Significance

Herpesviruses are ubiquitous human pathogens responsible for a wide spectrum of diseases, including severe infections in immunocompromised individuals and associations with neurodegenerative and autoimmune disorders. Despite the clinical importance of these viruses, current antiviral therapies are largely limited to the α-subfamily, with few effective options for β- and γ-subfamilies. The helicase-primase complex (HPC) is a critical enzyme for viral DNA replication and a validated antiviral target, yet its structural basis and inhibition mechanisms have remained elusive, hindering the rational development of next-generation therapeutics. This study provides the first high-resolution cryo-EM structures of the human herpesvirus 1 (HHV1) HPC bound to single-stranded DNA and two clinically relevant inhibitors, amenamevir and pritelivir. These structures reveal the modular organization of the HPC and identify a shared allosteric pocket where both inhibitors bind. Structural, biochemical, and molecular dynamics analyses demonstrate that these inhibitors lock the helicase in an open, catalytically inactive conformation, thereby blocking ATP binding and halting DNA unwinding. Fragment molecular orbital (FMO) calculations further elucidate the molecular determinants of inhibitor binding, explaining the α-subfamily specificity and the distinct antiviral spectra of amenamevir and pritelivir. The integrated approach combining cryo-EM, molecular dynamics, and quantum chemical calculations not only clarifies the mechanism of action of current clinical inhibitors but also establishes a robust framework for the rational design of novel antivirals. By providing detailed insights into the structural and dynamic properties of the HPC and its inhibition, this work paves the way for the development of new drugs with tailored specificity and improved pharmacokinetic properties, potentially expanding therapeutic options to currently untreatable herpesvirus infections. These findings have broad implications for antiviral drug discovery and for understanding the molecular mechanisms underlying DNA replication in herpesviruses.

Toru Sengoku
Associate Professor
Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan