered version of the ΔG-Rabies, the non-toxic self-inactivating (SiR) virus, represents the first tool for open-ended genetic manipulation of neural circuits."
It is not clear what the authors intend to be claiming with respect to "open-ended genetic manipulation of neural circuits" but it is clear that this assertion is overblown. There are numerous tools that are available for genetic manipulation of neural circuits. This is not the first, won't be the last, and it is arguably not the best."Interestingly, a fraction of tdTomato+ neurons survived in ΔG- Rab-CRE-injected brains, differing from what we observed when injecting ΔGRab-GFP, where no cells were detected at 3 weeks p.i. (Fig 3CD) (Ciabatti et al., 2017). " This is a known result (same as Chatterjee et al., 2018) with a known mechanism. GFP expression is not observed because the rabies virus transitions from transcription to replication resulting in the termination of GFP expression. But Cre-recombination of the genome permanently labels cells with TdTomato. This is how Chatterjee et al. demonstrated that half of the neurons infected with G-deleted rabies survive. They imaged cells and saw that the GFP disappeared but the cells marked by Cre-recombination and RFP expression remained healthy indefinitely. The consideration of this in the Introduction is strange. There is no reason to suppose that Cre expression would somehow protect cells from rabies infection and there is no need to propose any such mechanism to explain the observed results.
"Here we show that revertant-free SiR-CRE efficiently traces neurons in vivo without toxicity in cortical and subcortical regions for several months p.i.."
This wording is disingenuous and appears to be intentionally misleading. "Trace" implies that circuits were traced by transynaptic labeling, which they were not. -
Reviewer #2 (Public Review):
The study by Ciabatti et al examined the mutation issue for self-inactivating rabies (SiR), which was found by other labs. The authors identified the mutations in the rabies genome and showed that this mutation occurred more frequently after multiple passage of production cell lines with suboptimal TEVp expressions. The authors further showed that such mutation did not accumulate in vivo and that SiR-labeled cells remained alive across longitudinal imaging in vivo.
In this study, the rabies genome is rigorously examined by sequencing many viral particles from independent preparations. The rabies with point mutation in the PEST domain is directly engineered for sequencing and infection test. Overall, the mutation issue is well addressed by the authors and the conclusions are well supported, but some more …
Reviewer #2 (Public Review):
The study by Ciabatti et al examined the mutation issue for self-inactivating rabies (SiR), which was found by other labs. The authors identified the mutations in the rabies genome and showed that this mutation occurred more frequently after multiple passage of production cell lines with suboptimal TEVp expressions. The authors further showed that such mutation did not accumulate in vivo and that SiR-labeled cells remained alive across longitudinal imaging in vivo.
In this study, the rabies genome is rigorously examined by sequencing many viral particles from independent preparations. The rabies with point mutation in the PEST domain is directly engineered for sequencing and infection test. Overall, the mutation issue is well addressed by the authors and the conclusions are well supported, but some more aspects of discussion and data analysis need to be extended for an easier production of SiR in a condition not that optimal.
The authors stated that one should produce SiR from cDNA in order to avoid the potential mutation in SiR. From a practical point of view, it would be much better to amplify the rabies from a stock virus directly in the production cell lines. Any discussion or exploration on this direction would be appreciated in the field.
6 passages of production cell lines are not that extensive. In Fig.2C, there was already some level of TEVp activity reduction at 2nd passage. It is not clear to me that how the TEVp activity reduction naturally happens. Is there some room to play around puromycin concentration to maintain high TEVp activity?
-
Reviewer #3 (Public Review):
This paper is a response to the report by Lin et al., bioRxiv 2022 (DOI: https://doi.org/10.1101/550640) that mutations in the genome of SiR were identified, which could result in a canonical G-deleted Rabies virus.
Strengths:
First, the authors found that SiR production from cDNA leads to revertant-free viruses by analyzing a total of 400 individual viral particles obtained from 8 independent viral productions with Sanger sequencing. Next, they identified the molecular mechanisms of mutations in the SiR; they found that extensive amplification of packaging cells HEK-TGG leads to the selection of clones with suboptimal TEVp expression level, which leads to the accumulation of revertant mutants, where, as the authors discuss, the revertant mutants have a specific replication advantage. Based on these …
Reviewer #3 (Public Review):
This paper is a response to the report by Lin et al., bioRxiv 2022 (DOI: https://doi.org/10.1101/550640) that mutations in the genome of SiR were identified, which could result in a canonical G-deleted Rabies virus.
Strengths:
First, the authors found that SiR production from cDNA leads to revertant-free viruses by analyzing a total of 400 individual viral particles obtained from 8 independent viral productions with Sanger sequencing. Next, they identified the molecular mechanisms of mutations in the SiR; they found that extensive amplification of packaging cells HEK-TGG leads to the selection of clones with suboptimal TEVp expression level, which leads to the accumulation of revertant mutants, where, as the authors discuss, the revertant mutants have a specific replication advantage. Based on these observations, the authors recommend producing SiR freshly from cDNA with low passage packaging cells. Lastly, the authors observed that SiR-infected hippocampal and cortical neurons can survive for longer periods of time than the neurons infected with revertant mutants or a canonical G-deleted Rabies virus by combining next-generation sequencing of RNAs isolated from infected tissue and 2-photon in vivo longitudinal imaging of infected cortical neurons. Together, these findings support the idea that the degradation of N by PEST-mediated cellular mechanism results in the self-inactivation of SiR as suggested in the original SiR manuscript (Ciabatti et al., Cell 2017). Thus, SiR remains a powerful viral tool for the chronic investigation of neuronal circuitry and function as long as the virus is prepared in a way the authors recommend.
Weaknesses:
While most of the findings are solid, some conclusions are not fully supported by the data presented. The authors need to address the following points:
1. In Figure 3B-D, the authors concluded that SiR-CRE -infected cells did not show cell death in contrast to Rab-CRE and SiR-G453X, but it cannot be fully supported only by this experiment. The authors should consider the potential variance in infection efficiency in each experimental animal and show evidence of suppressed cell death. In addition, it needs to be confirmed that SiR-Cre is diminished in infected cells at later times. The authors should explain and address these concerns by conducting additional experiments, for example, cleaved caspase-3 staining and quantification of virus RNA levels in each time point as performed in their previous study Ciabatti et al., Cell 2017 (DOI: 10.1016/j.cell.2017.06.014).
2. In Figure 3E-F, to ensure the long-term stability of SiR-Cre in the vivo mouse brain, authors conducted SMRT sequencing 1 week after the virus infection. To test the potential slow accumulation of mutations at 1-month and 2-month, the authors should perform the same experiment at these time points. Only when SiR-Cre was undetected at 1-month and 2-month, would it be reasonable to show only 1-week data, however, such data is not presented.
3. In figure 4, the authors used only 2 mice for this experiment, although this is one of the most important experiments to ensure SiR-infected cells stay alive for the long term in vivo animals. It should be confirmed whether the conclusion remains the same by increasing the number of animals.
4. The legend in Table 3 doesn't match the contents.
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