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 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 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.
Department of Molecular and Medical Pharmacology, California Nano System Institute, UCLA, Los Angeles, CA 90095, USA.
Hsiang-I
HI
Liao
C Anders
CA
Olson
Seungmin
S
Hwang
Hongyu
H
Deng
Elaine
E
Wong
Ralph S
RS
Baric
Richard W
RW
Roberts
Ren
R
Sun
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000086462:Coronavirus Nucleocapsid Proteins; D005353:Fibronectins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D045169:Severe Acute Respiratory Syndrome; D014709:Vero Cells; D014779:Virus Replication
D000911:Antibodies, Monoclonal; D000086462:Coronavirus Nucleocapsid Proteins; D005353:Fibronectins; D019590:Nucleocapsid Proteins; D019151:Peptide Library; D012333:RNA, Messenger