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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., (© 2024. The Author(s).)

West Nile virus (WNV) is the most prevalent mosquito-borne virus and the leading cause of viral encephalitis in the continental United States. It belongs to the family Flaviviridae which includes other important human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV) and Zika viruses (ZIKV). Despite several decades of research, no specific antiviral drugs are available to treat flavivirus infections. The present study characterizes the interaction between the WNV NS3 and NS5 proteins for the purpose of identifying hotspots in the protein-protein interaction which could be targeted for the development of antiviral therapeutics. We previously developed an interaction model in silico based on data available in the literature. Here, potential interacting residues on NS3 and NS5 were mutated in a WNV replicon, and seven mutations in the NS3 protein were found to drastically reduce viral replication. In addition to being well conserved among mosquito-borne flaviviruses, these residues are located on the protein's surface in two clusters which might be interesting new targets for future drug development., Competing Interests: The author(s) declare that there are no conflicts of interest., (Copyright © 2024 The Authors.)

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.

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.

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.

Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surfaces and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with ∼97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S-pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:250 dilution, their infectivity was dramatically reduced by ∼98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses., (© The Author(s) 2022. Published by Oxford University Press.)

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV's ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that the M1 gene segment, encoding the μ2 protein, is the primary determinant of MRV's ability to alter AS, and that the amino acid at position 208 in μ2 is critical to induce these changes. Moreover, we showed that the expression of μ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of μ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and μ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The anti-apoptotic BAG-1 protein isoforms are known to be overexpressed in colorectal tumors and are considered to be potential therapeutic targets. The isoforms are derived from alternative translation initiations occuring at four in-frame start codons of a single mRNA transcript. Its 5'UTR also contains an internal ribosome entry site (IRES) regulating the cap-independent translation of the transcript. An RNA G-quadruplex (rG4) is located at the 5'end of the BAG-1 5'UTR, upstream of the known cis-regulatory elements. Herein, we observed that the expression of BAG-1 isoforms is post-transcriptionally regulated in colorectal cancer cells and tumors, and that stabilisation of the rG4 by small molecules ligands reduces the expression of endogenous BAG-1 isoforms. We demonstrated a critical role for the rG4 in the control of both cap-dependent and independent translation of the BAG-1 mRNA in colorectal cancer cells. Additionally, we found an upstream ORF that also represses BAG-1 mRNA translation. The structural probing of the complete 5'UTR showed that the rG4 acts as a steric block which controls the initiation of translation at each start codon of the transcript and also maintains the global 5'UTR secondary structure required for IRES-dependent translation., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Upon viral infection, a tug of war is triggered between host cells and viruses to maintain/gain control of vital cellular functions, the result of which will ultimately dictate the fate of the host cell. Among these essential cellular functions, alternative splicing (AS) is an important RNA maturation step that allows exons, or parts of exons, and introns to be retained in mature transcripts, thereby expanding proteome diversity and function. AS is widespread in higher eukaryotes, as it is estimated that nearly all genes in humans are alternatively spliced. Recent evidence has shown that upon infection by numerous viruses, the AS landscape of host-cells is affected. In this review, we summarize recent advances in our understanding of how virus infection impacts the AS of cellular transcripts. We also present various molecular mechanisms allowing viruses to modulate cellular AS. Finally, the functional consequences of these changes in the RNA splicing signatures during virus-host interactions are discussed. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing., (© 2019 The Authors. WIREs RNA published by Wiley Periodicals, Inc.)

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.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths around the world. Recent advances in genomic technologies have allowed the identification of various molecular signatures in HCC tissues. For instance, differential gene expression levels of various cytochrome P450 genes (CYP450) have been reported in studies performed on limited numbers of HCC tissue samples, or focused on a small subset on CYP450s. In the present study, we monitored the expression landscape of all the members of the CYP450 family (57 genes) in more than 200 HCC tissues using RNA-Seq data from The Cancer Genome Atlas. Using stringent statistical filters and data from paired tissues, we identified significantly dysregulated CYP450 genes in HCC. Moreover, the expression level of selected CYP450s was validated by qPCR on cDNA samples from an independent cohort. Threshold values (sensitivity and specificity) based on dysregulated gene expression were also determined to allow for confident identification of HCC tissues. Finally, a global look at expression levels of the 57 members of the CYP450 family across ten different cancer types revealed specific expression signatures. Overall, this study provides useful information on the transcriptomic landscape of CYP450 genes in HCC and on new potential HCC biomarkers., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

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.

[This corrects the article DOI: 10.1371/journal.pone.0193804.].

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.

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.

Flaviviruses, such as dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses, are serious human pathogens that cause significant morbidity and mortality globally each year. Flaviviruses are single-stranded, positive-sense RNA viruses, and encode two multidomain proteins, NS3 and NS5, that possess all enzymatic activities required for genome replication and capping. NS3 and NS5 interact within virus-induced replication compartments to form the RNA genome replicase complex. Although the individual enzymatic activities of both proteins have been extensively studied and are well characterized, there are still gaps in our understanding of how they interact to efficiently coordinate their respective activities during positive-strand RNA synthesis and capping. Here, we discuss what is known about the structures and functions of the NS3 and NS5 proteins and propose a preliminary NS3:NS5:RNA interaction model based on a large body of literature about how the viral enzymes function, physical restraints between NS3 and NS5, as well as critical steps in the replication process. WIREs RNA 2017, 8:e1437. doi: 10.1002/wrna.1437 For further resources related to this article, please visit the WIREs website., (© 2017 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) are small noncoding RNAs that repress the translation of their target genes. It has previously been shown that a target's availability to miRNA can be affected by its structure. G-quadruplexes (G4) are noncanonical structures adopted by G-rich nucleic acids that have been shown to have multiple biological functions. In this study, whether or not G4 structures' presence in the 3' UTRs of mRNAs can hinder miRNA binding was investigated. Putative G4 overlapping with predicted miRNAs' binding sites was searched for, and 44,294 hits were found in humans. The FADS2 mRNA/mir331-3p pair was selected as a model example. In-line probing and G4-specific fluorescent ligand experiments binding were performed and confirmed the presence of a G4 near the predicted miRNA binding site. Subsequent luciferase assays showed that the presence of the G4 prevents the binding of mir331-3p in cellulo. Together, these results served as proof of concept that a G4 structure present in a 3' UTR sequence should be taken into consideration when predicting miRNA binding sites., (© 2017 Rouleau et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

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 zinc mesoporphyrin 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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

G-quadruplexes (G4) are intricate RNA structures found throughout the transcriptome. Because they are associated with a variety of biological cellular mechanisms, these fascinating structural motifs are seen as potential therapeutic targets against many diseases. While screening of chemical compounds specific to G4 motifs has yielded interesting results, no single compound successfully discriminates between G4 motifs based on nucleotide sequences alone. This level of specificity is best attained using antisense oligonucleotides (ASO). Indeed, oligonucleotide-based strategies are already used to modulate DNA G4 folding in vitro. Here, we report that, in human cells, the use of short ASO to promote and inhibit RNA G4 folding affects the translation of specific mRNAs, including one from the 5'UTR of the H2AFY gene, a histone variant associated with cellular differentiation and cancer. These results suggest that the relatively high specificity of ASO-based strategies holds significant potential for applications aimed at modulating G4-motif folding., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Decapping scavenger (DcpS) assists in precluding inhibition of cap-binding proteins by hydrolyzing cap species remaining after mRNA 3'→5' degradation. Its significance was reported in splicing, translation initiation and microRNA turnover. Here we examine the structure and binding mode of DcpS from Caenorhabditis elegans (CeDcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that CeDcpS is a homodimer and propose high accuracy structural models of apo- and m(7) GpppG-bound forms. The analysis of CeDcpS regioselectivity uncovers that the only site of hydrolysis is located between the β and γ phosphates. Structure-affinity relationship studies of cap analogs for CeDcpS 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 m(7) Guo or an extended triphosphate chain. Docking analysis of cap analogs in the CeDcpS 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 CeDcpS with its well known human homolog provides general insights into DcpS-cap interactions., (© 2013 FEBS.)

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.

Mizoribine monophosphate (MZP) is a specific inhibitor of the cellular inosine-5'-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 5'-triphosphatase and RNA guanylyltransferase activities. HCE is involved in the synthesis of the cap structure found at the 5' 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 5'-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.

RNA possesses great potential for expanding the toolbox currently available to synthetic biologists. Here, the modulation of the Hepatitis Delta Virus ribozyme's activity with a series of rationally designed aptamers and effector RNA oligonucleotides is described. The ribozyme was initially fused with an 18-nucleotide hairpin structure that abolished its self-cleaving activity. The binding of a 14-mer oligonucleotide to the hairpin rescued the self-cleavage in a concentration-dependent manner. This modified ribozyme was inserted into the 5' UTR of a reporter gene, and the resulting construct was used to demonstrate that it is possible to modulate the ribozyme activity in cellulo with the oligonucleotide. Subsequently, ribozymes possessing specialized aptamers respecting other logic gates were also successfully designed and found to be functional in vitro. To our knowledge, this is the first example of HDV ribozyme regulation by oligonucleotides, as well as the first allosteric regulation of HDV ribozyme in mammalian cells.

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.

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.

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., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

A number of different human pathogens code for their own enzymes involved in the synthesis of the RNA cap structure. Although the RNA cap structures originating from human and microbial enzymes are often identical, the subunit composition, structure and catalytic mechanisms of the microbial-encoded enzymes involved in the synthesis of the RNA cap structure are often significantly different from those of host cells. As a consequence, these pathogenic cap-forming enzymes are potential targets for antimicrobial drugs. During the past few years, experimental studies have started to demonstrate that inhibition of the RNA capping activity is a reasonable approach for the development of antimicrobial agents. The combination of structural, biochemical, and molecular modeling studies are starting to reveal novel molecules that can serve as starting blocks for the design of more potent and specific antimicrobial agents. Here, we examine various strategies that have been developed to inhibit microbial enzymes involved in the synthesis of the RNA cap structure, emphasizing the challenges remaining to design potent and selective drugs., (Copyright © 2010 John Wiley & Sons, Ltd.)

The RNA guanylyltransferase (GTase) is involved in the synthesis of the (m7)Gppp-RNA cap structure found at the 5' 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.

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.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Decapping enzymes are required for the removal of the 5'-end (m7)GpppN cap of mRNAs to allow their decay in cells. While many cap-binding proteins recognize the cap structure via the stacking of the methylated guanosine ring between two aromatic residues, the precise mechanism of cap recognition by decapping enzymes has yet to be determined. In order to get insights into the interaction of decapping enzymes with the cap structure, we studied the vaccinia virus D10 decapping enzyme as a model to investigate the important features for substrate recognition by the enzyme. We demonstrate that a number of chemically modified purines can competitively inhibit the decapping reaction, highlighting the molecular features of the cap structure that are required for recognition by the enzyme, such as the nature of the moiety at positions 2 and 6 of the guanine base. A 3D structural model of the D10 protein was generated which suggests amino acids implicated in cap binding. Consequently, we expressed 17 mutant proteins with amino acid substitutions in the active site of D10 and found that eight are critical for the decapping activity. These data underscore the functional features involved in the non-canonical cap-recognition by the vaccinia virus D10 decapping enzyme.

The West Nile virus RNA helicase uses the energy derived from the hydrolysis of nucleotides to separate complementary strands of RNA. Although this enzyme has a preference for ATP, the bias towards this purine nucleotide cannot be explained on the basis of specific protein-ATP interactions. Moreover, the enzyme does not harbor the characteristic Q-motif found in other helicases that regulates binding to ATP. In the present study, we used structural homology modeling to generate a model of the West Nile virus RNA helicase active site that provides instructive findings on the interaction between specific amino acids and the ATP substrate. In addition, we evaluated both the phosphohydrolysis and the inhibitory potential of a collection of 30 synthetic purine analogs. A structure-guided alanine scan of 16 different amino acids was also performed to clarify the contacts that are made between the enzyme and ATP. Our study provides a molecular rationale for the bias of the enzyme for ATP by highlighting the specific functional groups on ATP that are important for binding. Moreover, we identified three new essential amino acids (Arg-185, Arg-202 and Asn-417) that are critical for phosphohydrolysis. Finally, we provide evidence that a region located upstream of motif I, which we termed the nucleotide specificity region, plays a functional role in nucleotide selection which is reminiscent to the role exerted by the Q-motif found in other helicases.

It is believed that peach latent mosaic viroid (PLMVd) strands of both the plus and minus polarities fold into similar secondary and tertiary structures. In order to verify this hypothesis, the behavior of both strands in three biophysical assays was examined. PLMVd transcripts of plus and minus polarity were found to exhibit distinct electrophoretic mobility properties under native conditions, to precipitate differently in the presence of lithium chloride, and to possess variable thermal denaturation profiles. Subsequently, the structure of PLMVd transcripts of minus polarity was elucidated by biochemical methods, thereby permitting comparison to the known structure of the plus polarity. Specifically, enzymatic probing, electrophoretic mobility shift assay, and ribonuclease H hydrolysis were performed in order to resolve the secondary structure of the minus polarity. The left domains of the strands of both polarities appear to be similar, while the right domain exhibited several differences even though they both adopted a branched structure. The pseudoknot P8 formed in the plus strand seemed not formed in the minus strands. The structural differences between the two polarities might have important implications in various steps of the PLMVd life cycle.

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.

The 5'-end of the flavivirus genome harbors a methylated (m7)GpppA(2'OMe) cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 2'-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery.

RNA triphosphatases (RTPases) are involved in the addition of the distinctive cap structure found at the 5' ends of eukaryotic mRNAs. Fungi, protozoa and some DNA viruses possess an RTPase that belongs to the triphosphate tunnel metalloenzyme family of enzymes that can also hydrolyze nucleoside triphosphates. Previous crystallization studies revealed that the phosphohydrolase catalytic core is located in a hydrophilic tunnel composed of antiparallel beta-strands. However, all past efforts to obtain structural information on the interaction between RTPases and their substrates were unsuccessful. In the present study, we used computational molecular docking to model the binding of a nucleotide substrate into the yeast RTPase active site. In order to confirm the docking model and to gain additional insights into the molecular determinants involved in substrate recognition, we also evaluated both the phosphohydrolysis and the inhibitory potential of an important number of nucleotide analogs. Our study highlights the importance of specific amino acids for the binding of the sugar, base and triphosphate moieties of the nucleotide substrate, and reveals both the structural flexibility and complexity of the active site. These data illustrate the functional features required for the interaction of an RTPase with a ligand and pave the way to the use of nucleotide analogs as potential inhibitors of RTPases of pathogenic importance.

Decapping enzymes are required for the removal of the 5'-end cap of mRNAs. These enzymes exhibit a specific hydrolase activity, resulting in cleavage between the alpha- and beta-phosphates of the m7GpppN cap to generate both m7GDP and monophosphorylated RNA products. Decapping enzymes have been found in humans, plants and yeasts, and have been discovered more recently in vaccinia virus (D10 protein). Although experimental evidences are lacking, three-metal- and two-metal-ion mechanisms have been proposed so far for the decapping enzymes. In the present study, we performed a biochemical characterization of the interaction of bivalent cations with the vaccinia virus D10 protein. Synergistic activation of the enzyme was observed in the presence of Mg2+ and Mn2+ ions, suggesting the existence of two metal-ion-binding sites on the D10 protein. Moreover, dual-ligand titration experiments using fluorescence spectroscopy demonstrated the presence of two metal-ion-binding sites on the enzyme. A three-dimensional structural model of the active site of the enzyme was generated which highlighted the importance of three glutamate residues involved in the co-ordination of two metal ions and a water molecule. Mutational analyses confirmed the role of two glutamate residues for the binding of metal ions. We demonstrate that one metal ion is co-ordinated by Glu132, while the second metal ion is co-ordinated by Glu145. Taken together, these results support the proposed two-metal-ion mechanistic model for the D10 decapping enzyme.

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.

Isothermal titration calorimetry was used to monitor the energetic landscape of a catalytic RNA, specifically that of the hepatitis delta virus ribozyme. Using mutants that isolated various tertiary interactions, the thermodynamic parameters of several ribozyme-substrate intermediates were determined. The results shed light on the impact of several tertiary interactions on the global structure of the ribozyme. In addition, the data indicate that the formation of the P1.1 pseudoknot is the limiting step of the molecular mechanism. Last, as illustrated here, isothermal titration calorimetry appears to be a method of choice for the elucidation of an RNA's folding pathway.

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.

The Chlorella virus RNA triphosphatase (cvRTPase) is involved in the formation of the RNA cap structure found at the 5'-end of the viral mRNAs and requires magnesium ions to mediate its catalytic activity. To extend our studies on the role of metal ions in phosphohydrolysis, we have used a combination of fluorescence spectroscopy, circular dichroism, denaturation studies and thermodynamic analyses to monitor the binding of magnesium ions to the cvRTPase. Using these techniques, the thermodynamic forces responsible for the interaction of metal ions with an RNA triphosphatase were also evaluated for the first time. Our thermodynamic analyses indicate that the initial association of magnesium with the cvRTPase is dominated by a favorable entropic effect and is accompanied by the release of eight water molecules from the enzyme. Moreover, both fluorescence spectroscopy and circular dichroism assays indicated that minor conformational changes were occurring upon magnesium binding. Mutational studies were also performed and confirmed the importance of three specific glutamate residues located in the active site of the enzyme for the binding of magnesium ions. Finally, in contrast to the yeast RNA triphosphatase, we demonstrate that the binding of magnesium ions to the cvRTPase does not lead to the stabilization of the ground state binding of the RNA substrate. Based on the results of the present study, we hypothesize that the binding of magnesium ions induces local conformational perturbations in the active site residues that ultimately positions the lateral chains of critical amino acids involved in catalysis. Our results highlight fundamental differences in the role of magnesium ions in the phosphohydrolase reactions catalyzed by the cvRTPase and the closely related yeast RNA triphosphatase.

The catalytic subunit of the human cytomegalovirus DNA polymerase is critical for the replication of the virus. In the present study, we report the expression and purification of a recombinant catalytic subunit of the human cytomegalovirus DNA polymerase expressed in bacteria which retains polymerase activity. As a first step towards elucidating the nature of the interaction between the enzyme, DNA and dNTPs, we have utilized endogenous tryptophan fluorescence to evaluate the binding of ligands to the enzyme. Using this technique, we demonstrate that the minimal DNA-binding site of the enzyme is 6 nt. We also report the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between the enzyme, DNA and dNTPs. Our thermodynamic analyses indicate that the initial formation of the enzyme-DNA binary complex is driven by a favourable entropy change, but is also clearly associated with an unfavourable enthalpic contribution. In contrast, the interaction of dNTPs to the binary complex was shown to depend on a completely different mode of binding that is dominated by a favourable enthalpy change and associated with an unfavourable entropy change. In order to provide additional insights into the structural modifications that occur during catalysis, we correlated the effect of DNA and dNTP binding on protein structure using CD. Our results indicate that the enzyme undergoes a first conformational change upon the formation of the protein-DNA binary complex, which is followed by a second structural modification upon dNTP binding. The present study provides a better understanding of the molecular basis of DNA and dNTP recognition by the catalytic subunit of the human cytomegalovirus DNA polymerase.

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-alpha 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.

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., ((c) 2007 John Wiley & Sons, Ltd.)

With the goal of gaining insight into the tertiary structure of the hepatitis delta virus ribozyme, cross-linking experiments using 4-thiouridine residues introduced in either the 5'-end portion of the substrate, or at seven strategic positions within the ribozyme, were performed. Mapping of the newly formed covalent bonds in cross-linked species obtained under various conditions, as well as using several mutated ribozymes, permitted monitoring of the formation of the ribozyme-substrate complex as the ribozyme proceeded along the folding pathway. In order to aid visualization of the tertiary structure transformation, an in silico animation of the "on" folding pathway was developed. In combination with those of the cleavage assays of structured substrates, these data shed light on the key contribution of the L3 loop in the formation of an active tertiary complex.