Anterograde Neuronal Circuit Tracers Derived from Herpes Simplex Virus 1: Development, Application, and Perspectives

Abstract

Herpes simplex virus type 1 (HSV-1) has great potential to be applied as a viral tool for gene delivery or oncolysis. The broad infection tropism of HSV-1 makes it a suitable tool for targeting many different cell types, and its 150 kb double-stranded DNA genome provides great capacity for exogenous genes. Moreover, the features of neuron infection and neuron-to-neuron spread also offer special value to neuroscience. HSV-1 strain H129, with its predominant anterograde transneuronal transmission, represents one of the most promising anterograde neuronal circuit tracers to map output neuronal pathways. Decades of development have greatly expanded the H129-derived anterograde tracing toolbox, including polysynaptic and monosynaptic tracers with various fluorescent protein labeling. These tracers have been applied to neuroanatomical studies, and have contributed to revealing multiple important neuronal circuits. However, current H129-derived tracers retain intrinsic drawbacks that limit their broad application, such as yet-to-be improved labeling intensity, potential nonspecific retrograde labeling, and high toxicity. The biological complexity of HSV-1 and its insufficiently characterized virological properties have caused difficulties in its improvement and optimization as a viral tool. In this review, we focus on the current H129-derived viral tracers and highlight strategies in which future technological development can advance its use as a tool.

A novel H129-based anterograde monosynaptic tracer exhibits features of strong labeling intensity, high tracing efficiency, and reduced retrograde labeling

“…Construction and propagation of the recombinant viruses: H129-dgK-G4, H129-dTK-G4, and AAV helpers H129-dgK-G4 was derived from the previously introduced H129-G4, which contains 2 tandem GFP expression cassettes, each with one GFP and one membrane-bound GFP (mGFP) coding gene [10,12]. Using the bacterial artificial chromosome (BAC) and homologous recombination technique, the gK gene (UL53) was knocked out by replacing it with an Ampicillin resistant gene (Amp R ) (Fig.…”

“…Using the bacterial artificial chromosome (BAC) technique and homologous recombination method, we knocked out the gK gene from H129-G4, the previously introduced polysynaptic tracer, by replacing it with the ampicillin-resistant gene (Amp R ) (Fig. 1A) [10,12]. The generated BAC DNA was transfected into Vero cells stably expressing wildtype gK (gK wt ) (Vero-gK wt ) to reconstitute the recombinant virus H129-dgK-G4 (gK wt ) (Fig.…”

“…During the past decade, several polysynaptic anterograde tracers have been developed based on H129 and have already contributed to revealing multiple neuronal circuits in a variety of animal models [7,[9][10][11]. Besides H129-G4, an anterograde polysynaptic tracer with high labeling intensity for mapping the output network, our group has also developed the first anterograde monosynaptic tracer H129-dTK-tdT [10,12]. But, its low labeling intensity requires immunostaining for signal amplification to visualize the labeled postsynaptic neurons, which strongly limits its broad application.…”

“…TK is essential for HSV-1 genome replication in nonsplitting cells, such as neurons [14]. Therefore knockout of the TK gene in H129 genome impairs viral genome replication in neurons, and in turn limits the fluorescent protein expression level, which is one of the intrinsic drawbacks of the H129-dTK monosynaptic tracers [12]. An additional drawback of current H129-derived tracers is the potential retrograde labeling caused by axon terminal invasion of virus, resulting in non-anterograde specific labeling and potential misleading of the tracing analysis [12,15,16].…”

“…The deficiency of the genes associated with viral genome replication, such as TK, limits the fluorescent protein expression. Therefore viral gene(s) expressed at the later phase after the viral genome replication, for example, the structural protein genes, is a promising alternative target [12]. Since the structural protein gene deficiency will not influence viral genome replication and viral protein synthesis, the fluorescent protein can be massively expressed, and in turn labels the postsynaptic neuron with strong intensity.…”