Abstract
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-gene-deleted 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-gene-replaced 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.
Highlights
Rabies virus (RV) is a nonsegmented negative-strand RNA-virus that belongs to the genus Lyssavirus of the family Rhabdoviridae
The first RV vector, G-gene-intact RV, was created by exchanging the G gene of the high egg passage-flury (HEP) strain with that of the Challenge Virus Standard (CVS) strain, which has been generally used for tracing experiments
At 6 dpi, the differences in the fluorescent intensity expanded over time after infection and the quantitative relation of the fluorescence intensity remained unchanged (p < 0.001, all pairs at 6 dpi). These results showed that the absence of G, after the G-gene deletion and its replacement, affected the expression level of the monomeric red fluorescent protein (mRFP) gene, which was inserted between the N and P genes in the RV genome, and the effect of the deletion was stronger than that of the replacement
Summary
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. Effects of G-gene Deletion and Replacement on Gene Expression of RV
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