RESUMEN
Coxsackievirus B3 is an enterovirus, with positive-sense single-stranded RNA genome containing 'Internal Ribosome Entry Site' (IRES) in the 5'UTR. Once sufficient viral proteins are synthesized in the cell from the input RNA, viral template switches from translation to replication to synthesize negative-strand RNA. Inhibition of translation is a key step in regulating this switch as the positive-strand RNA template should be free of ribosomes to enable polymerase movement. In this study, we show how a host protein hnRNP C1/C2 inhibits viral RNA translation. hnRNP C1/C2 interacts with stem-loop V in the IRES and displaces poly-pyrimidine tract binding protein, a positive regulator of translation. We further demonstrate that hnRNP C1/C2 induces translation to replication switch, independently from the already known role of the ternary complex (PCBP2-3CD-cloverleaf RNA). These results suggest a novel function of hnRNP C1/C2 in template switching of positive-strand from translation to replication by a new mechanism. Using mathematical modelling, we show that the differential affinity of hnRNP C1/C2 for positive and negative-strand RNAs guides the final ± RNA ratio, providing first insight in the regulation of the positive to negative-strand RNA ratio in enteroviruses.
Asunto(s)
Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , ARN Viral/genética , Regiones no Traducidas 5'/genética , Células HeLa , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Modelos Biológicos , Biosíntesis de Proteínas , Replicación Viral/genéticaRESUMEN
The 5' UTR of Coxsackievirus B3 (CVB3) contains internal ribosome entry site (IRES), which allows cap-independent translation of the viral RNA and a 5'-terminal cloverleaf structure that regulates viral replication, translation and stability. Here, we demonstrate that host protein PSF (PTB associated splicing factor) interacts with the cloverleaf RNA as well as the IRES element. PSF was found to be an important IRES trans acting factor (ITAF) for efficient translation of CVB3 RNA. Interestingly, cytoplasmic abundance of PSF protein increased during CVB3 infection and this is regulated by phosphorylation status at two different amino acid positions. Further, PSF protein was up-regulated in CVB3 infection. The expression of CVB3-2A protease alone could also induce increased PSF protein levels. Furthermore, we observed the presence of an IRES element in the 5'UTR of PSF mRNA, which is activated during CVB3 infection and might contribute to the elevated levels of PSF. It appears that PSF IRES is also positively regulated by PTB, which is known to regulate CVB3 IRES. Taken together, the results suggest for the first time a novel mechanism of regulations of ITAFs during viral infection, where an ITAF undergoes IRES mediated translation, sustaining its protein levels under condition of translation shut-off.
Asunto(s)
Enterovirus Humano B/genética , Interacciones Huésped-Patógeno , Factor de Empalme Asociado a PTB/genética , Biosíntesis de Proteínas , ARN Viral/genética , Ribosomas/genética , Regiones no Traducidas 5' , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Enterovirus Humano B/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Sitios Internos de Entrada al Ribosoma , Conformación de Ácido Nucleico , Factor de Empalme Asociado a PTB/antagonistas & inhibidores , Factor de Empalme Asociado a PTB/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Ribosomas/metabolismo , Transducción de Señal , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200µL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
Asunto(s)
Patógenos Transmitidos por la Sangre/aislamiento & purificación , Sangre/microbiología , Técnicas Microbiológicas/métodos , Patología Molecular/métodos , Sepsis/diagnóstico , Sepsis/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Candida tropicalis/genética , Candida tropicalis/aislamiento & purificación , Células Cultivadas , Recuento de Colonia Microbiana/métodos , ADN Bacteriano/sangre , ADN de Hongos/sangre , Enterococcus/genética , Enterococcus/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Hongos/clasificación , Hongos/genética , Hongos/aislamiento & purificación , Hongos/patogenicidad , Genoma Humano , Humanos , Límite de Detección , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Sepsis/sangre , Factores de Tiempo , beta 2 Glicoproteína I/administración & dosificaciónRESUMEN
Using anti-human CD45 antibody coated beads, we show a 98% reduction of WBCs from spiked blood samples in 1h, thereby enriching it for pathogens. This enrichment allowed the detection of <10CFU of Escherichia coli in 1mL blood using quantitative PCR; something not observed in unenriched samples.
Asunto(s)
Bacterias/aislamiento & purificación , Sangre/microbiología , Escherichia coli/aislamiento & purificación , Separación Inmunomagnética/métodos , Leucocitos , Reacción en Cadena de la Polimerasa/métodos , Bacterias/clasificación , Bacterias/genética , Bacterias/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Infecciones por Escherichia coli/diagnóstico , Humanos , Antígenos Comunes de Leucocito/inmunología , Límite de Detección , Sensibilidad y EspecificidadRESUMEN
Protein synthesis can be segmented into distinct phases comprising mRNA translation initiation, elongation, and termination. Translation initiation is a highly regulated and rate-limiting step of protein synthesis that requires more than 12 eukaryotic initiation factors (eIFs). Extensive evidence shows that the transcriptome and corresponding proteome do not invariably correlate with each other in a variety of contexts. In particular, translation of mRNAs specific to angiogenesis, tumor development, and apoptosis is altered during physiological and pathophysiological stress conditions. In cancer cells, the expression and functions of eIFs are hampered, resulting in the inhibition of global translation and enhancement of translation of subsets of mRNAs by alternative mechanisms. A precise understanding of mechanisms involving eukaryotic initiation factors leading to differential protein expression can help us to design better strategies to diagnose and treat cancer. The high spatial and temporal resolution of translation control can have an immediate effect on the microenvironment of the cell in comparison with changes in transcription. The dysregulation of mRNA translation mechanisms is increasingly being exploited as a target to treat cancer. In this review, we will focus on this context by describing both canonical and noncanonical roles of eIFs, which alter mRNA translation.
RESUMEN
Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES-40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES-RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA-protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES-RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S-3' UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES-RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.