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

Abstract

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.

Keywords: Capsid; HCMV; Replication; Structure; Tegument.

Fig. 1.

Structure-guided mutational analysis of the pp150-SCP interface. (A) Atomic model of the HCMV capsid with pp150 coat (left). The inset (right) shows zoomed-in view of subunits colored to emphasize different subunit interactions. (B) Sequence alignment of HCMV, murine CMV (MCMV), and simian CMV (SCMV) pp150 homologues between pp150 residues 240–276. (C) Surface interfaces between pp150 and SCP, with mutations colored by clusters to enhance detail (left). Surfaces with Hydrophilic, neutral, and hydrophobic areas colored in cyan white and gold respectively (middle) while positive, neutral, and negative electrostatic potentials are shown in blue, white, and red respectively (right). (D) Table listing cluster mutations, and their effect on virus replication, as observed in transfected cells. (E) Representative Fluorescence microscopy images of cells transfected with SCP mutant BACs, 1 (top) or 7 (bottom) days post transfection (DPT). (F) Same as (E) but from pp150 mutant BACs at 1 or 7 DPT.

Fig. 2.

Characterization of mutant pp150 and SCP interactions. (A) Immunofluorescence assays of ARPE-19 cells transfected with either WT or cluster mutations on pp150 or SCP showing colocalization between anti-HA (green) and anti-FLAG (red) with DAPI stained nuclei (blue). (B) Co-immunoprecipitation (CoIP) assays showing interactions of pp150 cluster mutants with WT SCP. Protein extracts were precipitated with anti-HA or anti-FLAG antibodies and resolved by SDS-PAGE. Protein-protein interactions were immunodetected using anti-HA and anti-FLAG antibodies.

Fig. 3.

Thin section TEM of ARPE-19 cells infected with WT and mutant HCMV (A) Representative TEM micrographs of WT (left) and AD169 pp150-C4 infected cells (right) with zoomed-in views highlighting different capsid types, with some A- (white arrowheads), B- (white with black outline arrowheads), and C-capsids (black arrowheads) indicated. (B) Distribution of A-, B-, and C-capsids as percentages of total counted and totals in accompanying table (right). (D) Representative micrograph of vAC from AD169 pp150-C4 infected cells with zoomed-in views of particles undergoing envelopment (right, top) along with dense bodies (DB) and examples of enveloped particles (right, bottom).

Fig. 4.

3D reconstructions of nucleocapsids reveal variable tegumentation. (A) Representative cryoEM images of A-, B-, and C-capsids purified from WT AD169 (left) and AD169 pp150-C4 mutant infected cells (right) and a virion particle (lower middle). Comparison of MCP towers in 2D electron micrographs (inset greyscale images, blue arrowhead) and empty spaces (black outlined arrowheads) to equivalent regions from whole capsid 3D reconstructions (insets) from B. (B) 3D Reconstruction of WT (left) and AD169 pp150-C4 A- and B-capsids colored by distance from center of capsid with MCP towers (blue arrowheads) and the empty spaces between them (black outlined arrowheads). (C) Capsid vertex sub-particle reconstruction from particles in B, colored by capsid proteins to convey nucleocapsid composition, viewed from outside the capsid and as a cross section (bottom). (D) Low resolution vertex sub-particle reconstruction from the WT C-capsids showing lacking pp150 density. (E) Representative cryoEM images demonstrating fuzzy tegument (black arrow) around B- (top) and A-capsids (bottom) with percentages from total B- and A-capsids observed with tegument and (F) a vertex sub-particle generated from particles in E (right) showing the presence of pp150 density (cyan) and asymmetric unit with distribution of pp150 and tRNA (right). (G) Representative images of pp150-C4 mutant capsids with fuzzy tegument (black arrows).