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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]

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]

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]

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]

RNA G-Quadruplexes (G4) have been shown to possess many biological functions, including the regulation of microRNA (miRNA) biogenesis and function. However, their impact on pri-miRNA processing remains unknown. We identified G4 located near the Drosha cleavage site in three distinct pri-miRNAs: pri-mir200c, pri-mir451a, and pri-mir497. The folding of the potential G4 motifs was determined in solution. Subsequently, mutations disrupting G4 folding led to important changes in the mature miRNAs levels in cells. Moreover, using small antisense oligonucleotides binding to the pri-miRNA, it was possible to modulate, either positively or negatively, the mature miRNA levels. Together, these data demonstrate that G4 motifs could contribute to the regulation of pri-mRNA processing, a novel role for G4. Considering that bio-informatics screening indicates that between 9% and 50% of all pri-miRNAs contain a putative G4, these structures possess interesting potential as future therapeutic targets. [ABSTRACT FROM PUBLISHER]

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]

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]

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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]

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]

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]

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]

The 3′ ends of eukaryotic mRNAs are characterized by the presence of a poly(A) tail, which plays a critical role in stability, transport, and translation of the mRNAs. In the present study, we report the expression, purification and enzymatic characterization of the poly(A) polymerase of Candida albicans, an important human pathogen. As a first step toward elucidating the nature of the interaction between RNA and the enzyme, fluorescence spectroscopy assays were also performed to monitor the binding of RNA to the protein. Our 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 the enzyme is eight nucleotides. Moreover, both the kinetics of real-time RNA binding and the contribution of electrostatic interactions to the overall binding energy were investigated. Finally, we also correlated the effect of RNA binding on protein structure, using both circular dichroism and guanidium hydrochloride-induced 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. In addition to the determination of the energetics of RNA binding, our study provides a better understanding of the molecular basis of RNA binding by poly(A) polymerases. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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]

In the present study, computer-assisted searches for sequence similarities were performed with amino acid sequences from mammalian reovirus proteins. These analysis revealed that many proteins of reovirus are partially similar to known viral or cellular proteins. Consensus sequences have been identified that are in accordance with already suspected functions of reovirus proteins. The analysis has also revealed unexpected similarities of some reovirus proteins with specific classes of proteins which sequences are present in the databases. This could suggest yet unidentified activities for some of the reovirus proteins. [ABSTRACT FROM AUTHOR]

As with most viruses, mammalian reovirus can be recognized and attacked by the host-cell interferon response network. Similarly, many viruses have developed resistance mechanisms to counteract the host-cell response at different points of this response. Reflecting the complexity of the interferon signaling pathways as well as the resulting antiviral response, viruses can—and often have—evolved many determinants to interfere with this innate immune response and allow viral replication. In the last few years, it has been evidenced that mammalian reovirus encodes many different determinants that are involved in regulating the induction of the interferon response or in interfering with the action of interferon-stimulated gene products. In this brief review, we present our current understanding of the different reovirus proteins known to be involved, introduce their postulated modes of action, and raise current questions that may lead to further investigations. [ABSTRACT FROM AUTHOR]

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]

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]