Abstract
Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.
Highlights
Influenza viruses are responsible for serious respiratory diseases and are deemed to be one of the biggest threats to human health
The HA1 genes were cloned in the pGEX-4T-1 expression vector (S1A Fig), transformed into Rosetta 2(DE3) cells, and GST-tagged proteins H1-HA1 and H5-HA1 were purified by glutathione-agarose affinity chromatography and Sephadex G-100 gel-filtration chromatography, as described in Supporting Information
The identification of the type of HA present on the viral surface could aid in the determination of the species that can be infected and the type of sialic acid that is necessary (Siaα2-6Gal or Siaα2-3Gal) for viral infection [34]
Summary
Influenza viruses are responsible for serious respiratory diseases and are deemed to be one of the biggest threats to human health. Belonging to the family Orthomyxoviridae, influenza virus is an enveloped virus with single-stranded negative-sense RNA consisting of eight segments [1]. Its two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [2], are highly expressed during viral infection and serve as targets for immune detection. 18 HA (H1–H18) and 11 NA (N1–N11) have been identified [3], and influenza is classified on the basis of the subtypes of HA and NA proteins. Subtype H5 is known as highly pathogenic in PLOS ONE | DOI:10.1371/journal.pone.0125060. Subtype H5 is known as highly pathogenic in PLOS ONE | DOI:10.1371/journal.pone.0125060 April 22, 2015
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