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Development of RNA aptamers targeting Ebola virus VP35.

ABSTRACT: Viral protein 35 (VP35), encoded by filoviruses, is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion, and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for nonenzymatic targets, are often challenging. Our previous work on filoviral VP35 proteins defined conserved basic residues located within its C-terminal dsRNA binding interferon (IFN) inhibitory domain (IID) as important for VP35 mediated IFN antagonism and viral polymerase cofactor functions. In the current study, we used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target for aptamer selection using SELEX. Select aptamers, representing, two distinct classes, were further characterized based on their interaction properties to eVP35 IID. These results revealed that these aptamers bind to distinct regions of eVP35 IID with high affinity (10-50 nM) and specificity. These aptamers can compete with dsRNA for binding to eVP35 and disrupt the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize the eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by the expression of select viral proteins. Taken together, our results support the identification of two aptamers that bind filoviral VP35 proteins with high affinity and specificity and have the capacity to potentially function as filoviral VP35 protein inhibitors.

SUBMITTER: Binning JM

PROVIDER: S-EPMC3909728 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Development of RNA aptamers targeting Ebola virus VP35.

Binning Jennifer M JM Wang Tianjiao T Luthra Priya P Shabman Reed S RS Borek Dominika M DM Liu Gai G Xu Wei W Leung Daisy W DW Basler Christopher F CF Amarasinghe Gaya K GK

Biochemistry 20131114 47

Viral protein 35 (VP35), encoded by filoviruses, is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion, and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for nonenzymatic targets, are often challenging ...[more]

PMID: 24067086

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Project description:Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.

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Development of RNA aptamers targeting Ebola virus VP35.

Publications

Development of RNA aptamers targeting Ebola virus VP35.

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