mRNA DISPLAY DESIGN OF FIBRONECTIN-BASED INTRABODIES THAT DETECT AND INHIBIT SARS-COV N PROTEIN

1 mRNA DISPLAY DESIGN OF FIBRONECTIN-BASED INTRABODIES THAT DETECT AND INHIBIT SARS-COV N PROTEIN Hsiang-I Liao, C. Anders Olson, Seungmin Hwang, Hongyu Deng, Elaine Wong, Ralph S. Baric, Richard W. Roberts, Ren Sun From Department of Department of Molecular and Medical Pharmacology, and California Nano System Institute, University of California, Los Angeles, 90095, Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125, Department of Epidemiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, 27599, and Department of Chemistry, Chemical Engineering, and Biology, University of Southern California, 3710 McClintock Avenue, Los Angeles, California, 90089-1211 These authors contributed equally Running Head: Fibronectin-based intrabodies that target SARS-CoV N Address correspondence to: Ren Sun, PhD, 650 Charles E. Young Drive South, CHS 23-120, University of California at Los Angeles, Los Angeles, CA 90095. Fax: 310-825-6267; Email: [email protected] The nucleocapsid (N) protein of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) plays important roles in both viral replication and modulation of host cell processes. New ligands that target 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 Kd = 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 11to 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.