Increased transgene expression level of rabies virus vector for transsynaptic tracing

RESEARCH ARTICLE Increased transgene expression level of rabies virus vector for transsynaptic tracing Shinya Ohara☯, Yasuhiro Sota☯, Sho Sato, Ken-Ichiro Tsutsui, Toshio Iijima* Division of Systems Neuroscience, Tohoku University Graduate School of Life Sciences, Sendai, Japan ☯ These authors contributed equally to this work. * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Ohara S, Sota Y, Sato S, Tsutsui K-I, Iijima T (2017) Increased transgene expression level of rabies virus vector for transsynaptic tracing. PLoS ONE 12(7): e0180960. https://doi. org/10.1371/journal.pone.0180960 Editor: Eric J Kremer, French National Centre for Scientific Research, FRANCE Received: November 30, 2016 Accepted: June 23, 2017 Published: July 10, 2017 Copyright: © 2017 Ohara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract 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 HEPFlury 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 rHEP5.0GctL 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. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by Grants-inAid for Scientific Research on Innovative Areas (#26119502), and by Grant-in-Aid for Scientific Research (KAKENHI) #15K18358 from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Transsynaptic tracers are useful tools to reveal the hierarchical connectivity in the central nervous system. Neurotropic viruses that can propagate within synaptically connected neural circuits and amplify signals through replication, such as the herpes simplex virus type 1, the pseudorabies virus, and the rabies virus (RV), have been used as such an anatomical tool [1– 3]. Among these viruses, RV is preferred owing to its ability to selectively infect neurons and low cytotoxicity [4–6]. PLOS ONE | https://doi.org/10.1371/journal.pone.0180960 July 10, 2017 1 / 16 Improved rabies virus vector for transsynaptic tracing Competing interests: The authors have declared that no competing interests exist. We previously developed a recombinant RV vector based on a vaccinated HEP-Flury strain (HEP) [7], since vaccinated strains show higher levels of transcription than pathogenic strains [8,9]. This vector was further developed by replacing the glycoprotein (G) gene of HEP with that of CVS, and adding an additional transgene insertion site between the N and P genes [10]. Since this recombinant RV vector (rHEP5.0-CVSG) efficiently propagated transsynaptically in a retrograde direction and expressed transgene in the infected neuron, this viral vector could be used as a potential tool for selective gene delivery in the central nervous system. Note that this RV vector differs from G-gene deleted RV vectors [11–15], which are now widely used in the field of neuroscience, in terms that this vector can propagate transsynaptically without supplying the G-gene in trans within the infected cells. By using this propagation-competent RV vector, we have revealed the mutlisynaptic connections in the medial temporal lobe memory system [16,17]. Although our RV vector was designed to express the transgene at high levels, the transgene expression level of this propagation-competent RV vector was significantly lower than that of the G-deleted RV vector, and expressed marker proteins must be immunostained to clearly visualize the morphological features of infected neurons [11]. Increasing the transgene expression level will expand the usability of this vector as a neurotracing tool since it will not only enable us to examine the morphology of targeted neurons without requiring any staining procedure, but may solve one of the pitfalls of transsynaptic tracing. In poly(trans)synaptic tracing, several samples with different survival times must be prepared to distinguish primary infected neurons (1st-order neurons) from secondary infected ones (2nd-order neurons). This requires the use of many experimental animals with accurate injection. This problem may be solved by using a propagation-competent RV vector with a high expression level of a fluorescent timer, which changes the color of its fluorescence over time [18], since it would enable the differentiation between the 1st- and the 2nd-order neurons in the same sample. To develop a recombinant RV vector with a high level of transgene expression, in this study we focused on two viral proteins: the large protein (L) and the matrix protein (M). The L gene encodes the viral RNA polymerase, and it has been reported that the overexpressed L gene increases viral-gene mRNA transcripts and the expression level of nucleoprotein (N) and phosphoprotein (P) [19,20]. Indeed, we have recently shown that the enhanced transgene expression of a G-deleted RV vector compared with a propagation-competent RV vector is partially due to the increased transcription level of the L gene [11,21]. M protein is mainly responsible for the assembly and budding of bullet-shaped viral particles [22], but is also a regulatory (...truncated)