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Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide and is a known etiological agent of hepatocellular carcinoma (HCC). The molecular mechanisms of HCV-mediated carcinogenesis are not fully understood. This review article focuses on the oncogenic potential of NS3, a viral protein with transformative effects on cells, although the precise mechanisms remain elusive. Unlike the more extensively studied Core and NS5A proteins, NS3's roles in cancer development are less defined but critical. Research indicates that NS3 is implicated in several carcinogenic processes such as proliferative signaling, cell death resistance, genomic instability and mutations, invasion and metastasis, tumor-related inflammation, immune evasion, and replicative immortality. Understanding the direct impact of viral proteins such as NS3 on cellular transformation is crucial for elucidating HCV's role in HCC development. Overall, this review sheds light on the molecular mechanisms used by NS3 to contribute to hepatocarcinogenesis, and highlights its significance in the context of HCV-associated HCC, underscoring the need for further investigation into its specific molecular and cellular actions. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide and is a known etiological agent of hepatocellular carcinoma (HCC). The molecular mechanisms of HCV-mediated carcinogenesis are not fully understood. This review article focuses on the oncogenic potential of NS3, a viral protein with transformative effects on cells, although the precise mechanisms remain elusive. Unlike the more extensively studied Core and NS5A proteins, NS3's roles in cancer development are less defined but critical. Research indicates that NS3 is implicated in several carcinogenic processes such as proliferative signaling, cell death resistance, genomic instability and mutations, invasion and metastasis, tumor-related inflammation, immune evasion, and replicative immortality. Understanding the direct impact of viral proteins such as NS3 on cellular transformation is crucial for elucidating HCV's role in HCC development. Overall, this review sheds light on the molecular mechanisms used by NS3 to contribute to hepatocarcinogenesis, and highlights its significance in the context of HCV-associated HCC, underscoring the need for further investigation into its specific molecular and cellular actions. [ABSTRACT FROM AUTHOR]

Many temperature-sensitive mutants have been isolated in early studies of mammalian reovirus. However, the biological properties and nature of the genetic alterations remain incompletely explored for most of these mutants. The mutation harbored by the tsI138 mutant was already assigned to the L3 gene encoding the λ1 protein. In the present study, this mutant was further studied as a possible tool to establish the role of the putative λ1 enzymatic activities in viral multiplication. It was observed that synthesis of viral proteins is only marginally reduced, while it was difficult to recover viral particles at the nonpermissive temperature. A single nucleotide substitution resulting in an amino acid change was found; the position of this amino acid is consistent with a probable defect in assembly of the inner capsid at the nonpermissive temperature. [ABSTRACT FROM AUTHOR]

Peach latent mosaic viroid (PLMVd) is a small, single-stranded, circular RNA pathogen that infects Prunus persica trees. As with all other known viroids, the PLMVd genome does not encode any proteins. Consequently, it must interact with host cellular factors in order to ensure its life cycle. With the objective of identifying cellular proteins that interact with PLMVd, Northwestern hybridizations were performed using partially purified peach leaf extracts. Mass spectrometric analysis of the detected RNA-protein complexes led to the identification of six putative RNA-binding proteins. One of these was found to be elongation factor 1-alpha (eEF1A), and because of its known involvement in the replication and translation of various RNA viruses, further characterizations were performed. Initially, the existence of this interaction received support from an experiment that immunoprecipitated the eEF1A from a crude extract of infected peach leaves, coupled with reverse transcription-PCR detection of the PLMVd. Subsequently, eEF1A interaction with PLMVd strands of both polarities was confirmed in vitro by electrophoresis mobility shift assays, fluorescence spectroscopy, and the prediction of an altered PLMVd RNase mapping profile in the presence of the protein. The potential contribution of eEF1A to the molecular biology of PLMVd, including for viroid replication, is discussed. [ABSTRACT FROM AUTHOR]

Abstract: West Nile virus (WNV) is a member of the Flaviviridae family which includes a number of important human pathogens. The WNV NS5 protein harbors an RNA-dependent RNA polymerase activity which is required both for replication and transcription of the viral genome. To extend our studies on the role of metal ions in the activity of flaviviral polymerases, we have used fluorescence spectroscopy, circular dichroism, and a combination of chemical and thermal denaturation assays to monitor the consequences of metal ion binding to the enzyme. We demonstrate that the binding of magnesium is not critical for the structural stabilization of the enzyme. Moreover, structural studies indicate that the protein does not undergo conformational change upon the binding of magnesium ions. Additional binding assays also indicate that the interaction of magnesium ions with the enzyme does not significantly stimulate the interaction with the RNA or NTP substrates. The inability of cobalt hexamine, an exchange-inert metal complex structurally analogous to magnesium hexahydrate, to support the catalytic activity also allowed us to demonstrate a direct role of magnesium ions in the catalytic activity of the enzyme. Finally, a three-dimensional structural model of the active center of the enzyme was generated which highlighted the importance of two aspartate residues involved in the coordination of two metal ions. Mutational analyses confirmed the importance of these two amino acids for the binding of magnesium ions. Our data provide further insight into the precise role of magnesium ions for the RNA polymerase activity of the protein, and more importantly, highlight key differences between the RNA polymerases of the Flaviviridae family. [Copyright &y& Elsevier]

The broad spectrum antiviral nucleoside ribavirin displays activity against a variety of RNA and DNA viruses. A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin, including the possibility that ribavirin might have a negative effect on the synthesis of the RNA cap structure of viral RNA transcripts. In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that ribavirin triphosphate can be used as a substrate by the capping enzyme and can form a covalent ribavirin monophosphate-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts that possess ribavirin as the blocking nucleoside are more stable than unblocked transcripts. However, in vitro translation assays indicate that RNA transcripts blocked with ribavirin are not translated efficiently. Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The biological implications of these findings for the proposed ribavitin-mediated inhibition of capping are discussed. [ABSTRACT FROM AUTHOR]

Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon-α for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae RNA triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5'-terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg[sup 2+], Mn[sup 2+], and Co[sup 2+] ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate. [ABSTRACT FROM AUTHOR]

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus–host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response. [ABSTRACT FROM AUTHOR]

As obligate intracellular parasites, viruses rely heavily on host cells for replication, and therefore dysregulate several cellular processes for their benefit. In return, host cells activate multiple signaling pathways to limit viral replication and eradicate viruses. The present study explores the complex interplay between viruses and host cells through next generation RNA sequencing as well as mass spectrometry (SILAC). Both the coding transcriptome and the proteome of human brain-derived U87 cells infected with Kunjin virus, Zika virus, or Yellow Fever virus were compared to the transcriptome and the proteome of mock-infected cells. Changes in the abundance of several hundred mRNAs and proteins were found in each infection. Moreover, the alternative splicing of hundreds of mRNAs was found to be modulated upon viral infection. Interestingly, a significant disconnect between the changes in the transcriptome and those in the proteome of infected cells was observed. These findings provide a global view of the coding transcriptome and the proteome of Flavivirus-infected cells, leading to a better comprehension of Flavivirus–host interactions. [ABSTRACT FROM AUTHOR]

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family that infects a large range of mammals, including humans. Recently, studies have shown that MRV alters cellular alternative splicing (AS) during viral infection. The structural protein μ2 appears to be the main determinant of these AS modifications by decreasing the levels of U5 core components EFTUD2, PRPF8, and SNRNP200 during infection. In the present study, we investigated the mechanism by which μ2 exerts this effect on the U5 components. Our results revealed that μ2 has no impact on steady-state mRNA levels, RNA export, and protein stability of these U5 snRNP proteins. However, polysome profiling and metabolic labeling of newly synthesized proteins revealed that μ2 exerts an inhibitory effect on global translation. Moreover, we showed that μ2 mutants unable to accumulate in the nucleus retain most of the ability to reduce PRPF8 protein levels, indicating that the effect of μ2 on U5 snRNP components mainly occurs in the cytoplasm. Finally, co-expression experiments demonstrated that μ2 suppresses the expression of U5 snRNP proteins in a dose-dependent manner, and that the expression of specific U5 snRNP core components have different sensitivities to μ2's presence. Altogether, these results suggest a novel mechanism by which the μ2 protein reduces the levels of U5 core components through translation inhibition, allowing this viral protein to alter cellular AS during infection. [ABSTRACT FROM AUTHOR]

Canine distemper virus (CDV) is a negative-sense, single-stranded RNA virus within the genus Morbillivirus and the family Paramyxoviridae. The Morbillivirus genome is composed of six transcriptional units that are separated by untranslated regions (UTRs), which are relatively uniform in length, with the exception of the UTR between the matrix (M) and fusion (F) genes. This UTR is at least three times longer and in the case of CDV also highly variable. Exchange of the M-F region between different CDV strains did not affect virulence or disease phenotype, demonstrating that this region is functionally interchangeable. Viruses carrying the deletions in the M 3' UTR replicated more efficiently, which correlated with a reduction of virulence, suggesting that overall length as well as specific sequence motifs distributed throughout the region contribute to virulence. [ABSTRACT FROM AUTHOR]

SUMMARY Knowledge of the structure of a viroid is critically important to elucidate the roles played by the various RNA motifs in the steps of the viroid's life cycle. A new technique, RNA-selective 2′-hydroxyl acylation analysed by primer extension (SHAPE), has recently been shown to be fast, reliable and applicable to the study of various RNA molecules. Consequently, this method was used to probe sequence variants of Peach latent mosaic viroid (PLMVd). Initially, probing data from RNA strands of both polarities of the Siberian C variant confirmed the secondary structures previously determined using both conventional and fastidious approaches. Subsequently, analysis of an Alberta variant showed an identical structure for the strand of (−) polarity, but the (+) polarity strand exhibited two differences from the Siberian C variant: the P11-L11 stem-loop domain formed a cruciform structure, and nucleotides from loops L1 and L11 were involved in the formation of a pseudoknot. The existence of both of these motifs was confirmed by site-directed mutagenesis. The subsequent probing of 12 natural sequence variants led to the elucidation of the criteria governing the formation of this novel pseudoknot. Importantly, this study revealed that the heterogeneity of a viroid is not limited to its nucleotide sequence, but may also occur at the structural level. [ABSTRACT FROM AUTHOR]

Summary: Both the role and the interacting partners of an RNA molecule can change depending on its tertiary structure. Consequently, it is important to be able to accurately predict the complete folding pathway of an RNA molecule. The hepatitis delta virus (HDV) ribozyme is a small catalytic RNA with the greatest number of folding intermediates making it the model of choice with which to address this problem. The tertiary structures of the known putative intermediates along the folding pathway of the HDV ribozyme were predicted using the Macromolecular Conformations Symbolic programming (MC-Sym) software. The structures obtained by this method received physical support from Selective 2′-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). The analysis of these structures elucidated several features of the HDV ribozyme. In addition, this report represents an application for MC-Sym that permits progression one step further toward the computer prediction of an RNA molecule-folding pathway. [ABSTRACT FROM AUTHOR]

An RNA guanylyltransferase activity is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mRNAs. The RNA guanylyltransferase activity is a two-step ping-pong reaction in which the enzyme first reacts with GTP to produce the enzyme-GMP covalent intermediate with the concomitant release of pyrophosphate. In the second step of the reaction, the GMP moiety is then transferred to a diphosphorylated RNA. Both reactions were previously shown to be reversible. In this study, we report a biochemical and thermodynamic characterization of both steps of the reaction of the RNA guanylyltransferase from Paramecium bursaria Chlorella virus 1, the prototype of a family of viruses infecting green algae. Using a combination of real-time fluorescence spectroscopy, radioactive kinetic assays, and inhibition assays, the complete kinetic parameters of the RNA guanylyltransferase were determined. We produced a thermodynamic scheme for the progress of the reaction as a function of the energies involved in each step. We were able to demonstrate that the second step comprises the limiting steps for both the direct and reverse overall reactions. In both cases, the binding to the RNA substrates is the step requiring the highest energy and generating unstable intermediates that will promote the catalytic activites of the enzyme. This study reports the first thorough kinetic and thermodynamic characterization of the reaction catalyzed by an RNA capping enzyme. [ABSTRACT FROM AUTHOR]

Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 µM for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication. [ABSTRACT FROM AUTHOR]

The RNA polymerase activity of the hepatitis C virus, a major human pathogen, has previously been shown to be supported by metal ions. In the present study, we report a systematic analysis of the effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase. Chemical and thermal denaturation assays revealed that the stability of the protein is increased significantly in the presence of metal ions. Structural analyses clearly established that metal ion binding increases hydrophobic exposure on the RNA polymerase surface. Furthermore, our denaturation studies, coupled with polymerization assays, demonstrate that the active site region of the polymerase is more sensitive to chemical denaturant than other structural scaffolds. We also report the first detailed study of the thermodynamic parameters involved in the interaction between the hepatitis C virus RNA polymerase and metal ions. Finally, a mutational analysis was also performed to investigate the importance of Asp220, Asp318, and Asp319 for metal ion binding. This mutational study underscores a strict requirement for each of the residues for metal binding, indicating that the active center of the HCV RNA polymerase is intolerant to virtually any perturbations of the metal coordination sphere, thereby highlighting the critical role of the enzyme-bound metal ions. Overall, our results indicate that metal ions play a dual modulatory role in the RNA polymerase reaction by promoting both a favorable geometry of the active site for catalysis and by increasing the structural stability of the enzyme. [ABSTRACT FROM AUTHOR]

Describes the characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase. Use of endogenous tryptophan fluorescent to evaluate the interactions of metal ions with the hepatitis C virus nonstructural 5B protein; Association of either magnesium or manganese ions with the ions; Decrease in the intensity of the tryptophan emission spectrum.

The 5' RNA cap structure (m7GpppRNA) is a key feature of eukaryotic mRNAs with important roles in stability, splicing, polyadenylation, mRNA export, and translation. Higher eukaryotes can further modify this minimal cap structure with the addition of a methyl group on the ribose 2'-O position of the first transcribed nucleotide (m7GpppNmpRNA) and sometimes on the adjoining nucleotide (m7GpppNmpNmpRNA). In higher eukaryotes, the DXO protein was previously shown to be responsible for both decapping and degradation of RNA transcripts harboring aberrant 5’ ends such as pRNA, pppRNA, GpppRNA, and surprisingly, m7GpppRNA. It was proposed that the interaction of the cap binding complex with the methylated cap would prevent degradation of m7GpppRNAs by DXO. However, the critical role of the 2’-O-methylation found in higher eukaryotic cap structures was not previously addressed. In the present study, we demonstrate that DXO possesses both decapping and exoribonuclease activities toward incompletely capped RNAs, only sparing RNAs with a 2’-O-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2’-O-methylation on the cap structure drastically reduces the affinity of DXO for RNA. Moreover, immunofluorescence and structure-function assays also revealed that a nuclear localisation signal is located in the amino-terminus region of DXO. Overall, these results are consistent with a quality control mechanism in which DXO degrades incompletely capped RNAs. [ABSTRACT FROM AUTHOR]

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein–Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection. [ABSTRACT FROM AUTHOR]

Background: Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. Results: Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. Conclusions: This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC. [ABSTRACT FROM AUTHOR]

Iron is an essential metal cofactor that is required for many biological processes. Eukaryotic cells have consequently developed different strategies for its acquisition. Until now, Schizosaccharomyces pombe was known to use reductive iron uptake and siderophore-bound iron transport to scavenge iron from the environment. Here, we report the identification of a gene designated shu1+ that encodes a protein that enables S. pombe to take up extracellular heme for cell growth. When iron levels are low, the transcription of shu1+ is induced, although its expression is repressed when iron levels rise. The iron-dependent down-regulation of shu1+ requires the GATA-type transcriptional repressor Fep1, which strongly associates with a proximal promoter region of shu1+ in vivo in response to iron repletion. HA4-tagged Shu1 localizes to the plasma membrane in cells expressing a functional shu1+-HA4 allele. When heme biosynthesis is selectively blocked in mutated S. pombe cells, their ability to acquire exogenous hemin or the fluorescent heme analog zincmesoporphyrin IX is dependent on the expression of Shu1. Further analysis by absorbance spectroscopy and hemin-agarose pulldown assays showed that Shu1 interacts with hemin, with a KD of ~2.2 µM. Taken together, results reported here revealed that S. pombe possesses an unexpected pathway for heme assimilation, which may also serve as a source of iron for cell growth. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a major public-health problem with 130–170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. Hepatocellular uptake of hLF was confirmed and correlated with qualitative HCV staining reduction. Quantitative dose–response inhibition assays confirmed an hLF-mediated and dose-dependent HCV replication reduction reaching up to 60%. The in cellulo anti-HCV activity of hLF was additive to both Ribavirin and Interferon-α-2b. Further investigation of hLF activity against the essential viral proteins involved in HCV genome replication revealed an inhibitory activity against the HCV ATPase/Helicase NS3 protein but not against the HCV RNA-dependent RNA polymerase (NS5B protein). NS3 inhibition was mediated by a direct and specific interaction between hLF and an allosteric binding site on NS3. Taken together, our findings reveal a new antiviral mechanism of action by which hLF inhibits intracellular HCV replication. [ABSTRACT FROM AUTHOR]

Decapping scavenger ( Dcp S) assists in precluding inhibition of cap-binding proteins by hydrolyzing cap species remaining after m RNA 3′→5′ degradation. Its significance was reported in splicing, translation initiation and micro RNA turnover. Here we examine the structure and binding mode of Dcp S from Caenorhabditis elegans ( Ce DcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that Ce DcpS is a homodimer and propose high accuracy structural models of apo- and m7 Gppp G-bound forms. The analysis of Ce Dcp S regioselectivity uncovers that the only site of hydrolysis is located between the β and γ phosphates. Structure-affinity relationship studies of cap analogs for Ce Dcp S reveal molecular determinants for efficient cap binding: a strong dependence on the type of substituents in the phosphate chain, and reduced binding affinity for either methylated hydroxyl groups of m7 Guo or an extended triphosphate chain. Docking analysis of cap analogs in the Ce DcpS active site explains how both phosphate chain mobility and the orientation in the cap-binding pocket depend on the number of phosphate groups, the substituent type and the presence of the second nucleoside. Finally, the comparison of Ce DcpS with its well known human homolog provides general insights into Dcp S-cap interactions. [ABSTRACT FROM AUTHOR]

Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine- 59-monophosphate dehydrogenase (IMPDH), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. MZP is a highly potent antagonistic inhibitor of IMPDH that blocks the proliferation of T and B lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 59-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 59 end of eukaryotic mRNAs, which is critical for the splicing of the cap-proximal intron, the transport of mRNAs from the nucleus to the cytoplasm, and for both the stability and translation of mRNAs. Our biochemical studies provide the first insight that MZP can inhibit the formation of the RNA cap structure catalyzed by HCE. In the presence of MZP, the RNA 59-triphosphatase activity appears to be relatively unaffected while the RNA guanylyltransferase activity is inhibited, indicating that the RNA guanylyltransferase activity is the main target of MZP inhibition. Kinetic studies reveal that MZP is a non-competitive inhibitor that likely targets an allosteric site on HCE. Mizoribine also impairs mRNA capping in living cells, which could account for the global mechanism of action of this therapeutic agent. Together, our study clearly demonstrates that mizoribine monophosphate inhibits the human RNA guanylyltransferase in vitro and impair mRNA capping in cellulo. [ABSTRACT FROM AUTHOR]

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses. [ABSTRACT FROM AUTHOR]

The RNA guanylyltransferase (GTase) is involved in the synthesis of the m7Gppp-RNA cap structure found at the 59 end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme- GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed. [ABSTRACT FROM AUTHOR]

Background/Aims: Infection with hepatitis C virus (HCV) is associated with alterations in body iron homeostasis by poorly defined mechanisms. To seek for molecular links, we employed an established cell culture model for viral replication, and assessed how the expression of an HCV subgenomic replicon affects iron metabolism in host Huh7 hepatoma cells. Methods: The expression of iron metabolism genes and parameters defining the cellular iron status were analyzed and compared between parent and replicon Huh7 cells. Results: By using the IronChip microarray platform, we observed replicon-induced changes in expression profiles of iron metabolism genes. Notably, ceruloplasmin mRNA and protein expression were decreased in replicon cells. In addition, transferrin receptor 1 (TfR1) was also downregulated, while ferroportin levels were elevated, resulting in reduced iron uptake and increased iron release capacity of replicon cells. These responses were associated with an iron-deficient phenotype, manifested in decreased levels of the “labile iron pool” and concomitant induction of IRE-binding activity and IRP2 expression. Furthermore, hemin-treated replicon cells exhibited a defect in retaining iron. The clearance of the replicon by prolonged treatment with interferon-α only partially reversed the iron-deficient phenotype but almost completely restored the capacity of cured cells to retain iron. Conclusions: We propose that Huh7 cells undergo genetic reprogramming to permit subgenomic viral replication that results in reduction of intracellular iron levels. This response may provide a mechanism to bypass iron-mediated inactivation of the viral RNA polymerase NS5B. [Copyright &y& Elsevier]

Abstract: The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5′ end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein. [Copyright &y& Elsevier]

Background: Alternative splicing (AS) is an important mRNA maturation step that allows increased variability and diversity of proteins in eukaryotes. AS is dysregulated in numerous diseases, and its implication in the carcinogenic process is well known. However, progress in understanding how oncogenic viruses modulate splicing, and how this modulation is involved in viral oncogenicity has been limited. Epstein-Barr virus (EBV) is involved in various cancers, and its EBNA1 oncoprotein is the only viral protein expressed in all EBV malignancies. Methods: In the present study, the ability of EBNA1 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in 1238 cancer-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by EBNA1. Results: Upon EBNA1 expression, we detected modifications to the AS profiles of 89 genes involved in cancer. Moreover, we show that EBNA1 modulates the expression levels of various splicing factors such as hnRNPA1, FOX-2, and SF1. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that EBNA1 immunoprecipitates specific cellular mRNAs, but not the ones that are spliced differently in EBNA1-expressing cells. Conclusion: The EBNA1 protein can modulate the AS profiles of numerous cellular genes. Interestingly, this modulation protein does not require the RNA binding activity of EBNA1. Overall, these findings underline the novel role of EBNA1 as a cellular splicing modulator. [ABSTRACT FROM AUTHOR]

Mammalian reovirus is an oncolytic virus that discriminates between parental and cancer cells. This is due in part to altered interferon-induced antiviral response in cancer cells. However, understanding of viral and cellular determinants involved remains largely incomplete. In our current work, two different approaches were used to identify reovirus variants exhibiting increased interferon sensitivity. (1) Chemical mutagenesis followed by selection of small plaques mutants in the presence of interferon; (2) Selection on interferon-deficient cells in order to relieve the pressure to keep interferon resistance. Virus variants were sequenced and plasmid-based reverse genetics was used to examine the effect of each mutation. A single amino acid substitution in the 2′-O methyltransferase of viral mRNA capping enzyme was responsible for 10–100 fold increased interferon sensitivity for one variant; work is under progress for additional viruses. In parallel, we took advantage of these well genetically-defined viruses to investigate host-cell response using RNA-Seq. A large number of induced genes was observed in wild-type virus-infected cells, to an extent that was not revealed by previously-used approaches. Most induced genes encode sensors or antiviral response determinants (ISG, IFIT, IRF, Mx). They were similarly induced by the wild-type and interferon-sensitive virus. This suggests that altered RNA capping increases virus’ sensitivity to interferon-induced antiviral factors rather than induction of interferon signaling. Similar analysis with other viruses of different interferon sensitivity, in parental and transformed cells, should allow to better comprehend the antiviral response during reovirus infection and could contribute to the design of better oncolytic viruses. [ABSTRACT FROM AUTHOR]

RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism. [ABSTRACT FROM AUTHOR]