The membrane-associated ubiquitin ligases MARCH2 and MARCH3 target TIM-1 to limit Zika virus infection

T-cell immunoglobulin mucin family member-1 (TIM-1, also known as HAVCR1/KIM-1) is a transmembrane glycoprotein that has been reported to act as an entry receptor for multiple flaviviruses including Zika virus (ZIKV). The post-translational regulation of TIM-1 and its effects on ZIKV infection are unclear. In this study, we identified the membrane-associated RING-CH-type finger (MARCH) E3 ubiquitin ligase family members MARCH2 and MARCH3 as critical negative regulators of TIM-1 under physiological conditions. MARCH2 and MARCH3 associate with TIM-1 and mediate its K48-linked polyubiquitination at K338 and K346 respectively, leading to subsequent proteasomal degradation. While deficiency of either MARCH2 or MARCH3 modestly increases TIM-1 levels and enhances ZIKV infectivity, double knockout of MARCH2/3 has a more dramatic effect. Double knockout of MARCH2/3 increased ZIKV infectivity in wild-type but not TIM-1 knockout cells, and reconstitution of TIM-1K338R/K346R into TIM-1-deficient cells increases ZIKV infectivity to a higher degree than reconstitution with wild-type TIM-1. Knockout of either MARCH2 or MARCH3 increased ZIKV infectivity and pathogenesis in mice, whereas double knockout of MARCH2/3 has a more dramatic effect. These findings suggest that MARCH2 and MARCH3 target TIM-1 for K48-linked polyubiquitination and proteasomal degradation, thereby acting as redundant host restriction factors to limit ZIKV infection and pathogenesis.