Structural and dynamic studies of viral capsid proteins

Jae Eun Suk, Purdue University

Abstract

West Nile virus(WN) is a member of the flavivirus Flaviviridae family and is one of the most significant human viral pathogens. The virus is comprised of a nucleocapsid core that consists of a viral RNA genome encapsidated by capsid protein, which is surrounded by a host-derived lipid bilayer and membrane-embedded envelope glycoproteins. The capsid protein (CP) plays key roles during the assembly process to produce infectious virus. The structure and dynamics of WN CP have been here investigated by multidimensional NMR techniques. The solution structure of WN CP was successfully determined using structure-based NOE assignment and restrained Molecular Dynamic simulations. Although two well-converged structures for WN CP were obtained by the structure-based calculation approach, careful examination of the calculated structures allows distinguishing the correct conformation of WN CP. The structure of WN CP is similar to that of Kunjin virus (KUN) and not that of dengue (DEN). (Both are members of the flavivirus family). The backbone dynamics of WN CP were characterized using ¹⁵N relaxation measurements. The overall correlation time estimated from the R₂/R₁ ratio indicates that WN CP is a dimer in solution. The order parameter of WN CP reveals that the structure of the protein is maintained by very rigid helices and tightly associated loops. Comparison of the internal mobility of WN CP with that of KUN and DEN CP shows that WN and KUN CP share the same characteristic backbone dynamics; however WN and DEN CP have distinct dynamic properties. The dynamic studies suggest that two flavivirus CP - WN CP and DEN CP indeed have different structures as well as different dynamic properties. Sindbis virus (SIN) is a member of the alphavirus family. Like flaviviruses, it has a relatively simple structure consisting of a nucleocapsid core, formed from genomic RNA and capsid proteins, surrounded by a membrane and envelope proteins. The capsid protein (CP) of SIN is responsible for genome packaging and budding of virus particles. Different functional regions of SIN CP have been identified. The structural and dynamic properties of residues 81-112 of CP(81-264) responsible for binding RNA were characterized here by NMR. Based on chemical shift indexes and ¹⁵N relaxation data, the residues 81-112 of SIN are largely disordered with a tentative helix around residue 103. Secondary structure analysis of C-terminal protease domain of residues 115 -264 of SIN CP indicates that the solution structure of this region is similar to that of the crystal structure. ¹⁵N relaxation data indicate that SIN CP(81-264) is a monomer in solution, suggesting that oligomerization of SIN CP requires RNA. NMR studies on a CP(81-112) peptide and encapsidation signal RNA show that interaction of the peptide and the RNA triggers conformational changes in both molecules. CP(81-112) originally unstructured becomes ordered upon interaction to the RNA while the RNA, which is folded without CP, appears unchanged except for chemical exchange of several imino protons. The hydrophobic pocket on the surface of SIN CP has been proposed as an interaction site for the cytoplasmic domain of the E2 envelope protein. Potential inhibitors targeting this site have been developed and screened. The binding properties of two active inhibitors and dioxane were examined using chemical shift perturbation. NMR study showed that two potential inhibitors as well as dioxane were found to bind the proposed hydrophobic pocket. However the estimated affinity associated with these interactions is low for all three compounds.^

Degree

Ph.D.

Advisors

Carol B. Post, Purdue University.

Subject Area

Chemistry, Biochemistry|Biology, Virology

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