Abstract
The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.
Highlights
Rabies virus (RV) selectively infects neurons and propagates between synaptically connected neurons in an exclusively retrograde direction [1,2,3,4]
It remains unclear whether the loss of G expression itself causes the change in the transgene expression level, the results indicate that the deletion of G gene has large effects on the RV vector resulting in the alteration of its nature, and makes this vector much suitable for anatomical and physiological studies
The number of cells infected with rHEP5.0-CVSG-monomeric red fluorescent protein (mRFP) increased significantly (P < 0.05, one-way ANOVA; post-hoc ttest with 1 dpi vs 3 dpi and 3 dpi vs 6 dpi, Bonferroni-corrected, all p < 0.016), and almost all cells were infected at 6 days postinfection (Figure 1C)
Summary
Rabies virus (RV) selectively infects neurons and propagates between synaptically connected neurons in an exclusively retrograde direction [1,2,3,4]. We have previously developed a recombinant RV vector that was derived from an avirulent vector (rHEP3.0) based on the HEP-Flury strain [5,6]. This recombinant RV vector (rHEP5.0-CVSG) propagated transsynaptically in a retrograde direction. The morphological features of the infected neurons were clearly visualized by using antibodies against the expressed marker protein, such as green fluorescent protein or β-galactosidase [7]. These features make this propagation-competent RV vector a useful trans-synaptic tracer. By using this viral vector, we have established a dual trans-synaptic tracing method that could be used to identify two different neuronal circuits in the same experiment and have revealed the organization of commissural connectivity in the hippocampus [7,8]
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