Matthias Gromeier

The Double-Stranded RNA Binding Protein 76:NF45 Heterodimer Inhibits Translation Initiation at the Rhinovirus Type 2 Internal Ribosome Entry Site

ABSTRACT Poliovirus (PV) plus-strand RNA genomes initiate translation in a cap-independent manner via an internal ribosome entry site (IRES) in their 5′ untranslated region. Viral translation is codetermined by cellular IRES trans-acting factors, which can influence viral propagation in a cell-type-specific manner. Engineering of a poliovirus recombinant devoid of neuropathogenic properties but highly lytic in malignant glioma cells was accomplished by exchange of the cognate poliovirus IRES with its counterpart from human rhinovirus type 2 (HRV2), generating PV-RIPO. Neuroblast:glioma heterokaryon analyses revealed that loss of neurovirulence is due to trans-dominant repression of PV-RIPO propagation in neuronal cells. The double-stranded RNA binding protein 76 (DRBP76) was previously identified to bind to the HRV2 IRES in neuronal cells and to inhibit PV-RIPO translation and propagation (M. Merrill, E. Dobrikova, and M. Gromeier, J. Virol. 80:3347-3356, 2006). The results of size exclusion chromatography indicate that DRBP76 heterodimerizes with nuclear factor of activated T cells, 45 kDa (NF45), in neuronal but not in glioma cells. The DRBP76:NF45 heterodimer binds to the HRV2 IRES in neuronal but not in glioma cells. Ribosomal profile analyses show that the heterodimer preferentially associates with the translation apparatus in neuronal cells and arrests translation at the HRV2 IRES, preventing PV-RIPO RNA assembly into polysomes. Results of this study suggest that the DRBP76:NF45 heterodimer selectively blocks HRV2 IRES-driven translation initiation in neuron-derived cells.

The hepatitis C virus 3′-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase

Enhancement of eukaryotic messenger RNA (mRNA) translation initiation by the 3′ poly(A) tail is mediated through interaction of poly(A)-binding protein with eukaryotic initiation factor (eIF) 4G, bridging the 5′ terminal cap structure. In contrast to cellular mRNA, translation of the uncapped, non-polyadenylated hepatitis C virus (HCV) genome occurs independently of eIF4G and a role for 3′-untranslated sequences in modifying HCV gene expression is controversial. Utilizing cell-based and in vitro translation assays, we show that the HCV 3′-untranslated region (UTR) or a 3′ poly(A) tract of sufficient length interchangeably stimulate translation dependent upon the HCV internal ribosomal entry site (IRES). However, in contrast to cap-dependent translation, the rate of initiation at the HCV IRES was unaffected by 3′-untranslated sequences. Analysis of post-initiation events revealed that the 3′ poly(A) tract and HCV 3′-UTR improve translation efficiency by enabling termination and possibly ribosome recycling for successive rounds of translation.

Poliovirus receptor CD155–targeted oncolysis of glioma

Cell adhesion molecules of the immunoglobulin superfamily are aberrantly expressed in malignant glioma. Amongst these, the human poliovirus receptor CD155 provides a molecular target for therapeutic intervention with oncolytic poliovirus recombinants. Poliovirus has been genetically modified through insertion of regulatory sequences derived from human rhinovirus type 2 to selectively replicate within and destroy cancerous cells. Efficacious oncolysis mediated by poliovirus derivatives depends on the presence of CD155 in targeted tumors. To prepare oncolytic polioviruses for clinical application, we have developed a series of assays in high-grade malignant glioma (HGL) to characterize CD155 expression levels and susceptibility to oncolytic poliovirus recombinants. Analysis of 6 HGL cases indicates that CD155 is expressed in these tumors and in primary cell lines derived from these tumors. Upregulation of the molecular target CD155 rendered explant cultures of all studied tumors highly susceptible to a prototype oncolytic poliovirus recombinant. Our observations support the clinical application of such agents against HGL.

Regulation of Eukaryotic Initiation Factor 4E (eIF4E) Phosphorylation by Mitogen-Activated Protein Kinase Occurs through Modulation of Mnk1-eIF4G Interaction

ABSTRACT The m7G cap binding protein eukaryotic initiation factor 4E (eIF4E) is a rate-limiting determinant of protein synthesis. Elevated eIF4E levels, commonly associated with neoplasia, promote oncogenesis, and phosphorylation of eIF4E at Ser209 is critical for its tumorigenic potential. eIF4E phosphorylation is catalyzed by mitogen-activated protein kinase (MAPK)-interacting serine/threonine kinase (Mnk), a substrate of Erk1/2 and p38 MAPKs. Interaction with the scaffolding protein eIF4G, which also binds eIF4E, brings Mnk and its substrate into physical proximity. Thus, Mnk-eIF4G interaction is important for eIF4E phosphorylation. Through coimmunoprecipitation assays, we showed that MAPK-mediated phosphorylation of the Mnk1 active site controls eIF4G binding. Utilizing a naturally occurring splice variant, we demonstrated that the C-terminal domain of Mnk1 restricts its interaction with eIF4G, preventing eIF4E phosphorylation in the absence of MAPK signaling. Furthermore, using a small-molecule Mnk1 inhibitor and kinase-dead mutant, we established that Mnk1 autoregulates its interaction with eIF4G, releasing itself from the scaffold after phosphorylation of its substrate. Our findings indicate tight control of eIF4E phosphorylation through modulation of Mnk1-eIF4G interaction.

PD-0332991, a CDK4/6 Inhibitor, Significantly Prolongs Survival in a Genetically Engineered Mouse Model of Brainstem Glioma

Diffuse intrinsic pontine glioma (DIPG) is an incurable tumor that arises in the brainstem of children. To date there is not a single approved drug to effectively treat these tumors and thus novel therapies are desperately needed. Recent studies suggest that a significant fraction of these tumors contain alterations in cell cycle regulatory genes including amplification of the D-type cyclins and CDK4/6, and less commonly, loss of Ink4a-ARF leading to aberrant cell proliferation. In this study, we evaluated the therapeutic approach of targeting the cyclin-CDK-Retinoblastoma (Rb) pathway in a genetically engineered PDGF-B-driven brainstem glioma (BSG) mouse model. We found that PD-0332991 (PD), a CDK4/6 inhibitor, induces cell-cycle arrest in our PDGF-B; Ink4a-ARF deficient model both in vitro and in vivo. By contrast, the PDGF-B; p53 deficient model was mostly resistant to treatment with PD. We noted that a 7-day treatment course with PD significantly prolonged survival by 12% in the PDGF-B; Ink4a-ARF deficient BSG model. Furthermore, a single dose of 10 Gy radiation therapy (RT) followed by 7 days of treatment with PD increased the survival by 19% in comparison to RT alone. These findings provide the rationale for evaluating PD in children with Ink4a-ARF deficient gliomas.

Cell-Type-Specific Repression of Internal Ribosome Entry Site Activity by Double-Stranded RNA-Binding Protein 76

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 polioviruss 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.

Activity of a type 1 picornavirus internal ribosomal entry site is determined by sequences within the 3′ nontranslated region

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.

Phosphorylation of Eukaryotic Translation Initiation Factor 4G1 (eIF4G1) by Protein Kinase Cα Regulates eIF4G1 Binding to Mnk1

ABSTRACT Signal transduction through mitogen-activated protein kinases (MAPKs) is implicated in growth and proliferation control through translation regulation and involves posttranslational modification of translation initiation factors. For example, convergent MAPK signals to Mnk1 lead to phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), which has been linked to malignant transformation. However, understanding the compound effects of mitogenic signaling on the translation apparatus and on protein synthesis control remains elusive. This is particularly true for the central scaffold of the translation initiation apparatus and ribosome adaptor eIF4G. To unravel the effects of signal transduction to eIF4G on translation, we used specific activation of protein kinase C (PKC)-Ras-Erk signaling with phorbol esters. Phospho-proteomic and mutational analyses revealed that eIF4G1 is a substrate for PKCα at Ser1186. We show that PKCα activation elicits a cascade of orchestrated phosphorylation events that may modulate eIF4G1 structure and control interaction with the eIF4E kinase, Mnk1.

Herpes Simplex Virus Proteins ICP27 and UL47 Associate with Polyadenylate-Binding Protein and Control Its Subcellular Distribution

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.

Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

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.