Cell-type specific innate immune responses shape rabies virus tropism

Viral tropism, or the specificity of a particular virus to infect a certain cell type, is crucial in determining virus replication, viral spread, and ultimately host survival. Rabies, one of the deadliest known zoonotic diseases, is still causing 60.000 human deaths annually. Upon central nervous system (CNS) entry, neurotropic rabies virus (RABV) preserves the neural network by limiting apoptosis and inflammation. To date, we do not fully understand the factors determining RABV tropism and why glial cells are unable to clear RABV from the infected brain. Here, we compare susceptibilities and innate immune responses of CNS cell types towards infection with virulent dog RABV Tha and less virulent Th2P-4M in vitro, highlighting differences in cellular susceptibility and antiviral responses. Less virulent Th2P-4M bears mutations introduced in viral phosphoprotein (P-protein) and matrix protein (M-protein) thereby hindering viral immune evasion of the host nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and Janus kinase (JAK) - signal transducer and activator of transcription protein (STAT) pathways. Our results reveal that human neural stem cell (hNSC)-derived neurons and astrocytes, in contrast to human iPSC-derived microglia, are highly susceptible to Tha and Th2P-4M infection in vitro. Surprisingly, Th2P-4M presents a stronger neurotropism in hNSC-derived CNS cultures compared to Tha suggesting that NF-{kappa}B- and JAK-STAT-mediated antiviral host responses are defining RABV replication and thereby its tropism. Further, we show that astrocyte-like (SVGp12) and microglia-like (HMC3) cells protect neuroblastoma cells (SK-N-SH) from Tha infection in vitro. Transcription profiles and quantification of intracellular protein levels revealed major differences in antiviral immune responses mediated by neurons, astrocytes (IFNB1, CCL5, CXCL10, IL1B, IL6, LIF), and microglia (CCL5, CXCL10, ISG15, MX1, IL6) upon virulent Tha infection. Overall, we provide evidence that RABV tropism depends on its capability to evade cell-type specific immune responses via P- and M-proteins. Author summaryRabies virus (RABV) neurotropism is widely reported as a unique feature of rabies, and still the exact mechanism underlying RABV susceptibility remains to be elucidated. Several receptors are known to accelerate RABV entry to the cell (1-4) and yet, none of them seems to be essential for successful infection (5,6) questioning a marked cellular tropism. Although RABV is classically reported as strictly neurotropic (7), recent studies report profound infection of glial cells in vivo depending on the viral strain and the infection route used (8,9). Here, we provide evidence that human neural stem cell (hNSC)-derived neurons (hiNeurons) and astrocytes (hiAstrocytes) are highly susceptible towards infection with the virulent field RABV strain Tha and less virulent Th2P-4M. In contrast, human iPSC-derived microglia-like cells (hiMicros) are resistant to viral replication in vitro. Whereas hiNeurons are immunologically quiescent upon Tha infection, fetal astrocytes and hiMicros establish strong antiviral responses. In contrast to Tha, Th2P-4M, which is unable to evade NF-{kappa}B and JAK-STAT pathways (10), shows a more profound neurotropism suggesting that cell-type specific responses shape RABV tropism. Hence, we conclude that viral evasion mechanisms mediated by P- and M-proteins partly determine Tha tropism of human CNS cell types in vitro.