P1-12 Development and Evaluation of Nucleic Acid Aptamers to a Novel Target Protein for Treatment and Detection of Human Norovirus

Monday, July 10, 2017
Exhibit Hall (Tampa Convention Center)
Matthew Moore , North Carolina State University , Raleigh , NC
Jeremy Faircloth , North Carolina State University , Raleigh , NC
Lee-Ann Jaykus , Department of Food, Bioprocessing, and Nutritional Sciences, North Carolina State University , Raleigh , NC
Introduction: One of the challenges for rapid control and detection of human noroviruses is their high degree of diversity, which makes generation of broadly-reactive ligands difficult, as these ligands target the viral capsid proteins. Noroviruses contain a more widely conserved protein, VPg, covalently linked to the 5’ end of the ssRNA genome. VPg is necessary for replication of the virus as it recruits host cell translation machinery at the beginning of the viral replication cycle, but has not been a main focus for generation of therapeutics or ligands for detection. Nucleic acid aptamers are emerging ligands that have shown promise as therapeutics and ligands for numerous viruses.

Purpose: The purpose of this study was to generate nucleic acid aptamers targeting the VPgs of human Norwalk virus (NV) and Tulane virus (TV), a cultivable surrogate.

Methods: The VPg regions of TV and NV were cloned into Escherichia coli, overexpressed, and used as targets for selection with aptamer pools containing a 40 nucleotide variable region. After aptamers were selected, their Gibbs energy values (DGs), secondary structures, and shared sequence motifs were evaluated. The aptamers effectiveness at reducing viral replication for TV were evaluated by plaque assay.

Results: After eight rounds of selection, 5 and 10 unique sequences out of 17 and 21 sequence pools (n) for TV and NV were identified, respectively. Numerous unique secondary structures with overlapping motifs were identified, with DGs ranging from -7.79 to -18.07 and -9.75 to -18.13, suggesting sequences form stable structures. Both TV (76%) and NV (38%) pools had a predominant sequence, suggesting good selection; and one similar sequence was shared between pools. Plaque assays showed TV VPg aptamers were dose-dependent, with log10reductions ranging from 0.12 to 1.05, which is comparable to other published therapeutic virus aptamers.

Significance: This work demonstrates the potential of a hitherto, under-investigated, protein target for human norovirus treatment and detection.