IQGAP1 and IQGAP3 are critical host factors for Marburg virus replication, nucleocapsid transport, and cell-to-cell spread.

The IQGAP protein family-comprising IQGAP1, IQGAP2, and IQGAP3-exhibits structural similarity but fulfils distinct cellular functions. We previously demonstrated that IQGAP1 is recruited to Marburg virus (MARV)-induced inclusion bodies (IBs) and associates with motile nucleocapsids. To further elucidate the roles of IQGAP proteins in the MARV life cycle, we generated Huh-7 cell lines with single, combined, or triple knockouts (KOs) of IQGAP isoforms. Loss of IQGAP proteins consistently reduced cellular permissiveness to MARV infection and impaired multiple key viral processes: (i) transcription and replication efficiency was diminished predominantly by IQGAP3 KO; (ii) virus release was most notably reduced in IQGAP3 KO cells, whereas cell-to-cell spread was more strongly impaired in IQGAP1 KO cells; and (iii) although actin tails continued to form at nucleocapsids in triple KO cells, long distance nucleocapsid transport was altered, with reduced spatial displacement efficiency observed in both IQGAP1 KO and IQGAP3 KO cells. The expression of individual IQGAPs in triple KO cells demonstrated their functionality and ability to partially restore the phenotype of wild-type cells. These findings identify IQGAPs as critical host factors that support MARV transcription/replication, nucleocapsid transport, and viral spread, likely through modulation of actin dynamics.