Increased transgene expression level of rabies virus vector for transsynaptic tracing

• Published in: PloS one Vol. 12; no. 7; p. e0180960
• Main Authors: Ohara, Shinya, Sota, Yasuhiro, Sato, Sho, Tsutsui, Ken-Ichiro, Iijima, Toshio
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.07.2017; Public Library of Science (PLoS)
• Subjects: Animals; Apoptosis; Attenuation; Biology and Life Sciences; Cell Line; Central nervous system; Circuits; Color; Differentiation; DNA-directed RNA polymerase; Expression vectors; Fluorescence; Gene expression; Gene order; Genes; Genetic Vectors - genetics; Life sciences; Low level; Matrix protein; Medicine and Health Sciences; Nervous system; Neural networks; Neurons; Neurosciences; Plasmids - genetics; Propagation; Propagation (polymerization); Proteins; Rabies; Rabies virus - genetics; Research and analysis methods; Ribonucleic acid; RNA; RNA polymerase; RNA synthesis; RNA viruses; Synapses - genetics; Transcription; Transgenes; Transgenes - genetics; Transport; University graduates; Viral Envelope Proteins - genetics; Viral proteins; Viral Proteins - genetics; Viruses; Wave propagation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0180960

Viral vectors that can infect neurons transsynaptically and can strongly express foreign genes are useful for investigating the organization of neural circuits. We previously developed a propagation-competent rabies virus (RV) vector based on a highly attenuated HEP-Flury strain (rHEP5.0-CVSG), which selectively infects neurons and propagates between synaptically connected neurons in a retrograde direction. Its relatively low level of transgene expression, however, makes immunostaining necessary to visualize the morphological features of infected neurons. To increase the transgene expression level of this RV vector, in this study we focused on two viral proteins: the large protein (L) and matrix protein (M). We first attempted to enhance the expression of L, which is a viral RNA polymerase, by deleting the extra transcription unit and shortening the intergenic region between the G and L genes. This viral vector (rHEP5.0-GctL) showed increased transgene expression level with efficient transsynaptic transport. We next constructed an RV vector with a rearranged gene order (rHEP5.0-GML) with the aim to suppress the expression of M, which plays a regulatory role in virus RNA synthesis. Although this vector showed high transgene expression level, the efficiency of transsynaptic transport was low. To further evaluate the usability of rHEP5.0-GctL as a transsynaptic tracer, we inserted a fluorescent timer as a transgene, which changes the color of its fluorescence from blue to red over time. This viral vector enabled us the differentiation of primary infected neurons from secondary infected neurons in terms of the fluorescence wavelength. We expect this propagation-competent RV vector to be useful for elucidating the complex organization of the central nervous system.