Enhancing mRNA Interactions by Engineering the Arc Protein with Nucleocapsid Domain

Abstract

Activity-regulated cytoskeleton-associated protein (Arc) forms virus-like capsids for mRNA transport between neurons. Unlike HIV-1 Group-specific Antigen (Gag), which uses its Nucleocapsid (NC) domain to bind HIV-1 genomic mRNA, mammalian Arc lacks the NC domain, and their direct mRNA binding interactions remain underexplored. This study examined rat Arc’s binding to rat Arc 5′ UTR (A5U), HIV-1 5′ UTR (H5U), and GFP mRNAs, revealing weak binding with no significant preference. Adding the HIV-1 NC domain to rArc’s C-terminus significantly improved binding to H5U, while also showing substantial binding to A5U at about 60% of its H5U level and exhibiting twice the affinity for A5U over GFP mRNA. Importantly, rArc-NC binds 3.4 times more A5U and H5U than GST-NC, indicating that rArc NTD-CA aids mRNA binding by HIV-1 NC. These findings suggest a conserved Gag protein–mRNA interaction mechanism, highlighting the potential for developing mRNA delivery systems that combine endogenous Gag NTD-CA with retroviral NC and UTRs.