Structure and inhibition mechanism of the helicase-primase complex from human herpesvirus 1

Human herpesviruses (HHVs) are widespread pathogens that can cause severe disease, particularly in immunocompromised individuals, and have been increasingly associated with chronic conditions such as Alzheimers disease and multiple sclerosis. Most antiviral therapies target the -subfamily of HHVs, while treatment options for the {beta}- and {gamma}-subfamilies remain limited. A key antiviral target in -subfamily is the helicase-primase complex (HPC), which is essential for viral DNA replication; however, its structure and inhibition mechanism have remained unclear. Here, we report the cryo-EM structures of the HPC from human herpesvirus 1 (HHV1) bound to single-stranded DNA(ssDNA) and either of two clinical inhibitors, amenamevir or pritelivir. The HPC comprises flexible helicase and primase modules, and both inhibitors engage a shared allosteric pocket near the ATPase site. Structural, biochemical, and molecular dynamics analyses reveal that these inhibitors lock the helicase in an open, inactive conformation, thereby preventing ATP binding and DNA unwinding. Furthermore, quantitative interaction energy analysis by fragment molecular orbital (FMO) calculation elucidates why these inhibitors are -subfamily selective and how subtle chemical differences between amenamevir and pritelivir lead to distinct antiviral spectra. Our findings clarify the inhibition mechanism of clinically relevant HPC inhibitors and provide a structural basis for their subfamily selectivity. This framework may guide the rational design of next-generation inhibitors with altered herpesvirus specificity and provide broader insights into the development of helicase-targeted therapeutics.