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

Abstract

HPV16 E2 protein is a key regulatory protein essential for viral replication, yet no enzymatic activity had 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 0.1074 {micro}M, mediated primarily by residues Q320 and H342. 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. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/676238v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@12fd0aforg.highwire.dtl.DTLVardef@2dc50forg.highwire.dtl.DTLVardef@e46f5eorg.highwire.dtl.DTLVardef@14b87da_HPS_FORMAT_FIGEXP M_FIG C_FIG E2 protein has traditionally been recognized primarily for its DNA-binding and transcriptional regulatory functions. This study provides the first evidence that E2 protein possesses intrinsic enzymatic activity, identifies a small-molecule inhibitor targeting this activity, and reveals a novel mechanism of E1-E2 interaction. O_LIHPV16 E2 Protein Exhibits ATPase and Helicase Activities C_LIO_LIIdentification of Key Amino Acid Residues for HPV16 E2 Protein Enzymatic Activity C_LIO_LIPPT Effectively Inhibits E2 Protein Helicase Activity In Vitro C_LIO_LIE2 Protein Exhibits Weaker Enzymatic Activity Than E1 and Inhibits E1 Helicase Activity C_LIO_LIE2 Inhibits E1 Helicase Activity Through Protein-Protein Interaction C_LI