Structure-guided mutagenesis targeting interactions between pp150 tegument protein and small capsid protein identify five lethal and two live-attenuated HCMV mutants.

Human cytomegalovirus (HCMV) replication relies on a nucleocapsid coat of the 150 kDa, subfamily-specific tegument phosphoprotein (pp150) to regulate cytoplasmic virion maturation. While recent structural studies revealed pp150-capsid interactions, the role of specific amino-acids involved in these interactions have not been established experimentally. In this study, pp150 and the small capsid protein (SCP), one of pp150's binding partners found atop the major capsid protein (MCP), were subjected to mutational and structural analyses. Mutations to clusters of polar or hydrophobic residues along the pp150-SCP interface abolished viral replication, with no replication detected in mutant virus-infected cells. Notably, a single amino acid mutation (pp150 K255E) at the pp150-MCP interface significantly attenuated viral replication, unlike in pp150-deletion mutants where capsids degraded outside host nuclei. These functionally significant mutations targeting pp150-capsid interactions, particularly the pp150 K255E replication-attenuated mutant, can be explored to overcome the historical challenges of developing effective antivirals and vaccines against HCMV infection.