Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM

Abstract

Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 Å resolution and describe the molecular interactions among the ~3000 protein molecules in the MCMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and β-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identifi ed as a "saddle loop" region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like fl oor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to “Megavirales”

“…Structural data for the entire protein within the assembled capsid, obtained by cryoEM, indicate that VP5 consists of the "floor" domain, which forms the icosahedral shell and is the only part of the protein that interacts with the capsid interior, as well as the "middle" and "top" domains protruding above the surface of the shell (Zhou et al, 2000). More recently, an assembly of additional domains has been recognized in the MCPs of HHV-1 (Hui et al, 2013) and of human cytomegalovirus (Human herpesvirus 5; Yu et al, 2017). A high-resolution X-ray structure is still available only for the top, or tower, domain of HHV-1, which is contiguous in the sequence of VP5, starting approximately at amino acid 500 (Bowman et al, 2003).…”

“…The structure of the tower domain of herpesvirus MCP is described as a pyramid with a square base (Bowman et al, 2003). Much attention has been given to the structural elements constituting the sides and the apex of the pyramid, as these sites appear to be involved in multiple protein-protein interactions (Hui et al, 2013). Less is known about the Table 2 Testing for relaxation of selection in the herpesvirus genes deposited with the assemblies of C. teleta and B. floridae.…”

“…The N-terminal region of MCPs in Herpesviridae was thought to have the HK97-like fold based on low-resolution cryo-EM (Baker et al, 2005;Hui et al, 2013). Very recently, the atomic structure of the entire MCP of HHV-5 has been determined by cryo-EM to 3.9 Å resolution, revealing that a modified HK97 fold is present in the structure, but it is assembled in space from several distal segments of the peptide chain.…”

Nature׳s favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold

“…The majority of capsids using the HK97-fold are of viruses infecting bacteria (bacteriophage or phage). However, this fold is also found in the coat protein floor domain of eukaryotic Herpesvirdae (shaded in Table 1 ), including Herpes simplex virus type 1 (HSV-1) (Baker et al, 2005; Zhou et al, 2000), murine cytomegalovirus (MCMV) (Hui et al, 2013), pseudorabies virus (PRV) (Homa et al, 2013), and rhesus monkey rhadinovirus (RRV) (Zhou et al, 2014), as well as the archaeal Haloarcula sinaiiensis tailed virus 1 (HSTV-1) (Pietila et al, 2013). …”

“…While no biochemical analysis has been performed, the location of these domains strongly suggests they are involved in capsomer stabilization (T4) or capsid stabilization ( 029). Herpesvirdae coat proteins have middle and upper domains protruding from the floor domain to form towers, which are involved in interactions with several outer capsid proteins, including the triplex proteins (VP19C and VP23), a capsid decorator protein (VP26), and tegument proteins (Homa et al, 2013; Hui et al, 2013; Zhou et al, 1999; Zhou et al, 2000). The crystal structure of the 65 kDa “upper domain’ of HSV-1 coat protein has been determined (Bowman et al, 2003).…”