Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression

May Effects of G-gene Deletion and Replacement on Rabies Virus Vector Gene Expression Sho Sato 0 1 Shinya Ohara 0 1 Ken-Ichiro Tsutsui 0 1 Toshio Iijima 0 1 0 Division of Systems Neuroscience, Tohoku University Graduate School of Life Sciences , Sendai , Japan 1 Academic Editor: Matthias Johannes Schnell, Thomas Jefferson University , UNITED STATES The glycoprotein-gene (G gene) -deleted rabies virus (RV) vector is a powerful tool to examine the function and structure of neural circuits. We previously reported that the deletion of the G gene enhances the transgene expression level of the RV vector. However, the mechanism of this enhancement remains to be clarified. We presume that there are two possible factors for this enhancement. The first factor is the glycoprotein of RV, which shows cytotoxicity; thus, may cause a dysfunction in the translation process of infected cells. The second possible factor is the enhanced expression of the L gene, which encodes viral RNA polymerase. In the RV, it is known that the gene expression level is altered depending on the position of the gene. Since G-gene deletion displaces the L gene in the genome, the expression of the L gene and viral transcription may be enhanced. In this study, we compared the transgene expression level and viral transcription of three recombinant RV vectors. The effect of glycoprotein was examined by comparing the viral gene expression of G-gene-intact RV and G-gene-replaced RV. Despite the fact that the L-gene transcription level of these two RV vectors was similar, the G-gene-replaced RV vector showed higher viral transcription and transgene expression level than the G-gene-intact RV vector. To examine the effect of the position of the L gene, we compared the viral gene expression of the G-genedeleted RV and G-gene-replaced RV. The G-gene-deleted RV vector showed higher L-gene transcription, viral transcription, and transgene expression level than the G-genereplaced RV vector. These results indicate that G-gene deletion enhances the transgene expression level through at least two factors, the absence of glycoprotein and enhancement of L-gene expression. These findings enable investigators to design a useful viral vector that shows a controlled desirable transgene expression level in applications. - Funding: This study was supported by Research fund for IIARE doctoral course students from the Institute for International Advanced Research and Education of Tohoku University, and by Grant-in-Aid for Scientific Research (KAKENHI) #24333004 from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The study was partly supported by Grants-in-Aid for Scientific Research on Innovative Areas (#25119001). The funders had no role in this study design, data collection and analysis, decision to publish, or preparation of the manuscript. Rabies virus (RV) is a nonsegmented negative-strand RNA-virus that belongs to the genus Lyssavirus of the family Rhabdoviridae. The genome of the RV encodes five viral proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L) [1]. This neurotropic virus selectively infects neurons, not glia cells, in the nervous system, and moves from neuron to neuron via synapse exclusively in a retrograde manner. This feature Competing Interests: The authors have declared that no competing interests exist. makes the RV and its vector a useful tool to reveal the hierarchical connectivity in complex neuronal circuits [2–6]. The infection properties of the RV are determined by its glycoprotein (RV-G), which forms spike-like projections on the surface of the viral particle that bind to neural receptors. The RV vector whose G gene was deleted from its genome (ΔG-RV) does not propagate trans-synaptically from the initial infected neurons because ΔG-RV-infected neurons do not produce infectious viral particles with RV-G [7–10]. This feature makes the ΔG-RV non-pathogenic and benefits investigators in terms of safety compared with the G-gene-intact RV. Another major advantage of G-gene deletion in the RV is that it enables selective infection of the RV vector to genetically targeted neurons by substituting the glycoprotein of the RV with that of the avian sarcoma/leukosis virus subtype A and supplying its receptor gene in trans within the targeted neurons. Furthermore, G-gene deletion also enables investigators to control trans-synaptic spread to presynaptic neurons by supplying the G gene within the initial infected neurons [11]. In addition to these useful features as a neural tracer, the ΔG-RV variants, which express a genetically encoded calcium indicator or optogenetic tool, have recently been developed for investigating the function of neural circuits [12]. For such purposes, a sufficient expression level of the transgene is required to detect enough signals from the ΔG-RV-infected cells or to manipulate their activity. We previously demonstrated that G-gene deletion from a G-gene-intact RV vector enhances the expression level of the transgene, which encodes a monomeric red fluorescent protein (mRFP) [13]. However, the reason of the enhancement remains to be clarified. We presume that there are at least two possible factors for the enhancement of the transgene expression level of the ΔG-RV. The first possible factor is the absence of the RV-G. We previously reported that the RV-G affects the cell viability and resting membrane potential of infected cells [13]. It is possible that the cytotoxicity of the RV-G causes some dysfunction in the translation process of infected cells. The other possible factor is the increased expression of the L gene, which encodes the viral polymerase. It was previously reported that increased L-gene expression enhances viral mRNA transcription [14]. In nonsegmented negative-strand RNA viruses, including the RV, it has been shown that the expression levels of the viral genes decrease monotonically as the distance increases from the start (3’ end) of the genome [15–17]. Since G-gene deletion displaces the L gene anteriorly in their genome, the expression level of the L gene in ΔG-RV-infected cells is presumed to be higher than that of the L gene in G-gene-intact RV-infected cells. This increase in the L-gene expression level might enhance the viral transgene expression in ΔG-RV-infected cells. In this study, we compared the transgene expression level and viral transcription of three RV vectors, G-gene-intact RV, G-gene-deleted RV and G-gene-replaced RV. The G-gene-replaced RV was created by replacing the RV-G with a fluorescent protein-encoding gene which was assumed that the fluorescent protein-encoding gene itself would not have any noteworthy effects on the viral gene expression. The aim of the replacement was to create G-gene-deleted RV without changing the position of the L gene in the genome. To examine the effect of the RV-G, we compared the viral gene expression of the G-gene-intact a (...truncated)