Atomic structure of the human cytomegalovirus capsid with its securing tegument layer of pp150.

INTRODUCTION Human cytomegalovirus (HCMV) is a leading cause of congenital defects and a major contributor to life-threatening complications in immunocompromised individuals. HCMV is a β-herpesvirus that more broadly belongs to Herpesviridae, whose members have long been in lockstep with humanity, responsible for ailments from chickenpox (varicella zoster virus, VZV) to the common cold sore (herpes simplex virus 1, HSV-1). Yet HCMV’s ability to establish relatively nontoxic lifelong latency in hosts, its high seroprevalence in human populations, and its large genetic capacity are characteristics shared among herpesviruses that give them desirable advantages over other viral candidates as tools in the development of gene delivery vehicles, oncolytic vectors, and vaccines against not just herpesviruses, but even HIV/AIDS. RATIONALE All human herpesviruses have a highly pressured nucleocapsid (up to tens of atmospheres) thanks to a large genome that packs tightly within a space-constrained capsid. HCMV’s 235-kb genome is by far the largest of any human herpesvirus at twice the size of VZV’s and >50% larger than HSV-1’s, although HCMV has a capsid that is similar in size to those of other herpesviruses. Previous evidence has suggested that the β-herpesvirus–specific tegument protein pp150 contributes to a netlike layer that may stabilize the HCMV capsid, but in the absence of an atomic description of HCMV particles, the exact mechanisms through which capsid stability is achieved have remained unclear. Despite recent advances in high-resolution studies of macromolecular complexes, an atomic structure of a herpesvirus has proved elusive because of the immense challenges posed by their size (more than 2000 A in diameter) and the associated fragility of such large assemblies. RESULTS By using an improved sample preparation strategy and electron-counting cryo–electron microscopy, we obtained a three-dimensional reconstruction of HCMV at 3.9-A resolution and derived an atomic structure for the herpesvirus-conserved capsid proteins MCP, Tri1, Tri2, and SCP and the HCMV-specific tegument protein pp150, totaling ~4000 molecules and 62 different conformers. MCPs manifest as a complex of domain insertions around a bacteriophage HK97 gp5–like “Johnson fold” domain, which gives rise to three classes of capsid floor–defining interactions beneath hexons and analogous, though less substantial, interactions beneath pentons. Triplexes, composed of two “embracing” Tri2 conformers and a “third-wheeling” Tri1, fasten the capsid floor. Whereas these stabilization mechanisms are likely conserved across herpesviruses, our structure also reveals HCMV-specific capsid stabilization strategies, including hexon channels that facilitate the packing of DNA and pp150 helix bundles that secure the capsid through a critical cysteine tetrad interaction with SCP, the smallest and least conserved capsid protein across Herpesviridae. CONCLUSION With an exceptionally large genome and high internal capsid pressure, HCMV achieves capsid stability through an extreme form of structural elaboration on a basic Johnson fold topology, relying on not only domain insertions into the major capsid protein and the inclusion of auxiliary heterotrimers, but also the recruitment of a tegumental layer of pp150 to secure its DNA-engorged capsid from without. Beyond providing an organizational blueprint to understand all other herpesviruses, our HCMV atomic structure should inform rational design of therapeutic strategies against HCMV, other herpesviruses, and, in light of recent findings in simian models, potentially HIV/AIDS.