Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome

Abstract

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.

Structural morphing in a symmetry-mismatched viral vertex

“…2c, d), even though the actual salt-bridge forming residues vary, thus depicting an extraordinary adaptation of portal's face while maintaining symmetry in the rest of the structure. Analysis of other portal structures suggest that the Nterminal region and loops located in the wing domain periphery are also flexible, implicating that similar symmetry-mismatch compensation mechanisms are likely employed by other viruses [15][16][17]35,36 .…”

“…However, despite more than five decades of intensive research and debate since discovery in 1965 [12][13][14] , the structure of the symmetry-mismatched viral portal vertex, the unique interactions at the interface of capsid and portal, and how these interactions drive virus morphogenesis remained mysterious. Even the most recent high resolution cryo-electron microscopy (cryo-EM) structures of phages or herpesviruses could not resolve the symmetry-mismatched interface [15][16][17] .…”

Cryo-EM analysis of a viral portal protein in situ reveals a switch in the DNA tunnel

“…Overall, this describes a packaging mechanism that is naturally safeguarded against genome loss. At full packaging, DNA can be held in place by constricted tunnel loops and reinforced by tail factors (5,16). The portal's high order of symmetry reconciles a symmetry mismatch.…”

“…The portal can therefore utilise the same few residues to interact around its circumference, which contrasts with the situation that would exist if the portal possessed C6, C3, or other lower symmetries matching that of tail components. The symmetry mismatch of the interaction is a general feature amongst all tailed bacteriophages and related viruses, including herpesviruses (5,16). In the case of bacteriophage 29 prohead (8,17), one of the structural roles of the pRNA appears to be equivalent to that of the capsid protein P-domain, in interacting with the outside of the portal Clip, with the 29 capsid protein Pdomain instead making contact with an N-terminal segment of the portal protein.…”

“…The influence of the properties of the internal tunnel, and how these can be modulated by external factors to coordinate DNA translocation, remains unclear. Cryo-EM studies on mesophilic herpesviruses characterised the shape of the portal protein tun-nel in the mature virion and showed how DNA can be locked inside (5,16). However, there are no detailed structural data on portal proteins in situ for unexpanded capsids, primed for DNA packaging.…”