The Hao-Fountain Syndrome Gene USP7 Restricts Neurotropic Orthoflavivirus Entry via Intrinsic Antiviral Activity

Inborn errors of immunity (IEI) can manifest with neurological disorders, indicating genetic overlap between immune and nervous system diseases. However, neurological disorder genes are rarely screened systematically for immune or antiviral functions. To address this, we performed arrayed RNA interference screens targeting neurodevelopmental disorder genes whose functions overlap with steps of the neurotropic flavivirus Zika virus (ZIKV) life cycle. From infected cells, we quantified viral replication and innate immune/inflammatory gene induction using quantitative RT-PCR (RT-qPCR). These screens identified ubiquitin-specific protease 7 (USP7) as a potent intrinsic inhibitor of multiple neurotropic orthoflaviviruses.Monogenic mutations in USP7 cause Hao-Fountain syndrome (HAFOUS), a rare autosomal-dominant disorder leading to developmental delay, autism spectrum disorders, and intellectual disabilities.In human cell lines, we found that USP7 potently restricted flavivirus infection independently of innate immune activation, as treatment with pan-JAK and TBK1 inhibitors did not abrogate this restriction.Using CRISPR/Cas9 base editing, we validated that introducing loss-of-function mutations in USP7’s catalytic core diminishes its antiviral activity. Virus internalization assays showed that USP7 primarily limits ZIKV entry into cells. As ZIKV enters via endocytosis, this restriction is reminiscent of USP7's function in endosomal trafficking, which is disrupted in HAFOUS. USP7 stabilizes the E3 ligase TRIM27, and together they regulate WASH ubiquitination to promote endosomal tubulation and recycling.Because TRIM27 also inhibited flavivirus entry, we hypothesized that impaired endosomal recycling flux promotes viral infection. Consistently, silencing core recycling regulators, including Rab11 and Rab35 GTPases, increased flavivirus infection. Identified in 2015, initial HAFOUS phenotyping studies did not describe immune dysregulation. However, patient record review identified previously undescribed rhinovirus and respiratory syncytial virus (RSV) infections requiring hospitalization and IgA deficiency among HAFOUS individuals. Ongoing investigations are exploring whether USP7 pathogenic mutations contribute to higher viral loads in patient primary cells and viral pathogenesis in vivo.This discovery aligns with recent virology studies showing that certain endosomal recycling pathways restrict viral entry by preventing access to downstream organelles. As endosomal recycling defects feature in multiple neurodevelopmental and neurodegenerative diseases, characterizing this intrinsic antiviral defense is particularly important for patients with such comorbidities.