Macrodomain catalytic activity modulates Chikungunya virus dissemination and transmission potential in Aedes mosquitoes

Abstract

Viral macrodomains are promising antiviral targets that counteract host ADP-ribosylation-mediated antiviral responses in mammals. However, their role in dual-host viruses within the mosquito vector remains largely unknown. Here, we investigated the role of the Chikungunya virus (CHIKV) nsP3 macrodomain catalytic activity by mutating the active-site asparagine 24 (N24). N24 mutant viruses rapidly selected for compensatory second-site mutations at aspartate 31 (D31) in mammalian Vero cells, while interferon-competent human cells favored reversion at position 24 instead, suggesting adaptive responses to macrodomain functional impairment. Catalytically inactive double-mutant viruses replicated poorly in interferon-competent human cells but comparably to wild-type in mosquito cells. In Aedes mosquitoes in vivo, macrodomain mutations modulated CHIKV replication and infectivity in a mutation-specific manner: one mutant reduced them while another enhanced them. Biochemical and structural analyses confirmed that these phenotypes arise in the context of a catalytically inactive macrodomain that retains ADP-ribose binding capacity, with D31 mutations lining the substrate exit path. These findings demonstrate that macrodomain catalytic activity modulates CHIKV dissemination and transmission potential in Aedes mosquitoes, revealing fundamental differences in host-dependent evolutionary advantage for a catalytically inactive viral enzyme.