Kinome-Wide RNA Interference Screening Identifies Mitogen-Activated Protein Kinases and Phosphatidylinositol Metabolism as Key Factors for Rabies Virus Infection

Abstract

Throughout the rabies virus (RABV) infectious cycle, host-virus interactions define its capacity to replicate, escape the immune response, and spread. As phosphorylation is a key regulatory mechanism involved in most cellular processes, kinases represent a target of choice to identify host factors required for viral replication. A kinase and phosphatase small interfering RNA (siRNA) high-content screening was performed on a fluorescent protein-recombinant field isolate (Tha RABV). We identified 57 high-confidence key host factors important for RABV replication with a readout set at 18 h postinfection and 73 with a readout set at 36 h postinfection, including 24 common factors at all stages of the infection. Amongst them, gene clusters of the most prominent pathways were determined. Up to 15 mitogen-activated protein kinases (MAPKs) and effectors, including MKK7 (associated with Jun N-terminal protein kinase [JNK] signalization) and DUSP5, as well as 17 phosphatidylinositol (PI)-related proteins, including PIP5K1C and MTM1, were found to be involved in the later stage of RABV infection. The importance of these pathways was further validated, as small molecules Ro 31-8820 and PD 198306 inhibited RABV replication in human neurons. IMPORTANCE Rabies virus relies on cellular machinery for its replication while simultaneously evading the host immune response. Despite their importance, little is known about the key host factors required for rabies virus infection. Here, we focused on the human kinome, at the core of many cellular pathways, to unveil a new understanding of the rabies virus infectious cycle and to discover new potential therapeutic targets in a small interfering RNA screening. The mitogen-activated protein kinase pathway and phosphatidylinositol metabolism were identified as prominent factors involved in rabies virus infection, and those findings were further confirmed in human neurons. While bringing a new insight into rabies virus biology, we also provide a new list of host factors involved in rabies virus infection.

Cell-type specific innate immune responses shape rabies virus tropism

“…Tha-eGFP and Th2P-4M-eGFP harbour the same genetic background except for two mutations introduced in viral P-protein (W265G, W287V) and four mutations introduced in the M protein (R77K, D100A, A104S, M110L) (10,31,32). For imaging purposes, the eGFP sequence was introduced after the M protein gene sequence as described previously (35). (B) Representative immunofluorescence pictures of SK-N-SH, SVGp12, and HMC3 cells upon infection with Tha-eGFP and Th2P-4M-eGFP.…”

“…The recombinant Th2P-4M virus harbours the same genetic background as Tha apart from bearing two mutations in the viral P-protein (W265G, M287V) and four mutations in the viral M-protein (R77K, D100A, A104S, M110L) which were previously described (10,31,32). To monitor viral infection, recombinant viral Tha-eGFP and Th2P-4M-eGFP constructs were used which were generated by cloning sequences of eGFP (recovered from pEGFP-C1 plasmid, Promega) into the genetic sequence of Tha (35) and Th2P-4M, respectively. Viral strains were sequenced before used for infection experiments.…”

SARS-CoV-2 Encodes a PPxY Late Domain Motif Known to Enhance Budding and Spread in Enveloped RNA Viruses

“…To sum up this section, proteins that interacts with this hot disordered loop are connected to lipids (membrane), particularly with phosphatidylinositol, and the regulation of bindings is made by phosphorylation. Recently, Besson et al (2019) identified that mitogen-activated protein kinase pathway and phosphatidylinositol metabolism are involved in the later stage of RABV infection.…”

Engagement of Neurotropic Viruses in Fast Axonal Transport: Mechanisms, Potential Role of Host Kinases and Implications for Neuronal Dysfunction

“…Specifically, mutant proteins linked to familial forms of neurodegenerative diseases including huntingtin, superoxide dismutase 1, tau, and spastin were all found to cause FAT deficits through a mechanism involving deregulation of selected kinase pathways (reviewed in Morfini et al, 2009a ; Brady and Morfini, 2010 ). Relevant to the topics discussed in this review, neurotropic viruses have been widely shown to modulate the activity of host kinases (Zachos et al, 1999 ; Piacentini et al, 2015 ; Bonjardim, 2017 ; Besson et al, 2019 ), including some involved in the regulation of FAT. A thorough description of these findings is beyond the scope of this review, but some relevant examples are provided below.…”

“…Given the number of kinases activated at different stages of the viral cycle and the complexity and crosstalk among kinase pathways, the identification of pathologically relevant ones may represent a major challenge for investigators (Besson et al, 2019 ). Nevertheless, a message of hope is provided by the successful development of highly specific, brain-permeable kinase inhibitors (Maphis et al, 2016 ), and increasing experimental evidence confirming a role of host kinases on the neuronal pathology triggered by some viruses (Beckham et al, 2007 ).…”