mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein
The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with K_d = 1.7 nM. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication.
Additional Information© 2009 the American Society for Biochemistry and Molecular Biology. Received for publication, March 6, 2009. Originally published In Press as doi:10.1074/jbc.M901547200 on April 13, 2009. We thank the University of Southern California Nanobiophysics core facility for Biacore use. This work was supported, in whole or in part, by National Institutes of Health Grant RO1 GM60416 (to R. W. R.). This work was also supported by a microbial pathogenesis training grant from Burroughs Wellcome Fund. The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods, additional references, and Figs. S1–S7.
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