Uncovering Conserved Viral Mechanisms that Target CNNM3 to Disrupt Magnesium Homeostasis

Magnesium (Mg2+) a key intracellular cation essential for metabolism and nucleic acid biochemistry, is tightly regulated by the TRPM7 channel and the CNNM family, with CNNM3 as a negative regulator. Teodoro lab work showed that CNNM3 knockdown in H1299 cells increases intracellular Mg2+ and protein translation. Recent observations indicate that viruses manipulate this pathway. The adenovirus was observed to elevates total and soluble Mg2+ partly via E4orf4-mediated CNNM3 dephosphorylation, and mass spectrometry identified additional CNNM3-binding viral proteins, including HIV gp160, SARS-CoV-2 ORF8, and EBV BALF2. Alignment revealed two conserved motifs, alpha and beta, and mutating these sites in E4orf4 or ORF8 was found to disrupt CNNM3 binding. We hypothesize that these motifs mediate viral interaction with CNNM3, raising intracellular Mg2+ to favor replication. To test this, we will use a dual-reporter luciferase assay to compare Mg2+ modulation by wild-type versus motif-mutant viral proteins. Also, to investigate ORF8 activity during infection, we will use a single-cycle replicon to measure Mg2+ changes and replication efficiency for wild-type, ORF8-deleted, and motif-mutant replicons. In parallel, we aim to generate a CNNM3 KO mouse model using CRISPR/Cas9 targeting exon 1 or exons 2-7 in L929 cells to assess viability and Mg2+-related phenotypes, and to determine whether an inducible model will be required. Once established, CNNM3 KO mice will enable investigation of CNNM3's role in Mg2+ homeostasis and viral pathogenesis in vivo using a mouse adenovirus. Overall, this project may define a previously unrecognized viral strategy to manipulate Mg2+ through CNNM3 and may identify CNNM3 or the conserved viral motifs as potential broad-spectrum antiviral targets.