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Journal of Virology, November 2009, p. 11902-11913, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.01378-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84{triangledown}

Nina Kaiser, Peter Lischka,{dagger} Nadine Wagenknecht, and Thomas Stamminger*

Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, 91054 Erlangen, Germany

Received 6 July 2009/ Accepted 31 August 2009

The UL84 open reading frame of human cytomegalovirus encodes an essential multifunctional regulatory protein that is thought to act in the nucleus as an initiator of lytic viral replication. Nuclear trafficking of pUL84 is facilitated by a complex nonconventional nuclear localization signal (NLS) that mediates its interaction with the cellular importin-{alpha}/β pathway. Since binding of pUL84 to importin-{alpha} proteins mechanistically differs from that of cellular proteins containing a classical NLS, we assumed that specific interference with the nuclear import of pUL84 might be possible and that this could constitute a novel principle for antiviral therapy. In order to test this hypothesis, we employed peptide aptamer technology and isolated several peptide aptamers from a randomized peptide expression library that specifically bind with high affinity to the unconventional pUL84 NLS under intracellular conditions. Coimmunoprecipitation experiments confirmed these interactions in mammalian cells, and the antiviral potential of the identified peptide aptamers was determined using three independent experimental approaches. (i) Infection experiments with a recombinant human cytomegalovirus expressing green fluorescent protein demonstrated 50 to 60% decreased viral replication in primary human fibroblasts stably expressing pUL84-specific aptamers. (ii) A 50 to 70% reduction of viral plaque formation, as well as a 70 to 90% inhibition of virus release in the presence of pUL84-specific aptamers, was observed. (iii) Immunofluorescence analyses revealed a shift from an almost exclusively nuclear pUL84 staining pattern to a nucleocytoplasmic distribution upon coexpression of the identified molecules, indicating that interference with the nuclear import of pUL84 contributes to the observed antiviral activity of the identified pUL84-binding aptamer molecules.

* Corresponding author. Mailing address: Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, 91054 Erlangen, Germany. Phone: 49 9131 8526783. Fax: 49 9131 8522101. E-mail: thomas.stamminger{at}viro.med.uni-erlangen.de

{triangledown} Published ahead of print on 9 September 2009.

{dagger} Present address: AiCuris GmbH & Co. KG, Friedrich Ebert-Str. 475, 42117 Wuppertal, Germany.

Journal of Virology, November 2009, p. 11902-11913, Vol. 83, No. 22
0022-538X/09/$08.00+0     doi:10.1128/JVI.01378-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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