Genomic stability of self-inactivating rabies

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.

Keywords: genetics; genomics; mouse; neural circuits; neuronal tracing; neuroscience; rabies; viral vector.

Figure 1.. SiR production from cDNA leads to revertant-free viral preparations.

(A) Scheme of experimental strategy to identify the emergence of “revertant” mutations during SiR production. 8 independent SiR preparations were rescued from cDNA and genomic RNA were extracted, treated with DNAse I, subjected to RT-PCR to amplify N-TEVs-PEST coding sequence and used to generate libraries for Sanger sequencing (50 clones per preparation were sequenced). (B) Example of sequencing results from one SiR preparation showing no mutations at the end of N. Symbols (#) show the position of previously identified mutations, marks on the sequences indicates the presence of mutations in different positions.

Figure 2.. High TEVp activity in packaging cells prevents accumulation of PEST-mutations.

(A) HEK-TGG packaging cells were amplified for several passages in absence or presence (1 or 2 μg/ml) of puromycin selection. (B) TEVp-dependent cleavage of TEVp-activity reporter was analysed by western blot in HEK-TGG at different amplification passages. (C) Quantification of TEVp-activity in packaging cells over time in presence or absence of antibiotic pressure. (mean ± SEM, n=3) (D) Experimental design to assess emergence of mutations in SiR preparations after multiple passages of amplification in high TEVp (HEK-TGG P0) or low TEVp HEK-TGG (HEK-TGG P8, without puromycin selection). (E) Quantification of frequency of the accumulation of PEST-targeting mutations over time that prevent translation of PEST domain (mean ± SEM, n=4 independent viral preparation). (F) Summary of the single nucleotide polymorphisms (SNPs) in the coding sequence (CDS) of N-TEVsPEST that reached threshold at P8 (mean ± SEM, n=4; n.d. indicates that the mutations were not detected above threshold). Top scheme shows the position of PEST-inactivating mutations.

Figure 2—figure supplement 1.. Western blots to test TEVp in packaging cells over time.

(A) Scheme of the TEVp activity reporter. (B) Original western blots stained with an anti-V5 antibody with the representative lanes used to generate Figure 2B.

Figure 2—figure supplement 2.. SMRT sequencing of SiR genomic libraries.

Scheme of the strategy to sequence SiR preparations using SMRT NGS technology from Pacbio. Amplicons of the entire coding sequence of N-TEVs-PEST gene are generated by RT-PCR. Unique Molecular Identifier (UMI) of 10 nucleotides is added during retrotranscription to each genomic molecule and sample specific barcodes of 16 nucleotides are added at the two ends during subsequent PCR. SMRT bell libraries are generated by ligating the provided adapters to generate circular DNA molecules that are sequenced continuously for multiple passages. Subreads are used to generate high-fidelity consensus sequences that are demultiplexed using the 16 nt barcodes, deduplicated using the UMIs and aligned to the reference for variant calling.

Figure 3.. Revertant-free SiR, but not PEST-mutant, is non-toxic and does not accumulate PEST-targeting mutations in vivo.

(A) Scheme of the engineered PEST-mutant SiR (SiR-G453X). (B) Experimental procedure. (C) Confocal images of hippocampal sections of Rosa26^LSL-tdTomato mice infected with SiR-CRE, Rab-CRE, SiR-G453X and imaged at 1 week, 1 month and 2 months p.i. Scale bar, 50 μm. (D) Number of tdTomato positive neurons at 1 week, 1 months, and 2 months p.i. normalized to 1 week time point (mean ± SEM, n=4 animals per virus per time point). (E) Experimental procedure for the sequencing of SiR particles from injected hippocampi at 1 week p.i. (F) List of PEST-inactivating mutations above 2% thresholds with relative frequency in each animal (n.d. indicates that the mutation was not detected above threshold; n=3 animals).

Figure 3—figure supplement 1.. SiR revertants lose functional TEVs and PEST domain.

(A) The conditional destabilization of N can be prevented by TEVp expression in the infected cells leading to cleavage of the TEVs-containing linker. (B) Engineered revertant SiR viruses containing the reporter PEST-inactivating substitutions in their cDNA. (C) Confocal images of HEK and HEK-TEVp at 48 hrs p.i. All images were acquired with same settings. Bottom panels have been equally adjusted in brightness in all conditions. Scale bar 100 μm.

Figure 3—figure supplement 2.. SiR RNA in injected hippocampi.

(A) Schematic of SiR-CRE injection in the hippocampus mice followed by total RNA extraction and RT-qPCR. (B) Levels of viral RNA normalized to 1 week RNA level (mean ± SEM, n=4 animals per time point).

Figure 4.. 2-photon in vivo longitudinal imaging of revertant-free SiR-infected cortical neurons reveals no toxicity and unaltered neuronal morphology after 5 months.

(A) Schematic of SiR-CRE or Rab-CRE injection in Rosa26^LSL-tdTomato mice in V1 followed by in vivo imaging. (B) Two-photon maximal projection of the same field in SiR-CRE and RabCRE injected cortices at 1, 4, and 21 weeks p.i. or 1, 4, and 9 weeks, respectively. Red arrowheads mark tdTomato positive neurons detected at 1 week that disappear in later recordings. Scale bar 50 μm. (C) Survival of the tdTomato-positive cells recorded at 1 week over time. (ROIs = 6 per virus. n=2 animals per virus). (D) Two-photon maximal projection of the same large field in SiR-CRE injected cortices at 1 week and 21 weeks p.i. Scale bar 50 μm.

Figure 4—figure supplement 1.. Two-photon in vivo longitudinal imaging of revertant-free SiR-infected cortical neurons.

Two-photon maximal projection of the same fields in SiR-CRE injected cortices at 1-2-3-4-21 weeks p.i.

Figure 5.. SiR vectors transsynaptic tracing of neural circuits in the central nervous system.

(A) Experimental design for the transsynaptic tracing of NAc inputs using EnvA-pseudotyped SiR-CRE or SiR-G453X-CRE in Rosa26^LSL-tdTomato mice. (B) Confocal images of BLA area of Rosa26^LSL-tdTomato mice infected with SiR-CRE or SiR-G453X-CRE. Arrows point to tdTomato⁺ microglia. (C) Number of tdTomato-positive neurons in the BLA at 1 month post SiR injection (mean ± SEM, n=4 animals per condition). (E) Number of tdTomato⁺ neurons in the BLA at 1 month post SiR injection (mean ± SEM, n=3 animals per condition). (F) Confocal images of BLA area of Rosa26^LSL-tdTomato mice infected with SiR-CRE or SiR-N2c-CRE. Scale bar, 100 μm.