As a cancer of endothelial origin that typically grows under the skin or mucous membranes, KS in AIDS patients mostly manifests as oral lesions. Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the gammaherpesvirus subfamily of the Herpesviridae family, has been shown to be an etiologic agent of all forms of KS, primary effusion lymphoma and multicentric Castleman's disease. Central to pathogenesis and spread of KSHV is lytic replication, a process that begins with the assembly of capsids inside host cells and ends with the release of infectious virions into the extra-cellular space for viral propagation. Currently, no drugs specifically targeting lytic replication of KSHV are available and no atomic capsid structures are available needed for rational design of anti-viral drugs and vaccines against KSHV infection. By cryo electron microscopy (cryoEM) in 1999, PI Zhou's group published the first three-dimensional (3D) structure of KSHV capsid isolated from AIDS patient-derived BCBL-1 cells, followed by 7-resolution structures of the murine (with PI Sun) and rhesus monkey gammaherpesviruses, revealing molecular interactions among gammaherpesvirus capsid proteins. Recently, the two PIs' collaborative efforts with improved resolution (4.5) cryoEM and bacterial artificial chromosome (BAC) mutagenesis of KSHV have mapped several important segments of the smallest capsid protein (SCP, ORF65) in cementing the major capsid protein (MCP) of KSHV. These structure results, together with published results from residue-scanning mutagenesis, have led to the hypotheses that the SCP-MCP interactions are vital to KSHV assembly and such interactions revealed in an atomic structure can be targeted for inhibitors against KSHV lytic infections. The studies described in this application will test the above hypotheses by taking advantage of technology breakthroughs in high-resolution cryoEM and KSHV BAC mutagenesis already established in the two PIs' labs.
In Aim 1, we will determine the structure of KSHV capsid to ~3 by cryoEM with the revolutionary direct electron counting technology. From this cryoEM map, we will derive an atomic model of the KSHV capsid and identify amino-acid residues within 6 of interacting capsid proteins - particularly those between SCP and MCP - i.e., residues vital to capsid assembly.
In Aim 2 a, we will refine our structural interpretation of interacting amino acids by correlating existing and new data from site-specific mutagenesis and assess their impact on capsid assembly. Next, key segments and specific bonds identified among SCP-SCP and SCP-MCP interactions will be targeted to design both dominant negative mutants (i.e., cell-expressed) and novel peptides (i.e., chemically-synthesized) to select potent inhibitors that can disrupt KSHV lytic replication (Aim 2b). Results from this research program will inform future development of therapeutics against KSHV infection and spread. The novel approach established will be generally applicable to other viruses and complexes.
The atomic model of the KSHV capsid resulting from this study will reveal protein-protein interactions at the level of specific chemical bonds and lead to mechanistic understanding of capsid assembly of this and other herpesviruses. By targeting interactions vital to KSHV capsid assembly, this study will design and optimize peptide inhibitors, thus informing future development efforts for anti-viral drugs against infections by KSHV and other herpesviruses.
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