Lipid-enveloped viruses replicate and bud from host cell membranes where they acquire their lipid coat. Understanding the budding processes of several viruses has had significant impact on elucidating the viral life cycle and identifying therapeutic targets. Filoviruses have a filamentous lipid- envelopeanddespitebeingdiscoveredmorethan30yearsago,notmuchisknownonhowtheyassemble and bud from the host cell plasma membrane. Filoviruses, which include Ebola virus (EBOV), have a high fatality rate and there is still a lack of FDA approved therapeutics or vaccines for treatment. Moreover, the EBOV glycoprotein, the prime target of antibody and vaccine therapy undergoes a high rate of mutation in animal and human studies and escape mutant of glycoprotein have been found as EBOV is passaged through animal models. Filoviruses encode seven genes including the viral matrix protein VP40, which regulates budding from the host cell. VP40 as the only filovirus protein expressed in mammalian cells is sufficient to produce virus like particles (VLPs) nearly indistinguishable from live virions. Thus, VP40 has served as a model to study viral budding outside of BSL-4 laboratories. VP40 has been shown to be a dimer, which is mediated by a?-helical interactions in its N-terminal domain (NTD). Mutation of residues in theNTDofVP40thatmediatedimerizationissufficienttoabrogateviralbuddinginmodelsystems.Todate, little is known about how VP40 monomer/dimer equilibrium and biophysics of oligomer assembly are regulatedaswellasifVP40isaviabledrugtargetinthevirallifecycle.ThecentralhypothesisofthisR21 proposalisthatgenerationofanewchemicaltoolkitbaseduponstapleda?-helicalpeptides canbeusedto study VP40 assembly and inhibit VP40 dimerization.
In specific aim 1, we will design and synthesize lead candidate stapled a?-helical peptides that target the VP40 dimer interface. We will elucidate the optimal amino acid sequences and chemical linker of stapled a?-helical peptides using computational analysis. We hypothesizethatoptimizationofthestapledhelicescanbeperformedtoblockVP40dimerformationinvitro and in cells. We will use computational analysis and a rapid chemical synthesis method to generate lead candidates for quantitative analysis.
Specific aim 2 will investigate the mechanism by which stapled a?- helicalpeptidesinteractwithVP40andinhibitVP40dimerizationandbuddingofVLPs. Quantitativeassays ofVP40dimerformation,VP40lipid-binding,andbuddingofVLPswillbeassessedtodeciphertheabilityof lead compounds to inhibit dimer formation and subsequent budding. Taken together, these studies should produce new and important mechanistic insight into the viability of VP40 as a drug target and a better biophysicalunderstandingofthepropertiesthatgovernVP40assembly.
To date there is a lack of FDA approved treatment options or preventive measures for infections by Filoviruses. The proposed studies will investigate a new pharmacological approach to inhibition of the Ebola virus assembly and replication. Specifically, chemically modified peptides will be synthesized and tested to potentially inhibit the Ebola virus budding from infected cells.
