Structures of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus virions reveal species-specific tegument and envelope features

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are classified into the gammaherpesvirus subfamily of Herpesviridae, which stands out from its alpha- and betaherpesvirus relatives due to the tumorigenicity of its members. Although structures of human alpha- and betaherpesviruses by cryogenic electron tomography (cryoET) have been reported, reconstructions of intact human gammaherpesvirus virions remain elusive. Here, we structurally characterize extracellular virions of EBV and KSHV by deep learning-enhanced cryoET, resolving both previously known monomorphic capsid structures and previously unknown pleomorphic features beyond the capsid. Through subtomogram averaging and subsequent tomogram-guided sub-particle reconstruction, we determined the orientation of KSHV nucleocapsids from mature virions with respect to the portal to provide spatial context for the tegument within the virion. Both EBV and KSHV have an eccentric capsid position and polarized distribution of tegument. Tegument species span from the capsid to the envelope and may serve as scaffolds for tegumentation and envelopment. The envelopes of EBV and KSHV are less densely populated with glycoproteins than those of herpes simplex virus 1 and human cytomegalovirus, representative members of alpha- and betaherpesviruses, respectively. This population density of glycoproteins correlates with their relative infectivity against HEK293T cells. Also, we observed fusion protein gB trimers exist within triplet arrangements in addition to standalone complexes, which is relevant to understanding dynamic processes such as fusion pore formation. Taken together, this study reveals nuanced yet important differences in the tegument and envelope architectures among human herpesviruses and provides insights into their varied cell tropism and infection.