HSV-1 strain H129 co-opts neuronal synaptic transmission machinery for its transsynaptic spread

The H129 strain (H129) of neurotropic herpes simplex virus-1 (HSV-1) is the only known HSV-1 strain that spreads transneuronally in a predominantly anterograde direction, leading to wide use for anterograde tracing in neural circuit mapping. However, it remains unclear whether H129 spreads in a synapse-specific manner or what mechanisms underlie its transmission. Here a microfluidic culture system for primary mouse cortical neurons and several engineered tool viruses were used to visualize the synapse-specific transmission of H129. We show that H129 particles are packaged into 'virion vesicles' that exploit the molecular machinery of Ca²⁺-dependent neurotransmitter release, including voltage-gated calcium channels, synaptotagmin-7 and the SNARE complex, to exit the presynaptic bouton. Subsequently, H129 enters the postsynaptic neuron through gD/nectin-1 and clathrin-mediated endocytosis at the perisynaptic site. These findings clearly illustrate the molecular mechanistic sequence of HSV-1 transsynaptic spread, advance our understanding of the viral infection in the nervous system and provide design principles for further optimizing H129-derived anterograde neural tracers.