Host and rabies virus gene expression is shaped by human brain cell type and reveals a preexisting pro-viral transcriptional state in astrocytes

Rabies virus (RABV) proteins play dual roles during the infection of complex neural tissue, the generation and spread of new virions and the active inhibition of cellular innate immune pathways, both contributing to rabies’ lethality. While spatially-distinct RABV protein residues specializing in virus-centric and immune-inhibitory functions have been identified, how these dual functions interact to shape infection outcomes across diverse types of host brain cells is unknown. To “unmask” and study how innate immune inhibition affects the transcriptional regulation of the human and viral genome with cellular resolution, we performed single-cell RNA sequencing of co-cultured human brain cell types, comparing infection dynamics of a wild-type RABV isolate virus to a mutant virus in which six critical point mutations in the phosphoprotein (P) and matrix (M) RABV proteins selectively neuter antagonism of interferon- and NF-κB- dependent cellular responses. Our analysis reveals that RABV gene expression is shaped by host cell type and that wild-type RABV infection induces small-scale, cell-type conserved transcriptional programs that may support infection by hijacking transcriptional feedback systems that control pro-viral host cell factors while minimizing anti-viral responses. In contrast to accepted models, disinhibited innate immune signaling increases RABV transcriptional output across infected cell types. Most strikingly, we discovered a subpopulation of astrocytes that supports an average of 6-fold higher viral mRNA expression through a massive host cell transcriptional change involving ∼38% of astrocyte expressed genes. Our analysis suggests that these astrocytes we term “pro-viral” are a rare subtype present in the human brain and are primed to play a protective role during viral infection in concert with interferon-sensitive microglia recalcitrant to infection.