Molecular mechanisms of enterovirus D68 viral entry mediated by the MFSD6 receptor

Nonpolio enteroviruses are ubiquitous viruses that generally cause mild respiratory and gastrointestinal symptoms but can, in rare cases, cause severe disease. Recent outbreaks of enterovirus D68 (EV-D68) have been associated with severe respiratory disease requiring hospitalization and neuropathogenesis, including polio-like paralysis syndrome. Despite the clinical relevance of EV-D68, there are no antiviral agents or licensed vaccines available, and EV-D68’s interactions with host proteins remains largely uncharacterized. In preliminary data, we have used genome-scale CRISPR screens and identified the poorly characterized multipass membrane transporter MFSD6 as a host entry factor for EV-D68. Knockout of MFSD6 expression abrogated EV-D68 infection of human cell lines derived from the lungs and the CNS. We show that MFSD6 is localized at the plasma membrane, and is required for viral internalization into the host cell. MFSD6 directly binds to EV-D68 particles via its third extracellular loop (L3). A decoy receptor, engineered by fusing MFSD6(L3) to Fc, blocked EV-D68 infection of human primary lung epithelial cells, and provided near complete protection against pathogenesis in a lethal mouse model of EV-D68 CNS infection. The preliminary data point to an essential role of MFSD6 as entry receptor, and its broad expression pattern in multiple tissues, including the spinal cord and lungs, suggest a role in pathogenesis. We hypothesize that the binding of EV-D68 with MFSD6 via molecular interactions results in the functional entry of EV-D68 RNA into the cell, and that this interaction is important for disease pathogenesis. The primary goals of this collaborative proposal between the Carette, Chiu, and Nagamine laboratories are: (1) to define the precise mechanism(s) by which MFSD6 facilitates EV-D68 entry into cells, (2) to gain high-resolution structural insight as to how MFSD6 engages the EV-D68 virion, and (3) to determine the detailed role of MFSD6 in the pathogenesis of EV-D68 infection in vivo using murine models. Our results will provide detailed insights into receptor-virus interactions and the fundamental entry mechanism of an emerging neurotropic picornavirus with pandemic potential. This information may facilitate the development of small molecules or biologicals that disrupt MFSD6 interaction with EV-D68, which could form the basis of future therapeutics that ameliorate disease. Project Number: 1R01AI193097-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Jan Carette | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $3,045,291 | Activity Code: R01 | Study Section: Viral Dynamics and Transmission Study Section [VDT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19309701