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. 2009 Feb;83(4):1911-9.
doi: 10.1128/JVI.02055-08. Epub 2008 Dec 10.

Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements

Adriane Marschalek  1 , Stefan Finke, Martin Schwemmle, Daniel Mayer, Bernd Heimrich, Lothar Stitz, Karl-Klaus Conzelmann
Affiliations

Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements

Adriane Marschalek et al. J Virol. 2009 Feb.

Abstract

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-beta) 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.

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Figures

FIG. 1.
FIG. 1.
Recombinant RV genomes. The genome of wt RV SAD L16 comprises five cistrons defined by transcription stop/restart signals (arrowheads) that give rise to five capped mRNAs as indicated. The IRES reporter virus SAD RL-IRES-FL contains an extra gene from which a bicistronic mRNA encoding RL and FL is transcribed. In SAD HRV2-P and SAD PV-P, here represented by the genome of SAD IRES-P, the transcription signal sequences of the N/P gene border are replaced by the IRES elements of HRV2 and PV, such that P protein expression depends on internal translation initiation by the IRES elements.
FIG. 2.
FIG. 2.
RV encoding bicistronic RL-IRES-FL reporter genes. (A) Growth of IRES reporter RVs in BSR T7/5 cells. Cells were infected with the indicated viruses at an MOI of 0.1, and infectious supernatant virus titers were determined at the indicated time points. As a control a virus containing an additional monocistronic gene downstream of G (SAD G GFP) was used for comparison. (B) Cell lines from nonprimate (upper panel) and primate species (lower panel), including cells of neuronal origin (underlined) were infected with the recombinant SAD RL-IRES-FL reporter viruses containing the IRES of PV Mahoney (light gray), HRV type 2 (black), or FMDV (dark gray). At 48 h p.i. RL and FL activities were measured using the dual luciferase reporter system (Promega). The ratio of activity of FL to RL with SAD RL-PV-FL was set as 100%. Data for every cell line are from at least two independent experiments, each of which included three parallel infections. Error bars indicate standard deviations. P ≤ 0.001 for all cells and viruses.
FIG. 3.
FIG. 3.
IRES-dependent expression of RV P. (A) BSR T7/5 cells were infected with the indicated RVs, and total RNA was isolated 48 h p.i. and analyzed by Northern blotting. Only bicistronic N/P mRNAs are apparent after substitution of the RV N/P gene border with PV or HRV2 IRES sequences. (B) Quantification of P protein levels by fluorescence imaging. The Western blot shows expression of N and P proteins in extracts from BSR T7/5 cells 48 h postinfection with the indicated viruses. Numbers below the lanes indicate relative levels of RV P after normalization with RV N and in comparison with P levels of SAD L16 (100%).
FIG. 4.
FIG. 4.
Growth of IRES-controlled RV in cell culture. IFN-incompetent BSR T7/5 cells (A) and IFN-competent NA cells (B) were infected with the indicated viruses at an MOI of 0.1, and infectious supernatant virus titers were determined at the indicated time points. Titers for every time point are from at least three independent experiments. Error bars indicate standard deviations.
FIG. 5.
FIG. 5.
Viral defects in counteracting IFN-β induction and JAK/STAT signaling. (A) HEK 293T cells were transfected with the IFN-β promoter-controlled plasmid p125-Luc and infected at an MOI of 3. FL expression was determined using the dual luciferase reporter system (Promega) at 24 h p.i. Both IRES-controlled RVs activate the IFN-β promoter more efficiently than wt RV SAD L16. SAD ΔPLP is a gene shift control virus expressing trace amounts of P (7). (B) Inhibition of IFN-stimulated gene expression by the indicated RV was analyzed in IFN-negative BSR T7/5 cells. Cells infected at an MOI of 1 and transfected 6 h later with the ISRE promoter-controlled reporter plasmid pISRE-Luc and pCMV-RL for normalization were treated 24 h p.i. with the indicated doses of IFN-α A/D. FL and RL activities were determined 48 h p.i. Only wt RV SAD L16 was able to almost completely abolish FL expression. Error bars indicate standard deviations from at least three parallel experiments.
FIG. 6.
FIG. 6.
Replication of IRES-controlled RVs in organotypic hippocampal slice cultures. Hippocampal slice cultures from newborn mice (C57/BL6) were infected with 2 × 104 FFU of SAD HRV2-P, SAD PV-P, or SAD L16. After the indicated time periods, slices were fixed and 50-μm sections were prepared and immunostained for the RV N protein (green) or calbindin (red), a marker protein of granule cells of the dentate gyrus. To visualize the cell nucleus and to evaluate tissue condition, the slices were counterstained with DAPI (blue). (A) Impact of RV infection on hippocampus damage. Left panels: survey of a DAPI-stained hippocampal culture infected with SAD HRV2-P and SAD PV-P for 8 days, showing intact cytoarchitecture similar to the uninfected control culture. Right graph: the extent of tissue damage induced by the different virus strains 8 days p.i. based on DAPI staining. (-), severe; (+/−), partial; (+), no loss of hippocampal organization. n = 16/group (the number of organotypic slice cultures investigated for each virus). (B) Virus spread of SAD HRV2-P, SAD PV-P, or SAD L16 in hippocampal slice cultures 3 to 4 days p.i. Note that cultures infected with SAD HRV2-P or SAD PV-P showed decreased viral spread. (C) Distribution of RV N antigen in hippocampal cultures. Left panel: SAD HRV2-P-infected slices show typical organization. A calbindin-positive granule cell layer and the mossy fiber projection are formed. At higher magnification few viral antigen signals are found on axonal profiles (arrowheads) and in calbindin-labeled granule cell somata (arrow). Middle panel: in SAD PV-P-infected cultures the cellular organization is maintained. At higher magnification many viral antigen signals are observed with a similar localization as in SAD HRV2-P-infected cultures. Right panel: cultures infected with SAD L16 are disorganized and only a few calbindin-positive granule cells are preserved. These cells also contain viral antigen. CA, cornu ammonis; dg, dentate gyrus; gc, granule cells; mf, mossy fiber projection; pcl, pyramidal cell layer. Bars, 200 μm (upper panels), 100 μm (middle panel, SAD HRV2-P and SAD PV-P), 50 μm (middle panel, SAD L16), or 15 μm (lower panels).
FIG. 7.
FIG. 7.
Survival of mice after i.c. infection with recombinant RVs. Three-week-old wt (A) or IFNAR−/− mice (C) were infected i.c. with 1 × 105 FFU/mouse, and newborn wt (B) or IFNAR−/− (D) mice were infected with 100 FFU/mouse of the indicated viruses. In adult wt and IFNAR−/− mice both SAD PV-P and SAD HRV2-P were completely nonpathogenic (A and C), in contrast to wt RV SAD L16. In newborn wt mice (B), SAD HRV2-P was still completely attenuated, whereas SAD PV-P caused mortality, although with a delay compared to SAD L16. Only newborn IFNAR−/− mice lacking IFNAR (D) succumbed to SAD HRV2-P.
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