key: cord-0681767-sb4dpfue
authors: Liao, Hsiang-I.; Olson, C. Anders; Hwang, Seungmin; Deng, Hongyu; Wong, Elaine; Baric, Ralph S.; Roberts, Richard W.; Sun, Ren
title: mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein
date: 2009-06-26
journal: J Biol Chem
DOI: 10.1074/jbc.m901547200
sha: b16c2d1b41a3dbcb2f912810468ebc1b2f810f10
doc_id: 681767
cord_uid: sb4dpfue

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.

. (A) Alignment of S. cerevisiae Tfg2p with its human homolog RAP30. The PY-NLS is underlined and the PY motif is in bold. Identical residues are highlighted in black and similar residues in gray. Beta strands are represented by gray arrows and alpha helices by black bars. Secondary structure labels are from the RAP30 crystal (1f3u) and NMR structures (1bby). Asterisks denote residues mutated in the DNA binding domain. (B) Crystal structure of RAP74 (gray)/ RAP30 (blue) homodimerization domains. The PY-NLS insertion (red dashed line) is between RAP30 residues 88 and 89 (red solid line). 

Tfg2 DBD (284-365) was subcloned from the full length construct into the pGexTev vector. All point mutations were cloned using the QuikChange method (Stratagene) and confirmed by nucleotide sequencing. GST-DBD proteins were expressed and purified using the same method as GST-Kapβ. Gel filtration chromatography assay-5.2 nmol HIV-2 promoter DNA (GATATACCCG CTGCTC) (3) was subjected to gel filtration chromatography over a Superdex 75 (GE Healthcare) in the presence and absence of wild type and mutant Tfg2p DBD (1:2 DNA:DBD molar ratio). Absorbance was measured at 260 nm.

Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution

The PyMOL User's Manual, DeLano Scientific

Structural homology between the Rap30 DNA-binding domain and linker histone H5: implications for preinitiation complex assembly