Organization of Capsid-Associated Tegument Components in Kaposi's Sarcoma-Associated Herpesvirus

Abstract

Capsid-associated tegument proteins have been identified in alpha-and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a ␤-sheet-rich triplexbinding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a longstanding mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein. IMPORTANCESeveral capsid-associated tegument proteins have been identified in the alpha-and betaherpesvirus subfamilies of the Herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

Gammaherpesvirus Tegument Protein ORF33 Is Associated with Intranuclear Capsids at an Early Stage of the Tegumentation Process

“…ORF25, which makes up the capsomere, has three domains: the floor, middle, and upper domains (35). Binding of the inner tegument protein(s) to the capsid is mediated by the upper domain of the major capsid proteins and the triplex protein(s) (1,35,36). We previously showed that capsid formation is not affected in the absence of ORF33, and ORF33 is an inner tegument protein of mature MHV-68 virions (8).…”

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

“…Although pUL77-mGFP and pUL93 could still interact when capsid formation was abrogated-for instance, upon disruption of the MCP ORF-proper nuclear localization of pUL77 seems to depend on additional factors. Whether pUL77 and pUL93 interact directly, as reported for the corresponding proteins of alpha-and gammaherpesviruses (23,24,75), remains to be tested by expressing the proteins independent of viral infection. Interestingly, in the accompanying article by P. Köppen-Rung and colleagues (84), direct interaction of pUL77 and pUL93 was confirmed by using such an approach, and the domain mediating the binding to UL93 was mapped to the N terminus of pUL77.…”

Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression

“…1D), indicating that they expressed an amount of RTA sufficient for lytic reactivation. It is important to note that the 3= end of the ORF31 coding sequence overlaps the 5= end of ORF32, a gene encoding a capsid-associated tegument protein that is homologous to members of the UL17 family of genes (23). UL17 of herpes simplex virus 1 (HSV-1) is essential for DNA packaging into the capsid and, thus, infectious virus production (24).…”