- Open Access
Combining genetic and biochemical approaches to identify functional molecular contact points
Biological Procedures Online volume 8, pages 77–86 (2006)
Protein-protein interactions are required for many viral and cellular functions and are potential targets for novel therapies. Here we detail a series of genetic and biochemical techniques used in combination to find an essential molecular contact point on the duck hepatitis B virus polymerase. These techniques include differential immunoprecipitation, mutagenesis and peptide competition. The strength of these techniques is their ability to identify contact points on intact proteins or protein complexes employing functional assays. This approach can be used to aid identification of putative binding sites on proteins and protein complexes which are resistant to characterization by other methods.
Ryan DP, Matthews JM. Protein-protein interactions in human disease. Curr Opin Struct Biol 2005; 15:441–446.
Arkin M. Protein-protein interactions and cancer: small molecules going in for the kill. Curr Opin Chem Biol 2005; 9:317–324.
Miller J, Stagljar I. Using the yeast two-hybrid system to identify interacting proteins. Methods Mol Biol 2004; 261:247–262.
Loregian A, Appleton BA, Hogle JM, Coen DM. Specific residues in the connector loop of the human cytomegalovirus DNA polymerase accessory protein UL44 are crucial for interaction with the UL54 catalytic subunit. J Virol 2004; 78:9084–9092.
Smith GP, Petrenko VA. Phage display. Chemical Reviews 1997; 97:391–410.
Lee WM. Hepatitis B virus infection. N Engl J Med 1997; 337:1733–1745.
Hu J, Toft D, Anselmo D, Wang X. In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins. J Virol 2002; 76:269–279.
Beck J, Nassal M. Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein. J Biol Chem 2003; 278:36128–36138.
Cao F, Badtke MP, Metzger LM, Yao E, Adeyemo B, Gong YH et al. Identification of an essential molecular contact point on the duck hepatitis B virus reverse transcriptase. J Virol 2005; 79:10164–10170.
Yao E, Gong Y, Chen N, Tavis JE. The majority of duck hepatitis B virus reverse transcriptase in cells is nonencapsidated and is bound to a cytoplasmic structure. J Virol 2000; 74:8648–8657.
Tavis JE, Ganem D. Evidence for the Activation of the Hepatitis B Virus Polymerase by Binding of Its RNA Template. J Virol 1996; 70:5741–5750.
Tavis JE, Massey B, Gong Y. The Duck Hepatitis B Virus Polymerase Is Activated by Its RNA Packaging Signal, Epsilon. J Virol 1998; 72:5789–5796.
Tavis JE, Perri S, Ganem D. Hepadnavirus Reverse Transcription Initiates within the Stem-Loop of the RNA Packaging Signal and Employs a Novel Strand Transfer. J Virol 1994; 68:3536–3543.
Wang GH, Seeger C. Novel Mechanism for Reverse Transcription in Hepatitis B Viruses. J Virol 1993; 67:6507–6512.
Gibbs RA, Posner BA, Filpula DR, Dodd SW, Finkelman MA, Lee TK et al. Construction and characterization of a single-chain catalytic antibody. Proc Natl Acad Sci USA 1991; 88:4001–4004.
Pollack JR, Ganem D. Site-specific RNA binding by a hepatitis B virus reverse transcriptase initiates two distinct reactions: RNA packaging and DNA synthesis. J Virol 1994; 68:5579–5587.
Kuo CC, Hsieh HP, Pan WY, Chen CP, Liou JP, Lee SJ et al. BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res 2004; 64:4621–4628.
O’Neill J, Manion M, Schwartz P, Hockenbery DM. Promises and challenges of targeting Bcl-2 antiapoptotic proteins for cancer therapy. Biochim Biophys Acta 2004; 1705:43–51.
Ho TY, Wu SL, Chen JC, Wei YC, Cheng SE, Chang YH et al. Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction. Antiviral Res 2006; 69:70–76.
Cohen EA, Gaudreau P, Brazeau P, Langelier Y. Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2. Nature 1986; 321:441–443.
Loregian A, Palu G. Disruption of protein-protein interactions: Towards new targets for chemotherapy. J Cellular Physiol 2005; 204:750–762.
Loregian A, Marsden HS, Palu G. Protein-protein interactions as targets for antiviral chemotherapy. Reviews in Medical Virol 2002; 12:239–262.
Loregian A, Palu G. Disruption of the interactions between the subunits of herpesvirus DNA polymerases as a novel antiviral strategy. Clin Microbiol Infection 2005;11:437–446.
Loregian A, Rigatti R, Murphy M, Schievano E, Palu G, Marsden HS. Inhibition of human cytomegalovirus DNA polymerase by C-terminal peptides from the UL54 subunit. J Virol 2003; 77:8336–8344.
Murray J, Loney C, Murphy LB, Graham S, Yeo RP. Characterization of monoclonal antibodies raised against recombinant respiratory syncytial virus nucleocapsid (N) protein: Identification of a region in the carboxy terminus of N involved in the interaction with P protein. Virology 2001; 289:252–261.
Morris MC, Robert-Hebmann V, Chaloin L, Mery J, Heitz F, Devaux C et al. A new potent HIV-1 reverse transcriptase inhibitor — A synthetic peptide derived from the interface subunit domains. J Biol Chem 1999; 274:24941–24946.
Maroun RG, Krebs D, Roshani M, Porumb H, Auclair C, Troalen F et al. Conformational aspects of HIV-1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide. Eur J Biochem 1999; 260:145–155.
Zhang ZY, Poorman RA, Maggiora LL, Heinrikson RL, Kezdy FJ. Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide. J Biol Chem 1991; 266:15591–15594.
About this article
Cite this article
Badtke, M.P., Cao, F. & Tavis, J.E. Combining genetic and biochemical approaches to identify functional molecular contact points. Biol. Proced. Online 8, 77–86 (2006). https://doi.org/10.1251/bpo121
- Hepatitis B Virus, Duck
- Protein Interaction Mapping