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Journal of Virology, February 2009, p. 1911-1919, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.02055-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Attenuation of Rabies Virus Replication and Virulence by Picornavirus Internal Ribosome Entry Site Elements{triangledown}

Adriane Marschalek,1 Stefan Finke,1,{dagger} Martin Schwemmle,2 Daniel Mayer,2 Bernd Heimrich,3 Lothar Stitz,4 and Karl-Klaus Conzelmann1*

Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University, Feodor-Lynen-Str. 25, 81377 Munich,1 Department of Virology, Institute for Medical Microbiology and Hygiene, Hermann-Herder-Strasse 11,2 Institute of Anatomy and Cell Biology, Albertstr. 23, University of Freiburg, 79104 Freiburg,3 Institute of Immunology, Friedrich-Loeffler-Institut Tübingen, Paul-Ehrlich-Strasse 28, 72076 Tübingen, Germany4

Received 30 September 2008/ Accepted 29 November 2008

Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5'-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-β) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-{alpha} receptor (IFNAR–/–). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR–/– mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.

* Corresponding author. Mailing address: Max von Pettenkofer Institute & Gene Center, Feodor-Lynen-Str. 25, D-81377 Munich, Germany. Phone: 49 89 2180 76851. Fax: 49 89 2180 76899. E-mail: conzelma{at}lmb.uni-muenchen.de

{triangledown} Published ahead of print on 10 December 2008.

{dagger} Present address: Institute for Molecular Biology, Friedrich-Loeffler-Institute, Südufer 10, 17493 Greifswald-Insel Riems, Germany.

Journal of Virology, February 2009, p. 1911-1919, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.02055-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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