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

Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University, Munich, Germany.
Journal of Virology (Impact Factor: 4.44). 02/2009; 83(4):1911-9. DOI: 10.1128/JVI.02055-08
Source: PubMed


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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Available from: Stefan Finke, Oct 01, 2014
    • "In vivo studies demonstrated that deletion of the dynein LC8 binding domain retained pathogenicity in suckling mice (Mebatsion 2001) but was nonpathogenic in adult mice (Tan et al. 2007). In another study, the internal ribosome entry site (IRES) from picornaviruses was introduced to control the expression of P, and an in vivo study showed reduction of replication in cell culture, increased activation of the beta interferon (IFN-β) promoter, and attenuation in mice infected intracerebrally (Marschalek et al. 2009). Taken together, targeting the P protein through either mutating the dynein LC8 binding domain or translational control of expression is a promising strategy to attenuate virus virulence of live RABV. "
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    ABSTRACT: Rabies is an ancient neurological disease that is almost invariably fatal once the clinical symptoms develop. Currently, prompt wound cleansing after exposing to a potentially rabid animal and vaccination using rabies vaccine combined with administration of rabies immune globulin are the only effective methods for post-exposure prophylaxis against rabies. Reverse genetic technique is a novel approach to investigate the function of a specific gene by analyzing the phenotypic effects through directly manipulating the gene sequences. It has revolutionized and provided a powerful tool to study the molecular biology of RNA viruses and has been widely used in rabies virus research. The attenuation of rabies virus virulence is the prerequisite for rabies vaccine development. Given the current challenge that sufficient and affordable high-quality vaccines are limited and lacking for global rabies prevention and control, highly cell-adapted, stable, and attenuated rabies viruses with broad cross-reactivity against different viral variants are ideal candidates for consideration to meet the need for human rabies control in the future. A number of approaches have been pursued to reduce the virulence of the virus and improve the safety of rabies vaccines. The application of reverse genetic technique has greatly advanced the engineering of rabies virus and paves the avenue for utilizing rabies virus for vaccine against rabies, viral vectors for exogenous antigen expression, and gene therapy in the future.
    Journal of NeuroVirology 05/2015; 21(4). DOI:10.1007/s13365-015-0350-2 · 2.60 Impact Factor
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    • "For example, removal of Dynein Light Chain 8 binding site motif substantially reduced viral transcription and replication in the central nervous system [32]. Another strategy was to make the expression of the essential phosphoprotein dependent on translation and not transcription [33]. The SAD dIND1 construct uses a different approach which is aimed at inducing an improved innate immune response in vaccinated animals. "
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    ABSTRACT: Different approaches have been applied to develop highly attenuated rabies virus vaccines for oral vaccination of mesocarnivores. One prototype vaccine construct is SAD dIND1, which contains a deletion in the P-gene severely limiting the inhibition of type-1 interferon induction. Immunogenicity studies in foxes and skunks were undertaken to investigate whether this highly attenuated vaccine would be more immunogenic than the parental SAD B19 vaccine strain. In foxes, it was demonstrated that SAD dIND1 protected the animals against a rabies infection after a single oral dose, although virus neutralizing antibody titres were lower than in foxes orally vaccinated with the SAD B19 virus as observed in previous experiments. In contrast, skunks receiving 10(7.5) FFU SAD dIND1 did not develop virus neutralizing antibodies and were not protected against a subsequent rabies infection.
    09/2011; 2011:898171. DOI:10.4061/2011/898171

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