Abstract
Human herpesviruses (HHVs) are widespread pathogens that cause severe disease. Their replication depends on the HHV protease (HHV Pr), an enzyme essential for capsid maturation. Because HHV Pr must dimerize to become catalytically active, disrupting dimer formation is a promising strategy for antiviral therapeutic development. We isolated a conformationally selective antibody inhibitor, Fab5, from a fully human naïve Fab-phage library that recognizes monomeric human cytomegalovirus protease (HCMV Pr). A 2.6 Å cryoelectron microscopy (cryo-EM) structure of the Fab5-HCMV Pr complex revealed that Fab5 binds a flexible loop distal from the active site and dimer interface which we call the latch loop. In HCMV Pr dimers, this loop secures the C-terminal tail to the protein core. Structure-guided mutagenesis confirmed that the latch loop is essential for HCMV Pr dimerization and activity. This loop is structurally conserved across all HHV Prs, and we show its functional role in Kaposi's Sarcoma-associated herpesvirus (KSHV) Pr as well. The latch loop plays a mechanistic role in the conformational transition required for HHV Pr activity, and it forms a cryptic site that presents a new avenue for future allosteric inhibitor development.