earticle

영어

Influenza NP is essential to viral replication, providing an ideal target for antiviral therapy. NP has been targeted at the protein level for antiviral therapy by a small molecule, but it is extremely difficult to be targeted by antibody since it is not exposed at the viral surface. Here we present a novel antibody-based antiviral strategy of targeting viral genomic RNA (vRNA) for degradation rather than neutralizing viral proteins. Based on the template of a seqeunce-nonspecific nucleic acid-hydrolyzing, single domain antibody of the light chain variable domain, 3D8 VL, we generated a synthetic library on the yeast surface by randomizing putative nucleic acid interacting residues. To target nucleocapsid protein (NP)- encoding viral genomic RNA (NP-vRNA) of H9N2 influenza virus, the library was screened against a 18- nucleotide single stranded nucleic acid substrate, dubbed asNP18, the sequence of which is unique to the NP- vRNA. We isolated a 3D8 VL variant, NP25, that had ~15-fold higher affinity (~54 nM) and ~3-fold greater selective hydrolyzing activity for the target substrate than for off targets. In contrast to 3D8 VL WT, asNP18 -selective NP25 constitutively expressed in the cytosol of human lung carcinoma A549 cells does not exhibit any signficant cytotoxicity and selectively degrades a reporter mRNA carrying the target asNP18 sequence in the stable cell lines. NP25 more potently inhibits the replication of H9N2 influenza virus than 3D8 VL WT in the stable cell lines. NP25 more selectively reduces the amount of the targeted NP-vRNA than 3D8 VL WT from the early stage of virus infection in the stable cell lines, without noticeable harmful effects on the endogenous mRNA, suggesting that NP25 indeed more specifically recognizes to hydrolyze the target NP-vRNA of H9N2 virus than off-targets. Our results provide a proof-of-concept of targeting viral genomic vRNAs by the sequence-specific nucleic acid hydrolyzing antibody for the prevention and control of influenza A virus infection in humans and animals.