Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons

Abstract

The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear. In this study, we constructed recombinant H129 variants with mCherry-labeled capsids and/or green fluorescent protein (GFP)-labeled envelopes and infected the cortical neurons to study axonal transport of H129 viral particles. We found that different types of viral particles were unevenly distributed in the nucleus, cytoplasm of the cell body, and axon. Most H129 progeny particles were unenveloped capsids and were transported as capsids rather than virions in the axon. Notably, capsids acquired envelopes at axonal varicosities and terminals where the sites forming synapses are connected with other neurons. Moreover, viral capsids moved more frequently in the anterograde direction in axons, with an average velocity of 0.62 ± 0.18 μm/s and maximal velocity of 1.80 ± 0.15 μm/s. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor. These findings support that H129-derived tracers map the neural circuit anterogradely and possibly transsynaptically. These data will guide future modifications and improvements of H129-based anterograde viral tracers. IMPORTANCE Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers.

Establishment of a Fosmid Library for Pseudorabies Virus SC Strain and Application in Viral Neuronal Tracing

“…anterograde movements were 0.71 ± 0.43 and 0.75 ± 0.49 µm/s. This is close to 0.62 ± 0.18 µm/s of HSV anterograde movement in cortical neuron axons by the same statistical analysis method (Dong et al, 2020). However, The average of PRV movement speed was 1.2 µm/s in the research by Hogue et al (2018), while the events less than 0.5 µm/s were deleted in the data of Gaussian distribution.…”

“…However, The average of PRV movement speed was 1.2 µm/s in the research by Hogue et al (2018), while the events less than 0.5 µm/s were deleted in the data of Gaussian distribution. The velocity changed at different times post-infection (Dong et al, 2020). Moreover, the virus strain, the kind of neuron, and statistical analysis method may all affect the reported average speed of capsid transport in axon retrograde.…”

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

“…Fetal mouse cortical neurons were isolated and cultured as described previously [25,26]. Briefly, the cerebral cortex was dissected from the forebrain of C57BL/6 mouse fetuses at embryonic day 18.5 (E18.5), and then dissociated with 0.25% trypsin (Gibco/Life Technologies)/DNase I (Sigma) for 15 min at 37 °C.…”

“…The microfluidic plate has been introduced previously and was fabricated following the described protocol [10,26]. In brief, it contains two isolated chambers connected by multiple microchannels (700 μm long, 10 μm wide, and 3 μm deep), which allows only the axons to grow through but not the somas or dendrites.…”

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

“…Different HSV-1 strains with different transmission properties, including F, 17, H129, and McIntyre-B, have been sequenced and compared for their transmission properties [ 106 ]. Dong et al recently showed that H129 capsid and envelope are transported in the axon separately, and notably, capsids are enveloped at axonal varicosity and terminals [ 107 ]. However, the detailed mechanism of HSV-1 transmission, especially why H129 gains the preferential anterograde transmission, remains unclear.…”