Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing

TECHNOLOGY REPORT ARTICLE published: 02 January 2014 doi: 10.3389/fnana.2013.00047 Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing Takuma Mori 1 * and Kinjiro Morimoto 2 1 2 Department of Informative Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan Department of Medical Pharmacy, Faculty of Pharmacy, Yasuda Women’s University, Hiroshima, Japan Edited by: Kathleen S. Rockland, Boston University School Medicine, USA Reviewed by: John Patrick Card, University of Pittsburgh, USA *Correspondence: Takuma Mori, Department of Informative Physiology, National Institute for Physiological Sciences, 5-1 Myodaiji-Higashiyama, Okazaki, Aichi 444-8787, Japan e-mail: Rabies virus (RV) has been widely used to trace multi-synaptic neuronal circuits. The recent development of glycoprotein-deficient rabies virus (RV-ΔG) expressing various proteins has enabled analyzes of both the structure and function of neuronal circuits.The main advantage of RV-ΔG is its ability to trace monosynaptic circuits by the complementation of rabies virus glycoprotein (RVG), but it has the disadvantage of cytotoxicity. Several strain variants of RV have different biological characteristics, such as synaptic spreading and cytotoxicity, mainly due to amino acid mutations in RVG. We developed an improved protocol for the production of a highly attenuated strain of RV-ΔG and assessed whether RVG variants affect rabies monosynaptic tracing and the health of infected neurons. We demonstrated that (1) rabies monosynaptic tracing with RVG variants traced different subsets of presynaptic partners, (2) RVG of the attenuated strain also labeled astrocytes, and (3) the cytotoxicity of RV-ΔG did not depend on RVG but on RV-ΔG. These findings indicate that RVG variants are an important determinant of rabies monosynaptic tracing. Keywords: trans-synaptic tracing, neuronal degeneration, neocortex, in utero electroporation, astrocytes INTRODUCTION Rabies virus (RV) has been used as a trans-synaptic neuronal tracer to reveal synaptic networks in the brain (Nassi et al., 2006; Ohara et al., 2009; Lyon et al., 2010). The gene encoding RV glycoprotein (RVG) is essential for trans-synaptic spreading (Mebatsion et al., 1996). Deletion of RVG from the genome made RV a useful retrograde viral tracer (Wickersham et al., 2007a). RVs with the glycoprotein deleted (RV-ΔG) have been developed to express fluorescent proteins as well as bio-tools such as channelrhodopsin-2, which helped reveal the anatomical and physiological structures of neural circuits (Osakada et al., 2011; Kiritani et al., 2012). RV-ΔG has also been utilized for monosynaptic restriction of trans-synaptic tracing (Wickersham et al., 2007b). This method has been used to trace hundreds of presynaptic neurons connected to a single cell (Marshel et al., 2010; Rancz et al., 2011), as well as certain neuron subtypes (Vivar et al., 2012; Watabe-Uchida et al., 2012; Wall et al., 2013). The number of traced presynaptic cells, however, was much smaller than the number of postsynaptic sites, suggesting that the rabies monosynaptic tracing may trace only part of the entire presynaptic population. Several highly attenuated strains of RV cannot spread efficiently in the nervous system, and certain amino acid mutations in RVG are correlated with the efficiency of spreading. One of the most studied mutations is a conversion of arginine to glycine at RVG position 333 (R333Q), which is associated with decreased virulence and less efficient spread of RV (Dietzschold et al., 1985; Takayama-Ito et al., 2006a). This R333Q mutation is frequently found in highly attenuated fixed strains of RV, such as HEPFlury, but is not contained in RVG of SADB19 (SADG) or CVS-11 (CVSG), RV strains frequently used in rabies monosynaptic tracing. Thus, it is believed that RVG of HEP-Flury (HEPG) would Frontiers in Neuroanatomy not be useful for tracing neuronal circuits, and HEPG has never been used in the rabies monosynaptic tracing. In this study we developed a protocol to produce HEP-Flury RV from which glycoprotein was deleted (HEP-ΔG), and investigated the effects of RVG variants on rabies monosynaptic tracing. We demonstrate that monosynaptic tracing with HEPG: (1) resulted in a different pattern of presynaptic neuronal distribution than tracing with SADcvsG, and (2) reliably traced not only neurons but also astrocytes. MATERIALS AND METHODS All protocols dealing with animals, transgenic bacteria, and virus were approved by the relevant committees of the National Institute for Physiological Sciences. CONSTRUCTION OF PLASMIDS The original plasmid, pcDNA-HEP (aka pHEP3.0), encodes the full length HEP-Flury antigenome flanked by Hammerhead (Ham) and hepatitis delta virus (HDV) ribozyme sequences under the control of CMV and T7 promoters (Inoue et al., 2003). To replace the original Ham with another variant, tHam (Le Mercier et al., 2002), the leader and nucleoprotein sequences of pcDNAHEP were amplified by PCR using primers that included the tHam sequence. These PCR products were ligated into pcDNAHEP, resulting in the plasmid pcDNA-tHEP. The HEPG gene was replaced by the GFP gene using the AflII and NheI restriction enzyme sites, yielding the plasmid pcDNA-tHEP-ΔG-GFP. To generate SADcvsG, the cytoplasmic domain of SADG was replaced by that of CVSG. The cytoplasmic domain of CVSG, rather than that of HEPG, was selected since the cytoplasmic domain of RVGs of attenuated strains, such as HEP-Flury, www.frontiersin.org January 2014 | Volume 7 | Article 47 | 1 “fnana-07-00047” — 2013/12/30 — 16:06 — page 1 — #1 Mori and Kinjiro Rabies monosynaptic tracing with RVG variants might induce cell apoptosis (Prehaud et al., 2010). PCR was utilized to replace the cytoplasmic domain of EnvARGCD glycoprotein, which originates from the SAD strain, with that of the CVS strain. RVG and its variants, RFP-f2a-TVA (RT) and EnvAcvsG, were ligated into the pCAGGS expression plasmid, yielding pCAGGS-RVG, pCAGGS-RT, and pCAGGS-EnvAcvsG, respectively. cells on 10-cm plates, followed 2 days later by transfection with 5 μg pCAGGS-RVG and incubation at 32◦ C in medium containing 2% fetal bovine serum (FBS). A cluster was defined as an isolated GFP cell (with no other GFP cells within approximately 1 mm) that became more than three cells within 100 μm 48 h after transfection. VIRAL TITRATION PRODUCTION OF GLYCOPROTEIN-DELETED RV We produced HEP-ΔG-GFP following protocols for the recovery of RV strains, (Inoue et al., 2003; Ito et al., 2003; Osakada et al., 2011). About 1.0 × 104 BHK-21 or BHK-T7/9 cells, the latter expressing T7 RNA polymerase, were seeded into each well of a 6-well plate. Cells were transfected with plasmid coding the full RV genome (2 μg pcDNA-HEP-ΔG-GFP) and helper plasmids encoding rabies nucleoprotein (RN; 1 μg pcDNA-RN), phosphoprotein (RP; 0.5 μg pcDNA-RP), and polymerase (RL; 0.25 μg pcDNA-RL) genes using TransIT-LT1, as des (...truncated)