Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma1,2, a cancer that commonly affects patients with AIDS3 and which is endemic in sub-Saharan Africa4. The KSHV capsid is highly pressurized by its double-stranded DNA genome, as are the capsids of the eight other human herpesviruses5. Capsid assembly and genome packaging of herpesviruses are prone to interruption6–9 and can therefore be targeted for the structure-guided development of antiviral agents. However, herpesvirus capsids—comprising nearly 3,000 proteins and over 1,300 Å in diameter—present a formidable challenge to atomic structure determination10 and functional mapping of molecular interactions. Here we report a 4.2 Å resolution structure of the KSHV capsid, determined by electron-counting cryo-electron microscopy, and its atomic model, which contains 46 unique conformers of the major capsid protein (MCP), the smallest capsid protein (SCP) and the triplex proteins Tri1 and Tri2. Our structure and mutagenesis results reveal a groove in the upper domain of the MCP that contains hydrophobic residues that interact with the SCP, which in turn crosslinks with neighbouring MCPs in the same hexon to stabilize the capsid. Multiple levels of MCP–MCP interaction—including six sets of stacked hairpins lining the hexon channel, disulfide bonds across channel and buttress domains in neighbouring MCPs, and an interaction network forged by the N-lasso domain and secured by the dimerization domain—define a robust capsid that is resistant to the pressure exerted by the enclosed genome. The triplexes, each composed of two Tri2 molecules and a Tri1 molecule, anchor to the capsid floor via a Tri1 N-anchor to plug holes in the MCP network and rivet the capsid floor. These essential roles of the MCP N-lasso and Tri1 N-anchor are verified by serial-truncation mutageneses. Our proof-of-concept demonstration of the use of polypeptides that mimic the smallest capsid protein to inhibit KSHV lytic replication highlights the potential for exploiting the interaction hotspots revealed in our atomic structure to develop antiviral agents.

Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes

“…As in HCMV and KSHV (20, 28), three types of MCP-MCP interactions form a hierarchical network of interactions on the HSV-1 capsid floor (Fig. 4, D and E, and Movie 5).…”

“…Instead, the Tri1 monomer inserts its N-terminal extension (N anchor) through the capsid floor and folds into a tri-lobed structure inside the capsid (Fig. 3, C, D, and L) (20, 28), anchoring the entire triplex. Selection pressure to mediate binding of divergent tegument proteins outside the capsid and to accommodate genomes of varying sizes inside the capsid could have also forced Tri1 to diverge on both its outer and inner sides.…”

“…Selection pressure to mediate binding of divergent tegument proteins outside the capsid and to accommodate genomes of varying sizes inside the capsid could have also forced Tri1 to diverge on both its outer and inner sides. Indeed, the Tri1 insertional arm, which emanates from a topologically similar location on one of the two β barrels as that of the Tri2 embracing arm, is the most externally located and is completely missing in HCMV Tri1 (20) and KSHV Tri1 (28). Likewise, the genome-facing N anchor of HSV-1 Tri1 is substantially longer than (102 versus 44 amino acids), and not as rigid as, that of HCMV (20).…”

“…Like that of HCMV or KSHV (20, 28), the HSV-1 MCP (VP5) subunit can be thought of as an extensive elaboration on a central bacteriophage HK97 gp5–like (“Johnson”) fold (amino acids 51 to 193, 232 to 298, 365 to 396, 1061 to 1113), with six additional domains, including the N lasso (1 to 50), dimerization (299 to 364), helix-hairpin (194 to 231), channel (407 to 480, 1335 to 1374), buttress (397 to 406, 1048 to 1060, 1114 to 1334) and upper (481 to 1047) domains (Fig. 4A).…”

Different capsid-binding patterns of the β-herpesvirus-specific tegument protein pp150 (pM32/pUL32) in murine and human cytomegaloviruses

“…Recent high-resolution cryoEM structures of human herpesviruses (12-14), particularly the demonstration of inhibitors designed based on the structure of small capsid protein (SCP) (12, 13), have opened the door to structure-guided design of new drugs and vaccines targeting HCMV capsid proteins and the β-herpesvirus-specific tegument protein pUL32 (or phosphoprotein pp150, see review 15). The cryoEM reconstruction of HCMV at 3.9 Å resolution (14) reveals that pUL32 forms a unique capsid-binding tegument layer, likely to secure encapsidation of its dsDNA genome of 235 kbp, which is the largest among all herpesviruses.…”