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mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein*

Open AccessPublished:April 13, 2009DOI:https://doi.org/10.1074/jbc.M901547200
      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.
      The ability to detect and inhibit protein function is central to molecular and cellular biology research. To date, phage display and monoclonal antibody production have been the most common routes to design reagents for protein detection and inhibition, antibodies and antibody-like reagents that serve as high affinity, high specificity molecular recognition tools (
      • Maynard J.
      • Georgiou G.
      ). Totally in vitro selection methods using alternative scaffolds are becoming more common to produce affinity reagents with improved and expanded functionality (
      • Lipovsek D.
      • Plückthun A.
      ,
      • Hosse R.J.
      • Rothe A.
      • Power B.E.
      ). For example, ribosome display and mRNA display enable creating 1–100 trillion-member peptide and protein libraries that surpass immunological and phage display diversities by 3–5 orders of magnitude (
      • Roberts R.W.
      • Szostak J.W.
      ).
      Antibodies or antibody-like molecules are important because they can serve as diagnostics, probes for studying proteins in vivo, and potential therapeutics (or surrogate ligands for therapeutic design/screening). Regarding biology, antibodies used inside living cells, denoted “intrabodies,” are appealing because they provide an alternative to genetic knock-outs, dominant negative mutations, and RNA interference strategies, enabling targeting proteins in a domain-, conformation-, and modification-specific fashion as well as identifying hot spots for protein interaction (
      • Carlson J.R.
      ,
      • Stocks M.
      ). For example, green fluorescent protein-labeled intrabodies can act as molecular beacons to determine real time, live cell localization of endogenous target proteins rather than non-native expression of green fluorescent protein target fusions (
      • Nizak C.
      • Monier S.
      • del Nery E.
      • Moutel S.
      • Goud B.
      • Perez F.
      ).
      Although antibodies often demonstrate laudable affinity and selectivity, these proteins are likely to be suboptimal as a general approach to create intracellular reagents. Most notably, antibodies contain disulfide bonds that are likely to be reduced in the cytosol, thus impeding their proper folding and function (
      • Rajpal A.
      • Turi T.G.
      ). To overcome the paucity of functional intrabodies generated by in vitro selection methods, in vivo screens may be employed at the expense of combinatorial diversity (
      • Visintin M.
      • Tse E.
      • Axelson H.
      • Rabbitts T.H.
      • Cattaneo A.
      ). On the other hand, it has been demonstrated that intracellular antibodies can generate aggresomes, which may inhibit the ubiquitin-mediated degradation pathway and promote apoptosis (
      • Vascotto F.
      • Campagna M.
      • Visintin M.
      • Cattaneo A.
      • Burrone O.R.
      ,
      • Cardinale A.
      • Filesi I.
      • Mattei S.
      • Biocca S.
      ,
      • Cardinale A.
      • Filesi I.
      • Biocca S.
      ).
      Ideally, intrabodies would be as follows: 1) easy to produce in a broad variety of cells; 2) stable; 3) specific; 4) high affinity; 5) highly selective; 6) functional in intracellular environments; and 7) noninterfering with normal cellular processes. Recently, ribosome display has been used to generate protein affinity reagents based on ankyrin domains (DARPins), which detect and inhibit kinase or proteinase function in vivo (
      • Kawe M.
      • Forrer P.
      • Amstutz P.
      • Plückthun A.
      ,
      • Amstutz P.
      • Binz H.K.
      • Parizek P.
      • Stumpp M.T.
      • Kohl A.
      • Grütter M.G.
      • Forrer P.
      • Plückthun A.
      ). Although this scaffold is powerful, it is structurally very different from antibodies as it utilizes a discontinuous binding surface rather than the continuous surface generated by the CDR loops in antibody VH and VL domains.
      Our approach here has been to use mRNA display to design disulfide-free antibody-like proteins that can be used to create general protein targeting tools. To do this, we used a protein library based on the 10th fibronectin type III domain of human fibronectin (10Fn3)
      The abbreviations used are: 10Fn3
      human fibronectin 10th fibronectin type III domain
      SARS-CoV
      severe acute respiratory disorder coronavirus
      N
      nucleocapsid protein
      s2m
      stem-loop 2 motif
      NTD
      N-terminal domain
      CTD
      C-terminal domain
      Ni-NTA
      nickel-nitrilotriacetic acid
      WT
      wild type.
      3The abbreviations used are: 10Fn3
      human fibronectin 10th fibronectin type III domain
      SARS-CoV
      severe acute respiratory disorder coronavirus
      N
      nucleocapsid protein
      s2m
      stem-loop 2 motif
      NTD
      N-terminal domain
      CTD
      C-terminal domain
      Ni-NTA
      nickel-nitrilotriacetic acid
      WT
      wild type.
      (
      • Olson C.A.
      • Roberts R.W.
      ,
      • Koide A.
      • Bailey C.W.
      • Huang X.
      • Koide S.
      ). The 10Fn3 domain was developed as an antibody mimetic by Koide et al. (
      • Koide A.
      • Bailey C.W.
      • Huang X.
      • Koide S.
      ) because of the following: 1) it is topologically analogous to the immunoglobulin VH domain; 2) it is exceptionally stable; 3) it presents a continuous protein interaction surface; and 4) it expresses well in both eukaryotic and bacterial cells (
      • Koide A.
      • Bailey C.W.
      • Huang X.
      • Koide S.
      ). We recently described construction and characterization of a 3 × 1013 member 10Fn3 library (
      • Olson C.A.
      • Roberts R.W.
      ) and validated this library by developing proteins and fluorescence resonance energy transfer sensors that recognize IκBα in a phosphoserine-specific fashion (
      • Olson C.A.
      • Liao H.I.
      • Sun R.
      • Roberts R.W.
      ). There the selected 10Fn3 functioned in vivo, blocking proteasome-mediated degradation of full-length IκBα efficiently.
      Here we have targeted the severe acute respiratory syndrome (SARS-CoV) nucleocapsid protein (N). SARS-CoV is a unique member of the Coronaviridae family with only 20% sequence identity to the closest homolog (
      • Rota P.A.
      • Oberste M.S.
      • Monroe S.S.
      • Nix W.A.
      • Campagnoli R.
      • Icenogle J.P.
      • Peñaranda S.
      • Bankamp B.
      • Maher K.
      • Chen M.H.
      • Tong S.
      • Tamin A.
      • Lowe L.
      • Frace M.
      • DeRisi J.L.
      • Chen Q.
      • Wang D.
      • Erdman D.D.
      • Peret T.C.
      • Burns C.
      • Ksiazek T.G.
      • Rollin P.E.
      • Sanchez A.
      • Liffick S.
      • Holloway B.
      • Limor J.
      • McCaustland K.
      • Olsen-Rasmussen M.
      • Fouchier R.
      • Günther S.
      • Osterhaus A.D.
      • Drosten C.
      • Pallansch M.A.
      • Anderson L.J.
      • Bellini W.J.
      ). There is a need for reagents and methods that can be used to detect new infectious entities as they arise. Indeed, the recent SARS epidemic was unexpected, reaching an 8% fatality rate despite the fact that coronaviruses typically are involved in ∼30% of common cold infections. N protein is 422 amino acids long, phosphorylated, and composed of two structured domains linked by a nonstructured domain. The N-terminal domain (NTD) is a putative RNA binding domain, and the C-terminal domain (CTD) mediates self-association (Fig. 1A) (
      • Surjit M.
      • Liu B.
      • Kumar P.
      • Chow V.T.
      • Lal S.K.
      ,
      • Saikatendu K.S.
      • Joseph J.S.
      • Subramanian V.
      • Neuman B.W.
      • Buchmeier M.J.
      • Stevens R.C.
      • Kuhn P.
      ). The unstructured middle domain interacts with the membrane (M) protein, anchoring M protein to the viral core. The two structured domains act in concert to bind genomic RNA, oligomerize, and form the final packaged ribonucleoprotein complex.
      Figure thumbnail gr1
      FIGURE 1mRNA display selection of 10Fn3 binders to N. A, representative diagram of dimerized N protein. B, 10Fn3 domain. Seventeen residues in the BC and FG loops are randomized in our trillion member combinatorial library. C, radiolabeled in vitro binding assay for monitoring binder enrichment in selection pool 3, 5, and 6. The data are represented as the percentage of radioactive 10Fn3 proteins bound to the beads with N protein (+N) or beads only (−N). The pulldown assays were performed at 4 °C. Pool 6 binding was also performed at 37 °C. D, radiolabeled in vitro binding assay for individual binders. 9 representative binders were chosen from pool 6 for the pulldown assay as described in C. E, sequence alignment of the nine chosen binders.
      We chose N as our target for several reasons. First, the N protein is the most abundant protein produced by SARS virus. Second, N plays multiple roles in vivo, including binding/packaging the viral genomic RNA, mediating interactions with the viral membrane (via the M protein), acting in genome replication, and exerting control over host cell processes (
      • Satija N.
      • Lal S.K.
      ,
      • Enjuanes L.
      • Almazán F.
      • Sola I.
      • Zuñiga S.
      ). Finally, no therapeutic reagents currently target N protein; therefore, new inhibitory N-directed ligands represent an important potential new route for developing anti-SARS drugs.
      After six rounds of selection, we were able to generate molecules that detect SARS N protein in vitro and modulate its SARS replication in vivo in a domain-specific manner. The selection yielded six high affinity molecules that recognize the CTD and two molecules that require the NTD for binding. We confirmed the interaction between the selected 10Fn3 proteins and N protein both in vitro and in vivo by pulldown, co-immunoprecipitation, and immunofluorescence microscopy. Seven of the 10Fn3-based intrabodies inhibit replication, ranging from 11- to 5900-fold, recognizing at least two nonoverlapping epitopes/hot spots in a synergistic manner. These molecules represent new tools for detecting SARS virus, assessing N function in living cells, and identifying regions of N critical for virus proliferation.

      DISCUSSION

      Our data demonstrate that directed evolution by mRNA display enabled designing of at least eight novel high affinity protein reagents targeting the SARS nucleocapsid protein (Fig. 1, D and E; Fn-N22, Fn-N17, Fn-N06, Fn-N06, Fn-N20, Fn-N08, Fn-N11, and Fn-N15). The pool showed a high fraction of binding (∼25%) even at physiologic temperature and salt. Biochemically, the best binder, Fn-N22, was the most abundant member of the cloned sequences (6 of 18 cloned) indicating that the pool was converging to a selection winner. None of the distinct clones are related by homology, indicating each is an independent solution to the binding problem we presented the library. Each of the loop sequences contains a high representation of aromatic and polar residues, typical of protein interaction surfaces (
      • Chakrabarti P.
      • Janin J.
      ). Given the relatively small number of pool members sequenced (18 total), it is likely that there are many more than eight independent binders in our round 6 pool.
      The selected 10Fn3 proteins were functional, well behaved, and able to be expressed in a number of formats. In addition to the reticulocyte lysate expression system used for library construction, 10Fn3 proteins were expressed in bacteria and mammalian cells, including 293T and VERO cells (FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6).
      Interestingly, our selection revealed two distinct binding sites on N for 10Fn3 recognition, the NTD and CTD (Fig. 1A). Six of the eight 10Fn3s bind the CTD alone (Fn-N06, Fn-N08, Fn-N10, Fn-N11, Fn-N15, and Fn-N22), whereas the other two (Fn-N17 and Fn-N20) require the NTD to bind. Structurally, the NTD and CTD are connected by unstructured regions (see Fig. 1A, schematic) and have been reported to be noninteracting (
      • Chang C.K.
      • Sue S.C.
      • Yu T.H.
      • Hsieh C.M.
      • Tsai C.K.
      • Chiang Y.C.
      • Lee S.J.
      • Hsiao H.H.
      • Wu W.J.
      • Chang W.L.
      • Lin C.H.
      • Huang T.H.
      ). On the other hand, three of our CTD binders (Fn-N08, Fn-N10, and Fn-N22) show markedly improved binding to N constructs lacking the N-terminal domain (Fig. 2D), indicating the NTD may partially occlude the binding site on the CTD.
      One CTD binder (Fn-N22) and one NTD-dependent binder (Fn-N17) were analyzed for affinity. The monovalent Fn-N22 and Fn-N17 bind to N protein with high affinity, similar to or better than the larger Fv region of monoclonal antibodies (Kd = 1.7 and 72 nm, respectively) (
      • Carter P.J.
      ). Even though this affinity is high, it is likely that affinity maturation and evolution could improve these affinities further, opening the potential that these binders could be used in ultra-sensitive detection platforms.
      A remarkable aspect of our work is that at least four of the selected 10Fn3 proteins (Fn-N10, Fn-N17, Fn-N20, and Fn-N22) specifically and dramatically inhibit SARS-CoV replication when transiently expressed in mammalian cell culture. Several points are worth noting. Generally, overexpression of the 10Fn3s is well tolerated inside the cell lines tested. 10Fn3 expression is relatively diffuse, with a higher density in the nucleus. Also, the 10Fn3 proteins are re-localized to the cytoplasm by N, demonstrating the potential for these molecules as tools for real time visualization (Fig. 4). Finally, the majority of the selected 10Fn3s (7/8 functional proteins) do not inhibit cellular function as measured in a relatively stringent assay, the ability to propagate an unrelated virus, herpesvirus.
      Functionally, ligands targeting the CTD (Fn-N10 and Fn-N22) appear to have a larger inhibitory effect compared with the NTD-dependent binders. Furthermore, combining an NTD and CTD binder results in synergistic inhibition (Fig. 5B). Our findings are reminiscent of the common antiviral mixture therapeutic strategy. Our findings are reminiscent of common antiviral mixture therapeutic strategies, where multiple small molecules drugs are used to target two or more key viral enzymatic functions. Here we have demonstrated that targeting two distinct protein surfaces of the nonenzymatic nucleocapsid protein also provides cooperative inhibition of virus production, a novel and intriguing result.
      Finally, we sought to characterize how our selected 10Fn3s might affect N function. The NTD of N is thought to mediate binding to the genomic RNA essential for the formation of ribonucleoprotein complex (
      • Saikatendu K.S.
      • Joseph J.S.
      • Subramanian V.
      • Neuman B.W.
      • Buchmeier M.J.
      • Stevens R.C.
      • Kuhn P.
      ). To determine whether 10Fn3 intrabodies inhibit SARS replication by effecting nucleic acid binding, we tested RNA band shifts using a portion of the SARS s2m. Both Fn-N10, the best inhibitor and a CTD binder, and Fn-N17, an NTD-dependent binder, do not compete with the RNA for binding to N. Both molecules have the effect of resolving the band formed by the complex. Although the band shift is well resolved, its low mobility makes it difficult to conclusively determine the effect of 10Fn3s on N oligomerization. Based on the data, our working hypothesis is that the 10Fn3 proteins reduce the structural and oligomeric heterogeneity of the N-RNA complexes.
      This study demonstrates the utility of using mRNA display selections to generate selective, high affinity 10Fn3-based proteins that target specific cellular components. 10Fn3 intrabodies are able to be expressed in bacteria for biophysical characterization and are stable in mammalian cells, advantages over the commonly used antibody scaffold. Importantly, no in vivo screen was required to filter the resulting pool for binders that function inside the cell. Of the eight intrabodies tested, seven function efficiently to block SARS replication and do not disrupt mammalian cellular function (Fig. 2B).
      Used inside cells, 10Fn3s provide a complementary tool to commonly used methods for the analysis of proteins in vivo such as gene knock-outs and small interfering RNA. The N binders described in this study may thus be useful for future studies of SARS virus. For example, N binders may shed light on key N protein-host cell protein interactions during various stages of the virus life cycle. Our results demonstrate that 10Fn3 intrabodies enable in vivo visualization of N. Therefore, the 10Fn3s may enable analyzing the fate of N and the ribonucleoprotein complex in real time during the various stages of the virus life cycle. This would be especially useful during the initial stages of virus entry, where reverse genetics cannot be easily applied because infectious viral particles may not be generated without functional viral capsid or envelope proteins. Finally, the biological neutrality of the 10Fn3s implies that combining two or more inhibitors may be useful clinically as an approach toward improving gene therapy-based preventative interventions for persistent viral diseases.

      Acknowledgments

      We thank the University of Southern California Nanobiophysics core facility for Biacore use.

      REFERENCES

        • Maynard J.
        • Georgiou G.
        Annu. Rev. Biomed. Eng. 2000; 2: 339-376
        • Lipovsek D.
        • Plückthun A.
        J. Immunol. Methods. 2004; 290: 51-67
        • Hosse R.J.
        • Rothe A.
        • Power B.E.
        Protein Sci. 2006; 15: 14-27
        • Roberts R.W.
        • Szostak J.W.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12297-12302
        • Carlson J.R.
        Mol. Cell. Biol. 1988; 8: 2638-2646
        • Stocks M.
        Curr. Opin. Chem. Biol. 2005; 9: 359-365
        • Nizak C.
        • Monier S.
        • del Nery E.
        • Moutel S.
        • Goud B.
        • Perez F.
        Science. 2003; 300: 984-987
        • Rajpal A.
        • Turi T.G.
        J. Biol. Chem. 2001; 276: 33139-33146
        • Visintin M.
        • Tse E.
        • Axelson H.
        • Rabbitts T.H.
        • Cattaneo A.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11723-11728
        • Vascotto F.
        • Campagna M.
        • Visintin M.
        • Cattaneo A.
        • Burrone O.R.
        J. Gen. Virol. 2004; 85: 3285-3290
        • Cardinale A.
        • Filesi I.
        • Mattei S.
        • Biocca S.
        Eur. J. Biochem. 2003; 270: 3389-3397
        • Cardinale A.
        • Filesi I.
        • Biocca S.
        Eur. J. Biochem. 2001; 268: 268-277
        • Kawe M.
        • Forrer P.
        • Amstutz P.
        • Plückthun A.
        J. Biol. Chem. 2006; 281: 40252-40263
        • Amstutz P.
        • Binz H.K.
        • Parizek P.
        • Stumpp M.T.
        • Kohl A.
        • Grütter M.G.
        • Forrer P.
        • Plückthun A.
        J. Biol. Chem. 2005; 280: 24715-24722
        • Olson C.A.
        • Roberts R.W.
        Protein Sci. 2007; 16: 476-484
        • Koide A.
        • Bailey C.W.
        • Huang X.
        • Koide S.
        J. Mol. Biol. 1998; 284: 1141-1151
        • Olson C.A.
        • Liao H.I.
        • Sun R.
        • Roberts R.W.
        ACS Chem. Biol. 2008; 3: 480-485
        • Rota P.A.
        • Oberste M.S.
        • Monroe S.S.
        • Nix W.A.
        • Campagnoli R.
        • Icenogle J.P.
        • Peñaranda S.
        • Bankamp B.
        • Maher K.
        • Chen M.H.
        • Tong S.
        • Tamin A.
        • Lowe L.
        • Frace M.
        • DeRisi J.L.
        • Chen Q.
        • Wang D.
        • Erdman D.D.
        • Peret T.C.
        • Burns C.
        • Ksiazek T.G.
        • Rollin P.E.
        • Sanchez A.
        • Liffick S.
        • Holloway B.
        • Limor J.
        • McCaustland K.
        • Olsen-Rasmussen M.
        • Fouchier R.
        • Günther S.
        • Osterhaus A.D.
        • Drosten C.
        • Pallansch M.A.
        • Anderson L.J.
        • Bellini W.J.
        Science. 2003; 300: 1394-1399
        • Surjit M.
        • Liu B.
        • Kumar P.
        • Chow V.T.
        • Lal S.K.
        Biochem. Biophys. Res. Commun. 2004; 317: 1030-1036
        • Saikatendu K.S.
        • Joseph J.S.
        • Subramanian V.
        • Neuman B.W.
        • Buchmeier M.J.
        • Stevens R.C.
        • Kuhn P.
        J. Virol. 2007; 81: 3913-3921
        • Satija N.
        • Lal S.K.
        Ann. N.Y. Acad. Sci. 2007; 1102: 26-38
        • Enjuanes L.
        • Almazán F.
        • Sola I.
        • Zuñiga S.
        Annu. Rev. Microbiol. 2006; 60: 211-230
        • Yount B.
        • Curtis K.M.
        • Fritz E.A.
        • Hensley L.E.
        • Jahrling P.B.
        • Prentice E.
        • Denison M.R.
        • Geisbert T.W.
        • Baric R.S.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12995-13000
        • Roberts R.S.
        • Yount B.L.
        • Sims A.C.
        • Baker S.
        • Baric R.S.
        Adv. Exp. Med. Biol. 2006; 581: 597-600
        • Ja W.W.
        • Adhikari A.
        • Austin R.J.
        • Sprang S.R.
        • Roberts R.W.
        J. Biol. Chem. 2005; 280: 32057-32060
        • Liu R.
        • Barrick J.E.
        • Szostak J.W.
        • Roberts R.W.
        Methods Enzymol. 2000; 318: 268-293
        • Milligan J.F.
        • Groebe D.R.
        • Witherell G.W.
        • Uhlenbeck O.C.
        Nucleic Acids Res. 1987; 15: 8783-8798
        • Chen C.Y.
        • Chang C.K.
        • Chang Y.W.
        • Sue S.C.
        • Bai H.I.
        • Riang L.
        • Hsiao C.D.
        • Huang T.H.
        J. Mol. Biol. 2007; 368: 1075-1086
        • Luo H.
        • Chen J.
        • Chen K.
        • Shen X.
        • Jiang H.
        Biochemistry. 2006; 45: 11827-11835
        • Yu I.M.
        • Gustafson C.L.
        • Diao J.
        • Burgner 2nd, J.W.
        • Li Z.
        • Zhang J.
        • Chen J.
        J. Biol. Chem. 2005; 280: 23280-23286
        • Chakrabarti P.
        • Janin J.
        Proteins. 2002; 47: 334-343
        • Chang C.K.
        • Sue S.C.
        • Yu T.H.
        • Hsieh C.M.
        • Tsai C.K.
        • Chiang Y.C.
        • Lee S.J.
        • Hsiao H.H.
        • Wu W.J.
        • Chang W.L.
        • Lin C.H.
        • Huang T.H.
        J. Biomed. Sci. 2006; 13: 59-72
        • Carter P.J.
        Nat. Rev. Immunol. 2006; 6: 343-357