Herpes Simplex Virus Proteins ICP27 and UL47 Associate with Polyadenylate-Binding Protein and Control Its Subcellular Distribution.
(2009)
Journal - Journal of virology (United States )
Abstract :
Human pathogenic viruses manipulate host cell translation machinery to ensure efficient expression of viral genes and to thwart host cell protein synthesis. Viral strategies include cleaving translation factors, manipulating translation factor abundance and recruitment into translation initiation complexes, or expressing viral translation factor analogs. Analyzing translation factors in herpes simplex virus type 1 (HSV-1)-infected HeLa cells, we found diminished association of the polyadenylate-binding protein (PABP) with the cap-binding complex. Although total PABP levels were unchanged, HSV-1 infection prompted accumulation of cytoplasmic PABPC1, but not its physiologic binding partner PABP-interacting protein 2 (Paip2), in the nucleus. Using glutathione S-transferase-PABP pull-down and proteomic analyses, we identified several viral proteins interacting with PABPC1 including tegument protein UL47 and infected-cell protein ICP27. Transient expression of ICP27 and UL47 in HeLa cells suggested that ICP27 and UL47 jointly displace Paip2 from PABP. ICP27 expression alone was sufficient to cause PABPC1 redistribution to the nucleus. ICP27 and UL47 did not alter translation efficiency of transfected reporter RNAs but modulated transcript abundance and expression of reporter cDNAs in transfected cells. This indicates that redistribution of PABPC1 may be involved in co- and posttranscriptional regulation of mRNA processing and/or nuclear export by HSV-1 gene regulatory proteins.
Competitive translation efficiency at the picornavirus type 1 internal ribosome entry site facilitated by viral cis and trans factors.
(2006)
Journal - Journal of virology (United States )
Abstract :
Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis- and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5'-proximal open reading frame and the 3' untranslated region, contribute to stimulation of IRES-mediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5' untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.
| ISSN : | 0022-538X |
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| Mesh Heading : | 3' Untranslated Regions Enterovirus Infections Hela Cells Humans Luciferases Open Reading Frames Picornaviridae RNA, Messenger Ribosomes metabolism metabolism genetics metabolism chemistry metabolism genetics |
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| Mesh Heading Relevant : | Protein Biosynthesis RNA, Viral metabolism metabolism |
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Structural determinants of insert retention of poliovirus expression vectors with recombinant IRES elements.
(2003)
Journal - Virology (United States )
Abstract :
Although picornaviruses provide attractive vectors for expression of foreign genes, poor genetic stability restricts their use for immunization purposes. A new prototype vector was generated to increase foreign insert retention, by shifting of the initiation codon to a cryptic AUG within the internal ribosomal entry site (IRES) and replacement of IRES domain VI with foreign ORFs. Using our strategy to replace regulatory noncoding sequences with unrelated foreign genetic material, we generated stable poliovirus-based expression vectors with robust long-term expression of foreign ORFs. Our studies revealed that size and predicted secondary structure formed by the heterologous sequences govern long-term retention and efficiency of expression of foreign inserts replacing IRES structures. These observations indicate that, with certain limitations imposed by structural preferences, foreign sequences can functionally replace IRES substructures in stable picornavirus immunization vectors.
| ISSN : | 0042-6822 |
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| Mesh Heading : | Base Sequence Gene Expression Genetic Vectors Hela Cells Humans Molecular Sequence Data Open Reading Frames Poliovirus RNA Stability Recombination, Genetic Regulatory Sequences, Nucleic Acid Ribosomes Thermodynamics Viral Vaccines genetics growth & development physiology genetics genetics |
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| Mesh Heading Relevant : | genetics genetics genetics genetics metabolism |
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Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3' nontranslated region.
(2003)
Journal - Proceedings of the National Academy of Sciences of the United States of America (United States )
Abstract :
We have proposed a cancer treatment modality based on poliovirus chimeras replicating under the translational control of an internal ribosomal entry site (IRES) derived from human rhinovirus type 2. Insertion of the heterologous IRES causes a neuron-specific propagation deficit and eliminates neurovirulence inherent in poliovirus without affecting viral growth in cells derived from malignant gliomas. We now report the elucidation of a molecular mechanism responsible for the cell type-specific defect mediated by the rhinovirus IRES. Rhinovirus IRES function in neuronal cell types depends on specific structural elements within the 3' non-translated region of the viral genome. Our observations suggest long-range interactions between the IRES and the 3' terminus that control IRES-mediated gene expression and virus propagation.
| ISSN : | 0027-8424 |
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| Mesh Heading : | Base Sequence Blotting, Western Hela Cells Humans Luciferases Molecular Sequence Data Nucleic Acid Conformation Picornaviridae RNA Recombination, Genetic Rhinovirus Ribosomes Time Factors metabolism metabolism genetics |
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| Mesh Heading Relevant : | 3' Untranslated Regions Gene Expression Regulation, Viral genetics metabolism chemistry |
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Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by Viral cis and trans Factors
(2006)
Journal - Journal of Virology
Abstract :
Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis- and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5'-proximal open reading frame and the 3' untranslated region, contribute to stimulation of IRES-mediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5' untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.
Cell-Type-Specific Repression of Internal Ribosome Entry Site Activity by Double-Stranded RNA-Binding Protein 76
(2006)
Journal - Journal of Virology
Abstract :
Translation of picornavirus plus-strand RNA genomes occurs via internal ribosomal entry at highly structured 5' untranslated regions. In addition to canonical translation factors, translation rate is likely influenced by supplementary host and viral trans-acting factors. We previously reported that insertion of a heterologous human rhinovirus type 2 internal ribosomal entry site (IRES) into the poliovirus (PV) genome, generating the chimeric virus PV-RIPO, selectively abrogates viral translation and propagation in neurons, which eliminate poliovirus's signature neuropathogenicity. While severely deficient in cells of neuronal lineage, the rhinovirus IRES promotes efficient propagation of PV-RIPO in cancer cells. Tumor-specific IRES function can be therapeutically exploited to direct viral cytotoxicity to cancer cells. Neuron-glioma heterokaryon analysis implicates neuronal trans-dominant inhibition in this effect, suggesting that host trans-acting factors repress IRES function in a cell-type-specific manner. We identified a set of proteins from neuronal cells with affinity for the rhinovirus IRES, including double-stranded RNA-binding protein 76 (DRBP76). DRBP76 associates with the IRES in neuronal but not in malignant glioma cells. Moreover, DRBP76 depletion in neuronal cells enhances rhinovirus IRES-driven translation and virus propagation. Our observations suggest that cell-type-specific association of DRBP76 with the rhinovirus IRES represses PV-RIPO translation and propagation in neuronal cells.
Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells
(2005)
Journal - Journal of Virology
Abstract :
The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.
Genetically Stable Picornavirus Expression Vectors with Recombinant Internal Ribosomal Entry Sites
(2002)
Journal - Journal of Virology
Abstract :
In many respects, picornaviruses are well suited for their proposed use as immunization vectors. However, their inherent genetic instability hinders application for prophylactic purposes. We demonstrate the improved expression and stability of a heterologous insert through a novel vector design strategy that partially replaces noncoding regulatory sequences with coding sequences for foreign gene products.