HPV16 E2 protein possesses intrinsic helicase activity and sterically hinders E1 function through direct interaction

HPV16 E2 protein is a key regulatory protein essential for viral replication, yet no enzymatic activity has been attributed to it until now. In this study, we report for the first time that E2 possesses intrinsic ATP-dependent DNA unwinding activity. Mutational analysis identified residues K299, Y303, and K306 as critical for this helicase function. We further demonstrate that podophyllotoxin directly binds to E2 and inhibits its unwinding activity with an IC₅₀ of 0.11±0.03 μM, mediated primarily by residues H320 and Q342. Comparative analysis revealed that the ATPase and helicase activities of E2 are considerably weaker than those of E1. Notably, we discovered that E2 potently inhibits the helicase activity of E1. This suppression is facilitated by the N-terminal domain of E2 (amino acids 1-245) through direct interaction with E1, with residue E39 playing a critical role. Our findings not only unveil a previously unrecognized enzymatic function of E2 but also suggest its role as a potential antiviral target. Moreover, the observed inhibitory effect of E2 on E1 highlights a novel regulatory mechanism for HPV DNA replication.