Herpesviruses possess a genome-pressurized capsid. The 235-kilobase genome of human cytomegalovirus (HCMV) is by far the largest of any herpesvirus, yet it has been unclear how its capsid, which is similar in size to those of other herpesviruses, is stabilized. Here we report a HCMV atomic structure consisting of 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 insertions around a bacteriophage HK97 gp5–like domain, which gives rise to three classes of capsid floor–defining interactions; triplexes, composed of two “embracing” Tri2 conformers and a “third-wheeling” Tri1, fasten the capsid floor. HCMV-specific strategies include using hexon channels to accommodate the genome and pp150 helix bundles to secure the capsid via cysteine tetrad–to-SCP interactions. Our structure should inform rational design of countermeasures against HCMV, other herpesviruses, and even HIV/AIDS.

Herpesviruses are enveloped viruses prevalent in the human population, responsible for a host of pathologies ranging from cold sores to birth defects and cancers. They are characterized by a highly pressurized, T (triangulation number) = 16 pseudo-icosahedral capsid encapsidating a tightly packed dsDNA genome1–3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package, and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Though this process has been studied in dsDNA phages6–9—with which herpesviruses bear some similarities—a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. Thus, to better define the structural basis of genome packaging and organization in the prototypical herpesvirus, herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryoEM images of HSV-1 virions, enabling us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we show in situ structures of the unique portal vertex, genomic termini, and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex not observed in phages, indicative of adaptations in the DNA-packaging process specific to herpesviruses. Finally, our atomic models of portal vertex elements reveal how the five-fold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal—long a mystery in icosahedral viruses—and inform possible DNA sequence-recognition and headful-sensing pathways involved in genome packaging. Our work represents the first fully symmetry-resolved structure of a portal vertex and first atomic model of a portal complex in a eukaryotic virus.

Graphical Abstract Highlights d Genome-packing portal and capsid-associated tegument complexes (CATCs) resolved d Partial and asymmetric CATC occupancy introduces structural variability d CATC binding introduces 120 counterclockwise rotation of triplex Ta on the capsid d Structure-based mutageneses reveal binding hotspot for antiviral development SUMMARYAssembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an $150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.