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Approaches for the Generation of New Anti-cytomegalovirus Agents: Identification of Protein–Protein Interaction Inhibitors and Compounds Against the HCMV IE2 Protein

  • Beatrice Mercorelli
  • Giorgio Gribaudo
  • Giorgio Palù
  • Arianna LoregianEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1119)

Abstract

Human cytomegalovirus (HCMV) infection is responsible for severe, often even fatal, diseases in immunocompromised subjects and also represents the major cause of viral-associated congenital malformations in newborn children. The few drugs licensed for anti-HCMV therapy suffer from many drawbacks and none of them have been approved for the treatment of congenital infections. Furthermore, the emergence of drug-resistant viral strains represents a major concern for disease management. Thus, there is a strong need for new anti-HCMV drugs. Here we describe three different assays for the discovery of novel anti-HCMV compounds: two are in vitro assays, i.e., a fluorescence polarization (FP)-based assay and an enzyme-linked immunosorbent assay (ELISA), which are designed to search for compounds that act by disrupting the interactions between the HCMV DNA polymerase subunits, but in general can be employed to find inhibitors of any protein–protein interaction of interest; the third is a cell-based assay designed to identify inhibitors of the viral immediate-early 2 (IE2) protein activities.

Key words

Antivirals Cell-based assay DNA polymerase inhibitors Enzyme-linked immunosorbent assay Fluorescence polarization Human cytomegalovirus IE2 protein Protein–protein interaction 

Notes

Acknowledgments

The research in our laboratories was supported by MURST EX60%, Progetto di Ricerca di Ateneo 2007 (grant no. CPDA074945), and PRIN 2008 (grant no. 20085FF4J4) to A.L., by PRIN and the Piedmont Region (Ricerca Sanitaria Finalizzata) to G.G., and by Regione Veneto and Progetto Strategico di Ateneo 2008 to G.P.

References

  1. 1.
    Mercorelli B, Sinigalia E, Loregian A, Palù G (2008) Human cytomegalovirus DNA replication: antiviral targets and drugs. Rev Med Virol 18:177–210PubMedCrossRefGoogle Scholar
  2. 2.
    Loregian A, Coen DM (2006) Selective anti-cytomegalovirus compounds discovered by screening for inhibitors of subunit interactions of the viral polymerase. Chem Biol 13:191–200PubMedCrossRefGoogle Scholar
  3. 3.
    Mercorelli B, Lembo D, Palù G, Loregian A (2011) Early inhibitors of human cytomegalovirus: state-of-art and therapeutic perspectives. Pharmacol Ther 131:309–329PubMedCrossRefGoogle Scholar
  4. 4.
    Lundblad JR, Laurance M, Goodman RH (1996) Fluorescence polarization analysis of protein-DNA and protein-protein interactions. Mol Endocrinol 10:607–612PubMedGoogle Scholar
  5. 5.
    Moerke NJ (2009) Fluorescence polarization (FP) assays for monitoring peptide-protein or nucleic acid-protein binding. Curr Protoc Chem Biol 1:1–15PubMedGoogle Scholar
  6. 6.
    Wu J, O’Neill J, Barbosa MS (1998) Transcription factor Sp1 mediates cell-specific trans-activation of the human cytomegalovirus DNA polymerase gene promoter by immediate-early protein IE86 in glioblastoma U373MG cells. J Virol 72:236–244PubMedCentralPubMedGoogle Scholar
  7. 7.
    Asmar J, Wiebusch L, Truss M, Hagemeier C (2004) The putative zinc finger of the human cytomegalovirus IE2 86-kilodalton protein is dispensable for DNA binding and autorepression, thereby demarcating a concise core domain in the C-terminus of the protein. J Virol 78:11853–11864PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Luganini A, Caposio P, Mondini M, Landolfo S, Gribaudo G (2008) New cell-based indicator assays for the detection of human cytomegalovirus infection and screening of inhibitors of viral immediate-early 2 protein activity. J Appl Microbiol 105:1791–1801PubMedCrossRefGoogle Scholar
  9. 9.
    Azad RF, Driver VB, Tanaka K, Crooke RM, Anderson KP (1993) Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region. Antimicrob Agents Chemother 37:1945–1954PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Loregian A, Mercorelli B, Muratore G, Sinigalia E, Massari S, Gribaudo G, Gatto B, Tabarrini O, Palumbo M, Cecchetti V, Palù G (2010) The 6-aminoquinolone WC5 inhibits human cytomegalovirus replication at an early stage by interfering with the transactivating activity of viral immediate-early 2 protein. Antimicrob Agents Chemother 54:1930–1940PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Loregian A, Appleton BA, Hogle JM, Coen DM (2004) Residues of human cytomegalovirus DNA polymerase catalytic subunit, UL54, that are necessary and sufficient for interaction with the accessory protein UL44. J Virol 78:158–167PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Loregian A, Rigatti R, Murphy M, Schievano E, Palù G, Marsden HS (2003) Inhibition of human cytomegalovirus DNA polymerase by C-terminal peptides from the UL54 subunit. J Virol 77:8336–8344PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Stammers DK, Tisdale M, Court S, Parmar V, Bradley C, Ross CK (1991) Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope. FEBS Lett 283:298–302PubMedCrossRefGoogle Scholar
  14. 14.
    Jault FM, Spector SA, Spector DH (1994) The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlates with permissiveness of the cells for each virus. J Virol 68:959–973PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Beatrice Mercorelli
    • 1
  • Giorgio Gribaudo
    • 2
  • Giorgio Palù
    • 1
  • Arianna Loregian
    • 1
    Email author
  1. 1.Department of Molecular MedicineUniversity of PaduaPaduaItaly
  2. 2.Department of Life Sciences and Systems BiologyUniversity of TurinTurinItaly

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