Predicted transcription factors for ASNS.

CMV/HIV coinfection markedly exacerbates disease progression, elevates treatment failure risk, and worsens patient outcomes, yet the underlying molecular mechanisms remain incompletely understood—creating an urgent need for targeted host-focused research. This study identifies asparagine synthetase (ASNS) as a pivotal metabolic-signaling hub in coinfection pathogenesis, with critical interactions with the PI3K-AKT-mTOR pathway. Using integrated bioinformatics analyses of transcriptomic data, ASNS emerged as a central hub in protein-protein interaction networks, with robust positive co-expression alongside key PI3K-AKT-mTOR components (PIK3CA, MTOR, AKT2, AKT3), while machine learning validated AKT2 as a critical node. ASNS was consistently upregulated 48 hours following CMV infection and across all HIV disease stages, while single-cell RNA sequencing localized ASNS and MDM2 to plasma cells in HIV-positive individuals—implicating their role in virus-driven immune responses. Transcription factor analysis identified RUNX1 as a central regulator: bioinformatics predictions confirmed RUNX1 binds to the ASNS promoter, and validation studies identified RUNX1 as the top biomarker for HIV treatment resistance (AUC = 0.714). Molecular docking and 200-ns dynamics simulations showed that cidofovir—an approved antiviral agent—binds ASNS with high affinity (−6.61 kcal/mol) through nine hydrogen bonds, forming a more stable complex than ASNS-ONL, with VAL-51 and ASN-74 as key residues. Collectively, these findings establish ASNS as a host metabolic-signaling hub exploited by CMV and HIV, highlighting its potential as a novel therapeutic target. Targeting ASNS, particularly at residues VAL-51 and ASN-74, may offer a promising host-directed strategy to improve coinfection treatment outcomes. This work lays the groundwork for experimental validation and the development of targeted therapies for CMV/HIV coinfection.