Kaposi's sarcoma (KS) is the most common malignancy associated with infection by human immunodeficiency virus (HIV). 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. Currently, no drugs specifically targeting lytic replication of KSHV are available. Additionally, atomic structures of KSHV viral genome packaging/ejection machinery and fusion-mediating glycoproteins needed for rational design of antiviral drugs and vaccines are unavailable. The prior four-years' funding of this multiple principal investigator (MPI) R01 project has led to the publication of the atomic structure of the KSHV capsid in Nature. Structure-guided mutagenesis studies have identified amino acid interactions among capsid proteins that are essential to capsid assembly and informed the design of small peptide mimics that inhibited viral maturation. Jointly, the MPIs' groups also published the first atomic model of the KSHV DNA-packaging portal complex and capsid associated tegument complexes in Cell. Preliminary data for this renewal application have established the feasibility of obtaining in situ structures of genome-packaging portal-terminase complex and the cell-entry glycoprotein B (gB) in both pre-fusion and post-fusion conformations. These portal protein and envelope glycoprotein structures and structure-guided mutagenesis results have led to three hypotheses: (1) the portal- associated proteins and terminase interactions are vital to KSHV genome encapsidation; (2) the interactions and conformational changes among envelope glycoproteins are required for KSHV fusion with host cells during cell entry; and (3) such interactions revealed in atomic structures can help design inhibitors and vaccines against KSHV lytic infection. The studies described in this application will test the above hypotheses by taking advantage of technology breakthroughs in high-resolution cryoEM and KSHV mutagenesis already demonstrated in the two MPIs' labs in the current funding cycle.
In Aim 1, we will determine the in situ structures of KSHV portal-associated proteins and the terminase to ~3 by cryoEM. From these structures, we will derive atomic models and identify amino- acid residues within 6 of interacting proteins (i.e., residues vital to DNA packaging and ejection).
In Aim 2, we will determine the structures of major envelope glycoproteins to ~3. Specifically, we will characterize the pre- fusion and post-fusion states of gB and the interactions of gH/gL, gM/gN, and K8.1A with their binding partners.
In Aim 3, we will refine our structural interpretation through structure-guided mutagenesis and identify target sites for inhibition of genome encapsidation and membrane fusion. Results from this research program will inform future development of drugs and novel vaccines against KSHV infection and spread. The novel approach established will be generally applicable to other viruses and complexes.
Kaposi's sarcoma-associated herpesvirus (KSHV) causes several types of cancer, including lymphomas and AIDS-associated malignancies. The atomic models of the KSHV portal-associated proteins, terminase, and envelope glycoproteins from this study will reveal protein-protein interactions at the level of specific chemical bonds and lead to a mechanistic understanding of genome packaging and membrane fusion?two steps in KSHV infection and spread. By targeting these interactions, this study will inform future development efforts for antiviral drugs against infections by KSHV and other herpesviruses.
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