Structure and mutagenesis reveal essential capsid protein interactions for KSHV replication.

Area of Science:

• Structural Biology
• Virology
• Biochemistry

Background:

• Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer prevalent in AIDS patients and sub-Saharan Africa.
• Herpesvirus capsids are large, complex structures under high pressure from their DNA genome, making them challenging targets for antiviral development.
• Understanding capsid assembly and genome packaging is crucial for targeting herpesvirus replication.

Purpose of the Study:

• To determine the high-resolution atomic structure of the KSHV capsid.
• To map the molecular interactions involved in capsid stability and assembly.
• To identify potential targets for antiviral drug development.

Main Methods:

• Cryo-electron microscopy (cryo-EM) with electron counting to achieve 4.2 Å resolution.
• Atomic model building of the KSHV capsid, including major capsid protein (MCP), smallest capsid protein (SCP), and triplex proteins (Tri1, Tri2).
• Mutagenesis studies to verify the functional roles of identified protein interaction sites.

Main Results:

• A detailed atomic model of the KSHV capsid was generated, revealing 46 unique protein conformers.
• A critical groove on the MCP was identified, mediating interactions with SCP to stabilize the capsid structure.
• Multiple interaction interfaces between MCP subunits, including stacked hairpins, disulfide bonds, and N-lasso/dimerization domains, contribute to capsid robustness.
• Triplex proteins (Tri1/Tri2) were shown to anchor the capsid floor, plugging holes and reinforcing the structure via Tri1 N-anchor interactions.
• Mutagenesis confirmed the essential roles of MCP N-lasso and Tri1 N-anchor domains.

Conclusions:

• The high-resolution KSHV capsid structure elucidates intricate protein-protein interactions essential for capsid integrity.
• Identified interaction hotspots, particularly involving MCP and SCP, provide a basis for structure-guided antiviral drug design.
• Polypeptides mimicking SCP demonstrated potential in inhibiting KSHV lytic replication, validating the therapeutic strategy.