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...
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