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Acute antiviral functions of all nucleated cells rely on type I interferon (IFN-I) pathways triggered upon viral infection. Host responses encompass the sensing of incoming viruses, the activation of specific transcription factors that induce the transcription of IFN-I genes, the secretion of different IFN-I types and their recognition by the heterodimeric IFN-α/β receptor, the subsequent activation of JAK/STAT signaling pathways, and, finally, the transcription of many IFN-stimulated genes (ISGs)., Epstein-Barr virus (EBV), a human herpesvirus, encodes 44 microRNAs (miRNAs), which regulate many genes with various functions in EBV-infected cells. Multiple target genes of the EBV miRNAs have been identified, some of which play important roles in adaptive antiviral immune responses. Using EBV mutant derivatives, we identified additional roles of viral miRNAs in governing versatile type I interferon (IFN) responses upon infection of human primary mature B cells. We also found that Epstein-Barr virus-encoded small RNAs (EBERs) and LF2, viral genes with previously reported functions in inducing or regulating IFN-I pathways, had negligible or even contrary effects on secreted IFN-α in our model. Data mining and Ago PAR-CLIP experiments uncovered more than a dozen previously uncharacterized, direct cellular targets of EBV miRNA associated with type I IFN pathways. We also identified indirect targets of EBV miRNAs in B cells, such as TRL7 and TLR9, in the prelatent phase of infection. The presence of epigenetically naive, non-CpG methylated viral DNA was essential to induce IFN-α secretion during EBV infection in a TLR9-dependent manner. In a newly established fusion assay, we verified that EBV virions enter a subset of plasmacytoid dendritic cells (pDCs) and determined that these infected pDCs are the primary producers of IFN-α in EBV-infected peripheral blood mononuclear cells. Our findings document that many EBV-encoded miRNAs regulate type I IFN response in newly EBV infected primary human B cells in the prelatent phase of infection and dampen the acute release of IFN-α in pDCs upon their encounter with EBV.

The discovery of a new coronavirus (CoV) as the causative agent of the severe acute respiratory syndrome (SARS) pandemic outbreak in 2003 has stimulated a number of studies on the molecular biology of SARS-CoV and related viruses. This research has provided significant new insight into functions and activities of the CoV replication-transcription complex, a multi-protein complex that directs coordinated processes of both continuous and discontinuous RNA synthesis to replicate and transcribe the large CoV genome, a single-stranded, positive-sense RNA of ∼30 kilobases. In this review, we summarize current understanding of the expression and functions of key replicative enzymes, such as RNA polymerases, ribonucleases, methyltransferases and other replicase gene encoded proteins involved in genome expression, virus-host interactions and other processes. Collectively, these recent studies reveal fascinating details of a huge enzymatic machinery unique in the RNA virus world.

RNA viruses encode dedicated protein machinery required through the viral life cycle. Some enzymatic activities are generally associated with RNA viruses such as RNA- or DNA-dependent RNA polymerases, RNA helicases or proteases. Some viral enzyme activities are however unique to some viral families. This is the case of two 3'-5' exoribonuclease activities identified in arenavirus and coronavirus proteomes. Arenaviruses have a segmented ambisense single stranded RNA genome of negative polarity while coronaviruses have a positive single-stranded genomic RNA. Although both enzymes belong to the same exo(ribo)nuclease superfamily, available data indicate that they are involved in very different pathways. Indeed, the exoribonuclease activity carried by the arenavirus nucleoprotein seems to counteract the innate immunity antiviral response while the exoribonuclease activity carried by the coronavirus nsp14 protein is likely involved in a unique RNA repair mechanism. In this review, we present our current knowledge about these two viral enzymes and their functions in the viral life cycle.

The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV’s multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV’s prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established., Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells.

Epstein-Barr virus (EBV) infects and activates resting human B-lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cellsin vitroare known, but their functions in the early, pre-latent phase of infection have not been investigated systematically. In studies during the first eight days of infection using derivatives of EBV with mutations in single genes of EBVs we found only EBNA2 to be essential for activating naïve human B-lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, LMP2A and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding EBER RNAs had no discernable phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the pre-latent phase. Even EBNA1 which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a critical parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B-lymphocytes from energetically quiescent to activated cells.Author summaryThe preferred target of Epstein-Barr virus (EBV) are human resting B-lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume followed by cellular DNA synthesis after three days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to three days later the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains we investigated the individual contributions of EBV’s multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV’s pre-latent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B-lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming viral latent genes other than EBNA2 are dispensable but some, EBNA-LP for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

EBV reduces the activation of cytotoxic CD4+ effector T cells by inducing a state of reduced immunogenicity in infected B cells. EBV-derived miRNAs suppress release of proinflammatory cytokines, interfere with peptide processing and presentation on HLA class II, repress differentiation of naive CD4+ T cells to Th1 cells, and ultimately avoid killing of infected B cells., Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4+ T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4+ effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4+ T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection.

Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4(+) T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8(+) T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8(+) T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8(+) T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8(+) T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4(+) but also by antiviral CD8(+) T cells.

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1–16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3′ to 5′ direction as well as a single mismatched nucleotide at the 3′-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3′-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2′-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism.

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

International audience; The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.

Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential.

International audience; To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress.

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Flaviviruses are the causative agents of severe diseases such as Dengue or Yellow fever. The replicative machinery used by the virus is based on few enzymes including a methyltransferase, located in the N-terminal domain of the NS5 protein. Flaviviral methyltransferases are involved in the last two steps of the mRNA capping process, transferring a methyl group from S-adenosyl-l-methionine onto the N7 position of the cap guanine (guanine-N7 methyltransferase) and the ribose 2?O position of the first nucleotide following the cap guanine (nucleoside-2?O methyltransferase). The RNA capping process is crucial for mRNA stability, protein synthesis and virus replication. Such an essential function makes methyltransferases attractive targets for the design of antiviral drugs. In this context, starting from the crystal structure of Wesselsbron flavivirus methyltransferase, we elaborated a mechanistic model describing protein/RNA interaction during N7 methyl transfer. Next we used an in silico docking procedure to identify commercially available compounds that would display high affinity for the methyltransferase active site. The best candidates selected were tested in vitro to assay their effective inhibition on 2?O and N7 methyltransferase activities on Wesselsbron and Dengue virus (Dv) methyltransferases. The results of such combined computational and experimental screening approach led to the identification of a high-potency inhibitor.

Municipal solid waste incinerator (MSWI) bottom ash may be used as a road construction material; it potentially contains however a sizable quantity of heavy metals, which under the effect of rainfall infiltration through the road structure can be leached out from the material and infiltrate into the underlying soil. An eco-compatibility assessment of MSWI bottom ash reuse in road construction applications necessitates examining the solubility and retention of heavy metals in road soils. This study is dedicated to Pb transfer, sorption and desorption (NEN 7341 standard test) within various soils. These experiments yield results relative to the interaction between road soils and an MSWI bottom ash leachate representative of a “fresh” product, with a high leaching potential. For the soils investigated, the sorption of lead varies between 90% and 99%. For an extraction at pH 7, Pb release is very low (

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses., Author Summary A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ∼930 Å2 activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.

SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5′ end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2′O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2′O-methylated by the 2′O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2′OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design., Author Summary In 2003, an emerging coronavirus (CoV) was identified as the etiological agent of severe acute respiratory syndrome (SARS). SARS-CoV replicates and transcribes its large RNA genome using a membrane-bound enzyme complex containing a variety of viral nonstructural proteins. A critical step during RNA synthesis is the addition of a cap structure to the newly produced viral mRNAs, ensuring their efficient translation by host cell ribosomes. Viruses generally acquire their cap structure either from cellular mRNAs (e.g., “cap snatching” of influenza virus) or employ their own capping machinery, as is supposed to be the case for coronaviruses. mRNA caps synthesized by viruses are structurally and functionally undistinguishable from cellular mRNAs caps. In coronaviruses, methylation of mRNA caps seems to be essential, since mutations in viral methyltransferases nsp14 or nsp16 render non-viable virus. We have discovered an unexpected key role for SARS-CoV nsp10, a protein of previously unknown function, within mRNA cap methylation. Nsp10 induces selective 2′O-methylation of guanine-N7 methylated capped RNAs through direct activation of the otherwise inactive nsp16. This finding allows the full reconstitution of the SARS-CoV mRNA cap methylation sequence in vitro and opens the way to exploit the mRNA cap methyltransferases as targets for anti-coronavirus drug design.