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

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-␤) 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-␣ 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.The order Mononegavirales, also known as nonsegmented negative-strand (NNS) RNA viruses, includes the Rhabdoviridae, Paramyxoviridae, Filoviridae, and Bornaviridae families (46). Though diverse in host range, tropism, and pathogenesis, these viruses share a highly conserved mode of gene expression and gene order, which is 3Ј-N-P-M-G-L-5Ј (the nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and "large" polymerase genes, respectively) in the prototype rhabdoviruses, such as rabies virus (RV; genus Lyssavirus) or vesicular stomatitis virus (genus Vesiculovirus). The hallmarks of their gene expression are (i) obligatory sequential transcription of monocistronic genes from a single 3Ј-terminal promoter, (ii) attenuation of transcription at conserved gene borders, giving rise to an mRNA transcript gradient, and (iii) 5Ј-cap-dependent translation of the mRNAs. Accordingly, the gene order and the steepness of the transcript gradient predetermine the level of individual mRNAs and of viral protein synthesis (for a recent review see reference 58).Once inside a cell, RNA synthesis of mammalian NNS RNA viruses appears not to be restricted by host species or cell type, invariably leading to the canonical transcript gradient, i.e., their 3Ј promoters for RNA synthesis and the polymerase (P plus L) are always "on." The possibilities of manipulating the expression of individual viral genes on the l...

Genetic Dissection of Interferon-Antagonistic Functions of Rabies Virus Phosphoprotein: Inhibition of Interferon Regulatory Factor 3 Activation Is Important for Pathogenicity

“…In RV-infected cells, RIG-I is activated by viral triphosphate RNAs (11,18); however, IRF3 phosphorylation, dimerization, and nuclear import are pre-vented in the presence of P (4). The importance of the IFNantagonistic functions of P has been illustrated by recombinant RV expressing reduced amounts of P, either by shifting the P gene to a promoter-distal position from which it is transcribed poorly (4) or by using picornavirus internal ribosome entry site (IRES) elements to limit the translation rate of P (26). Apart from a modest defect in replication, such viruses have severe defects in their ability to counteract both IFN induction and IFN signaling and are therefore highly attenuated in vivo.…”

Neuroattenuation of Vesicular Stomatitis Virus through Picornaviral Internal Ribosome Entry Sites

“…In contrast, the replication of the VSV M mutant (VSV ⌬51 ) was severely restricted due to its inability to turn off host gene expression. Although primary neurons were not completely resistant to the IRES-controlled viruses in vitro, animal studies revealed a high degree of protection against lethal encephalitis, as observed earlier for rabies virus (30). At the dose level of 10 4 TCID 50 s, wild-type VSV administered intracranially killed 80% of immunocompetent mice, while the IRES-controlled viruses did not cause any morbidity or mortality.…”

“…Following this incubation, supernatant was removed, the monolayer was washed, and fresh growth medium was added. Supernatant was collected at predetermined time points (6,12,18,24,30, and 36 h), and the cell pellets were washed with PBS and stored at Ϫ80°C. Virus titer was determined by the TCID 50 method.…”

“…It was previously demonstrated that the IRES elements of HRV2 and FMDV attenuated the neurovirulence of poliovirus and Theiler's murine encephalomyelitis virus (TMEV), respectively, by impairing their ability to replicate in neuronal tissues (27,36). Also, Marschalek et al (30) suggested that HRV2 IRES insertion might provide a basis for the neuroattenuation of rabies virus. We therefore characterized the protein expression and growth properties of our IRES-controlled VSVs in primary human cortical neuronal cells (HCN-2) infected at an MOI of 10 ( Fig.…”

“…Thus, it was shown that depletion of DRBP76 from neuronal cells enhances rhinovirus IRES-driven translation and virus propagation (27). Placing essential viral genes under the control of an HRV2 IRES can attenuate the neurovirulence of poliovirus (28), herpesvirus (29), and rabies virus (30). We therefore sought to determine whether this neuron-selective inhibition of IRES elements could be exploited to control the expression level of vesicular stomatitis viral proteins in neurons, thereby ameliorating neurotoxicity.…”