ATF4-2, a variant of the activating transcription factor 4 (ATF4), belongs to the bZIP family and acts as a key mediator of cellular stress responses. Encoded by the ATF4 gene, it harbors a basic DNA-binding region and a leucine zipper dimerization motif, regulating target gene transcription by binding to cAMP response elements (CREs). It is ubiquitously expressed and central to pathways like amino acid metabolism, oxidative stress adaptation, and endoplasmic reticulum unfolded protein response. Dysregulated ATF4-2 is linked to metabolic disorders, neurodegenerative diseases, and cancers, while its role in viral infections involves hijacking by pathogens. ATF4-2 interacts with HIV1/gag, the viral structural polyprotein, potentially facilitating viral assembly and enhancing HIV-1 replication through transcriptional activation.To detect the activity of recombinant ATF4-2, a functional ELISA assay was performed to evaluate the interaction between recombinant human ATF4-2 and recombinant human HIV/gag. Briefly, biotin-linked ATF4-2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to HIV/gag-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of ATF4-2 and HIV/gag was shown in Figure 1, the EC50?for this effect is 0.0087μg/mL.