Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing

Takuma Mori,Kinjiro Morimoto

doi:10.3389/fnana.2013.00047

Abstract

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.

Highlights

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)
We demonstrated that (1) rabies monosynaptic tracing with RV glycoprotein (RVG) variants traced different subsets of presynaptic partners, (2) RVG of the attenuated strain labeled astrocytes, and (3) the cytotoxicity of RV-ΔG did not depend on RVG but on RV-ΔG
OPTIMIZATION OF PRODUCTION OF GLYCOPROTEIN-DELETED HEP-FLURY RV; EFFECTS OF T7 RNA POLYMERASE, RIBOZYME AND RVG VARIANTS The HEP-Flury strain was selected as a “core” of RV-ΔG, because it has been successfully pseudotyped with more than two variants of RVG (Takayama-Ito et al, 2006a; Ohara et al, 2009)

Summary

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). RV-ΔG has 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). 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. 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 neurons and astrocytes

MATERIALS AND METHODS

RESULTS

DISCUSSION