Combined Disruption of Multiple Cytokine Signaling Pathways Enables One-Step Anterograde Tracing with Vesicular Stomatitis Virus

Defining the direct postsynaptic targets of selected neuronal populations remains a major challenge for neural circuit mapping. Vesicular stomatitis virus (VSV) spreads efficiently in the anterograde direction, but replication-competent VSV undergoes multistep spread and therefore cannot distinguish direct from indirect downstream targets. Here, we developed a glycoprotein-deleted VSV (VSVdG)-based strategy for one-step anterograde tracing using AAV-mediated trans-complementation with several adaptations. In this system, VSVdG was engineered to encode Cre, allowing a Cre-dependent AAV to express VSV-G only after VSVdG infected the same cells, thereby limiting VSV-G expression to a short time window. To reduce VSV-M-mediated cytotoxicity, we introduced the M33A/M51R double-mutant VSV-Md variant. Using the basal ganglia circuit as a model, these adaptations enabled VSVdG spread from the striatum to expected downstream targets in mice of both sexes. Efficient VSVdG-based one-step spread required loss of type I interferon signaling in IFNAR1-knockout mice and additional suppression of cytokine-mediated antiviral responses that were independent of type I and type II interferon signaling. This was achieved either by AAV-mediated delivery of rabies virus phosphoprotein from the CVS-N2c strain or by a cytokine-blocking antibody cocktail. Although cells labeled by VSV transmission were confined to expected brain regions, the downstream labeled cells included both neurons and glia, revealing an important limitation for interpreting this approach as strictly neuron-to-neuron monosynaptic anterograde spread. Overall, this study provides a proof-of-concept VSVdG strategy for one-step anterograde circuit tracing and defines viral toxicity, innate immunity, and cell-type specificity constraints that must be addressed to develop a monosynaptic anterograde viral tracer. Significance StatementMapping direct downstream targets of defined neuronal populations is essential for understanding neural circuit function, but reliable monosynaptic anterograde viral tracers remain limited. We developed a VSVdG-based strategy that uses AAV-mediated trans-complementation to restrict VSV-G expression to starter cells in a short time window and incorporates a VSV-M variant to reduce toxicity. In the mouse basal ganglia, this system enabled one-step spread from the striatum to expected output regions when innate antiviral barriers were suppressed. Our results identify type I interferon and additional type I/type II interferon-independent cytokine signaling as major restrictions on VSVdG spread. This study establishes proof of principle for VSV-based one-step anterograde tracing while defining viral toxicity, innate immunity, and cell-type specificity constraints for further improvement.