O-methyltransferase Activity Research Articles

Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes

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.

Salt stress-induced seedling growth inhibition coincides with differential distribution of serotonin and melatonin in sunflower seedling roots and cotyledons.

Indoleamines regulate a variety of physiological functions during the growth, morphogenesis and stress-induced responses in plants. Present investigations report the effect of NaCl stress on endogenous serotonin and melatonin accumulation and their differential spatial distribution in sunflower (Helianthus annuus) seedling roots and cotyledons using HPLC and immunohistochemical techniques, respectively. Exogenous serotonin and melatonin treatments lead to variable effect on hypocotyl elongation and root growth under NaCl stress. NaCl stress for 48 h increases endogenous serotonin and melatonin content in roots and cotyledons, thus indicating their involvement in salt-induced long distance signaling from roots to cotyledons. Salt stress-induced accumulation of serotonin and melatonin exhibits differential distribution in the vascular bundles and cortex in the differentiating zones of the primary roots, suggesting their compartmentalization in the growing region of roots. Serotonin and melatonin accumulation in oil body rich cells of salt-treated seedling cotyledons correlates with longer retention of oil bodies in the cotyledons. Present investigations indicate the possible role of serotonin and melatonin in regulating root growth during salt stress in sunflower. Effect of exogenous serotonin and melatonin treatments (15 μM) on sunflower seedlings grown in the absence or presence of 120 mM NaCl substantiates their role on seedling growth. Auxin and serotonin biosynthesis are coupled to the common precursor tryptophan. Salt stress-induced root growth inhibition, thus pertains to partial impairment of auxin functions caused by increased serotonin biosynthesis. In seedling cotyledons, NaCl stress modulates the activity of N-acetylserotonin O-methyltransferase (HIOMT; EC 2.1.1.4), the enzyme responsible for melatonin biosynthesis from N-acetylserotonin.

Recognition at the leaf surface.

Recognition at the leaf surface.

Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~3.8Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.

Association between ASMT and autistic-like traits in children from a Swedish nationwide cohort

Individuals with autism spectrum disorders often show low levels of melatonin, and it has been suggested that this decrease may be because of the low activity of the acetylserotonin O-methyltransferase (ASMT), the last enzyme in the melatonin-synthesis pathway. Also, genetic variants in ASMT have been associated with autism, as well as with low ASMT activity and melatonin levels, suggesting that the low ASMT activity observed in autism may partly be because of variations within the ASMT gene. In this study, we present a symptom-based approach to investigate possible associations between ASMT and autistic-like traits in the general population. To this end, continuous measures of autistic-like traits were assessed in a nationally representative twin cohort (n=1771) from Sweden and six single nucleotide polymorphisms (SNPs), and a duplication of exons 2-8 in ASMT were genotyped. Our results show a nominally significant association, in girls, between one single nucleotide polymorphism (rs5949028) in the last intron of ASMT and social interaction impairments. No significant association, however, was observed with traits related to language impairment or restricted and repetitive behavior. In conclusion, our results support the possible involvement of the ASMT gene in autism spectrum disorders, and our finding that only one of the three traits shows association suggests that genetic research may benefit from adopting a symptom-specific approach to identify genes involved in autism psychopathology.

Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity.

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern respiratory syndrome CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2'-O-methyltransferase (2'-O-MTase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection and develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both in vitro and in vivo. While viruses lacking 2'-O-MTase activity had enhanced sensitivity to type I interferon (IFN), they were not completely restored in their absence in vivo. However, the absence of either MDA5 or IFIT1, IFN-responsive genes that recognize unmethylated 2'-O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscore the necessity of 2'-O-MTase activity for SARS-CoV pathogenesis and identify host immune pathways that mediate this attenuation. In addition, we describe novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2'-O-MTase activity to subvert the immune system. Preventing recognition by the host immune response represents a critical aspect necessary for successful viral infection. Several viruses, including SARS-CoV, utilize virally encoded 2'-O-MTases to camouflage and obscure their viral RNA from host cell sensing machinery, thus preventing recognition and activation of cell intrinsic defense pathways. For SARS-CoV, the absence of this 2'-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. The results indicate that both MDA5, a recognition molecule, and the IFIT family play an important role in mediating this attenuation with restored virulence observed in their absence. Understanding this virus-host interaction provided an opportunity to design a successful live-attenuated vaccine for SARS-CoV and opens avenues for treatment and prevention of emerging CoVs and other RNA virus infections.

Attenuation and Restoration of Severe Acute Respiratory Syndrome Coronavirus Mutant Lacking 2'-O-Methyltransferase Activity

Attenuation and Restoration of Severe Acute Respiratory Syndrome Coronavirus Mutant Lacking 2'-O-Methyltransferase Activity

Sequestration by IFIT1 impairs translation of 2'O-unmethylated capped RNA.

Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.

Production of pinostilbene compounds by the expression of resveratrol O-methyltransferase genes in Escherichia coli

Resveratrol (3,4′,5-trans-trihydroxystilbene) is a polyphenolic phytoalexin that belongs to a family of naturally occurring stilbenes. It has been reported that the health-promoting activities of certain methylated resveratrol derivatives are more effective than those of unmodified resveratrol. In this study, we isolated two candidate genes with resveratrol O-methyltransferase (ROMT) activity from grape (Vitis riparia) and sorghum (Sorghum bicolor). To assess their ROMT activities in vivo, we synthesized VrROMT and SbROMT3 following codon-optimization and expressed the VrROMTsyn and SbROMT3syn genes using a dual expression vector system. Furthermore, we attempted to produce pterostilbene from resveratrol as a substrate by the expression of two putative ROMT proteins in Escherichia coli. Unexpectedly, expression of the SbROMT3syn gene in E. coli led to the production of mono-methylated stilbene (3,4′-dihydroxy-5-methoxy-trans-stilbene, pinostilbene) from resveratrol compounds. However, a very small amount of di-methylated stilbene (3,5-dimethoxy-4′-hydroxy-trans-stilbene, pterostilbene) was also detected. Consistently, we found that in vitro methylation assays of resveratrol by recombinant SbROMT3syn produced pinostilbene as the major product besides a very small amount of pterostilbene. By contrast, very small amounts of methylated resveratrol derivatives were detected in E. coli expressing the VrROMTsyn protein. This suggests that the SbROMT3syn is more useful in the production of pinostilbene compounds than pterostilbene from resveratrol in E. coli.

Essential oil and volatile emissions of basil (Ocimum basilicum) leaves exposed to NaCl or Na2SO4 salinity

AbstractThe volatile compounds emitted by living leaves of basil (Ocimum basilicum L. cv. Genovese) plants under saline conditions were investigated by means of headspace–solid phase microextraction (HS‐SPME) and gas chromatography coupled with mass spectrometry (GC–MS). Furthermore, the composition of the essential oil obtained by hydrodistillation of the leaves was studied. Plants were grown for 15 d without salt or with an equimolar concentration of Na+ in the form of Na2SO4 (25 mM) and NaCl (50 mM), after which the growth, the essential oil, and the volatile constituents of the leaves were determined. Fifty‐four components were identified belonging to different chemical classes. Under control conditions, the essential oil was rich in linalool (45.9%), 1,8‐cineole (16.7%), eugenol (10.3%), trans‐α‐bergamotene, and epi‐α‐cadinol (4.9%). The main volatiles detected in the headspace of leaves of untreated basil plants were linalool (29.8%), followed by 1,8‐cineole (19.2%), trans‐α‐bergamotene (10.0%), and eugenol (7.0%). Under saline conditions, leaf growth was more depressed by 25 mM Na2SO4 than 50 mM NaCl, and essential oil concentration increased by 22% in the NaCl, but decreased by 18% in the Na2SO4 treatment, respectively. Both salts caused some changes in the essential oil and composition of volatile compounds. Most prominent was a strong negative correlation between eugenol and methyleugenol proportions, which may indicate an enhancement of the O‐methyltransferase activity. In comparison to the essential oil, the composition of volatiles showed some variations in their emission profile under both salts, the most important was an increase of eugenol. It is therefore concluded that the decrease of eugenol occurring in basil essential oil caused by both salts could be due to the enhancement of the eugenol O‐methyltransferase activity, an enzyme that accepts eugenol as substrate, generating methyl eugenol, and also to the increase of the eugenol emission as shown by the SPME profile.

Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Renalase regulates renal dopamine and phosphate metabolism

Renalase is a kidney-secreted catecholamines-degrading enzyme whose expression and activity are downregulated by increased dietary phosphate. A renalase knockout (KO) mouse model was used to explore the mechanisms mediating renalase's effect on phosphate excretion. Compared with wild-type (WT) mice maintained on a regular diet, KO mice show decreased serum PO4(-) (KO = 5.3 ± 0.2 vs. WT = 6.0 ± 0.1, n = 6; P < 0.04) and increased urinary PO4(-) excretion (urine PO4(-)/creatinine: KO = 7.7 ± 0.3 vs. WT = 6.1 ± 0.3, n = 6; P < 0.02). However, both WT and KO mice respond similarly to PO4(-) restriction by increasing renal COMT-1 activity and markedly decreasing PO4(-) excretion, which excludes an intrinsic renal defect in the KO. Renal sodium-phosphate cotransporter Npt2a, sodium proton exchanger NHE3 expression, and MAO-A and B activity did not differ between WT and KO. Only catechol-O-methyl transferase (COMT) expression and activity were significantly increased in KO mice. Despite that, urinary dopamine increased by twofold, whereas urinary l-DOPA excretion decreased by twofold in the KO mouse, indicating an upregulation of renal dopamine (DA) synthesis. These data indicate that renalase deficiency is associated with increased renal DA synthesis, stimulated PO4(-) excretion, and moderately severe hypophosphatemia. The signal to increase renal DA synthesis is strong since it overcomes a compensatory increase in COMT activity.

Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves

The effects of two sodium salts on growth, fatty acids, and essential oil compositions were investigated in a medicinal and aromatic plant, Ocimum basilicum cultivated in hydroponic medium. Plants were subjected to an equimolar concentration of Na2SO4 (25 mM) and NaCl (50 mM) for 15 days. Our results showed that leaf growth rate was more depressed by 25 mM Na2SO4 than by 50 mM NaCl. The total fatty acid contents did not show any change in plants. α-Linolenic, palmitic, and linoleic acids were the major fatty acids. The identification of basil leaf fatty acids has not been previously studied and this work revealed the predominance of polyunsaturated fatty acids. Under both salts, leaf fatty acid composition remained unchanged. Regarding the essential oil yield, it decreased significantly by 28 % under 25 mM Na2SO4 and showed an increase by 27 % under 50 mM NaCl. The major volatile compound in leaves was linalool with 34.3 % of total essential oil constituents, followed by eugenol (19.8 %), 1.8-cineole (14.4 %) and methyl eugenol (5.2 %). Further, levels of eugenol and methyl eugenol were most modulated by salt, and the negative correlation between these two compounds reflects the stimulation of O-methyltransferase activity under both salts.

Veratrole Biosynthesis in White Campion

White campion (Silene latifolia) is a dioecious plant that emits 1,2-dimethoxybenzene (veratrole), a potent pollinator attractant to the nocturnal moth Hadena bicruris. Little is known about veratrole biosynthesis, although methylation of 2-methoxyphenol (guaiacol), another volatile emitted from white campion flowers, has been proposed. Here, we explore the biosynthetic route to veratrole. Feeding white campion flowers with [(13)C9]l-phenylalanine increased guaiacol and veratrole emission, and a significant portion of these volatile molecules contained the stable isotope. When white campion flowers were treated with the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid, guaiacol and veratrole levels were reduced by 50% and 63%, respectively. Feeding with benzoic acid (BA) or salicylic acid (SA) increased veratrole emission 2-fold, while [(2)H5]BA and [(2)H6]SA feeding indicated that the benzene ring of both guaiacol and veratrole is derived from BA via SA. We further report guaiacol O-methyltransferase (GOMT) activity in the flowers of white campion. The enzyme was purified to apparent homogeneity, and the peptide sequence matched that encoded by a recently identified complementary DNA (SlGOMT1) from a white campion flower expressed sequence tag database. Screening of a small population of North American white campion plants for floral volatile emission revealed that not all plants emitted veratrole or possessed GOMT activity, and SlGOMT1 expression was only observed in veratrole emitters. Collectively these data suggest that veratrole is derived by the methylation of guaiacol, which itself originates from phenylalanine via BA and SA, and therefore implies a novel branch point of the general phenylpropanoid pathway.

A lignan O-methyltransferase catalyzing the regioselective methylation of matairesinol in Carthamus tinctorius

Lignans are a group of plant phenolic compounds with various biological activities, including antitumor and antioxidant properties. O-Methylation is a critical step in biosynthesis of these compounds. However, little is known about the O-methyltransferase (OMT) enzymes that catalyze lignan O-methylation. We discovered a highly regioselective OMT activity in safflower (Carthamus tinctorius) seeds that catalyzed the methylation of matairesinol, a dibenzylbutyrolactone lignan, into 4′-O-methylmatairesinol (arctigenin) but not 4-O-methylmatairesinol (isoarctigenin). By examining such OMT activity in correlation with OMT transcript abundances during seed development, we cloned a few putative OMT cDNAs and produced their recombinant proteins in Escherichia coli. Among them, one protein exhibited O-methylation activity for matairesinol with the regioselectivity identical to that of the plant protein, and was named C. tinctorius matairesinol OMT (CtMROMT). CtMROMT did not show any detectable OMT activities towards phenylpropanoid monomers under the reaction conditions tested, while it methylated flavonoid apigenin efficiently into 4′-O-methylapigenin (acacetin). However, quantitative real-time polymerase chain reaction analysis demonstrated that expression of the CtMROMT gene was synchronized with the CtMROMT activity profile and arctigenin accumulation in the plant. These results demonstrated that CtMROMT is a novel plant OMT for lignan methylation.

Tricin biosynthesis during growth of wheat under different abiotic stresses

In plants, O-methylation is mediated by an enzyme family of O-methyltransferases (OMTs) that transfer the methyl groups from the methyl donor, S-adenosyl-L-methionine (AdoMet) to suitable phenolic acceptor molecules. In a previous study [1], a flavonoid OMT (TaOMT2) was isolated and characterized from wheat (Triticum aestivum L.) leaves. Its novel gene product catalyzes three sequential O-methylations of the flavone tricetin (5,7,3′,4′,5′-pentahydroxyflavone) to its 3′-monomethyl-(selgin)→3′,5′-dimethyl-(tricin)→3′,4′,5′-trimethyl (TMT) ether derivatives, with tricin being the major product of the reaction. In this report, the biological significance of tricetin methylation was investigated by measuring the OMT activity, its expression level, and the accumulation of its major product (tricin) at different stages of development of wheat plants exposed to different abiotic stresses such as cold, salt and drought. The results showed that tricin accumulates mostly in wheat inflorescences under normal conditions compared to leaves and other developmental stages. Tricin accumulation was associated with increased TaOMT2 expression level and its enzyme activity, suggesting a possible de novo synthesis of the enzyme at this important developmental stage. This phenomenon may be attributed to the putative role of tricin in protecting seeds against biotic and abiotic stresses. The functions of tricin during growth and development of wheat and the importance of tricetin methylation during abiotic stresses are discussed.

Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations.

Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV . The docking simulations using GOLD, Glide, and Dock give consistent binding modes and binding affinities of adamantanes in the AdoMet-binding site of NS5MTaseDV and, in particular, yield similar positions for the previously found inhibitors. Combined, they perfectly correspond to the bioassay measurements of nucleoside-2'O-methyltransferase activity of NS5TaseDV , which confirmed inhibitory properties of the active urea adamantane but did not show inhibitory activity for amantadine and rimantadine. We also employed microscopic molecular dynamics (MD) simulations and a linear interaction energy (LIE) method to verify the docking results. The MD/LIE binding free energies of selected protein-inhibitor complexes agree overall with the binding affinities from docking and demonstrate that amantadine and rimantadine only weakly bind at the explored site. The MD simulations also demonstrated the flexible character of a protein loop that is located between the β2 and β3 strands and is part of the AdoMet-binding pocket of NS5MTaseDV .

ISG56/IFIT1 is primarily responsible for interferon-induced changes to patterns of parainfluenza virus type 5 transcription and protein synthesis

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2′-O-methyltransferase activity, an enzyme that methylates the 2′-hydroxyl group of ribose sugars in the 5′-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2′-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

Dengue virus nonstructural protein 5 adopts multiple conformations in solution.

Dengue virus (DENV) nonstructural protein 5 (NS5) is composed of two globular domains separated by a 10-residue linker. The N-terminal domain participates in the synthesis of a mRNA cap 1 structure ((7Me)GpppA(2'OMe)) at the 5' end of the viral genome and possesses guanylyltransferase, guanine-N7-methyltransferase, and nucleoside-2'O-methyltransferase activities. The C-terminal domain is an RNA-dependent RNA polymerase responsible for viral RNA synthesis. Although crystal structures of the two isolated domains have been obtained, there are no structural data for full-length NS5. It is also unclear whether the two NS5 domains interact with each other to form a stable structure in which the relative orientation of the two domains is fixed. To investigate the structure and dynamics of DENV type 3 NS5 in solution, we conducted small-angle X-ray scattering experiments with the full-length protein. NS5 was found to be monomeric and well-folded under the conditions tested. The results of these experiments also suggest that NS5 adopts multiple conformations in solution, ranging from compact to more extended forms in which the two domains do not seem to interact with each other. We interpret the multiple conformations of NS5 observed in solution as resulting from weak interactions between the two NS5 domains and flexibility of the linker in the absence of other components of the replication complex.

Microarray and Growth Analyses Identify Differences and Similarities of Early Corn Response to Weeds, Shade, and Nitrogen Stress

Weed interference with crop growth is often attributed to water, nutrient, or light competition; however, specific physiological responses to these stresses are not well described. This study's objective was to compare growth, yield, and gene expression responses of corn to nitrogen (N), low light (40% shade), and weed stresses. Corn vegetative parameters from V2 to V12 stages, yield parameters, and gene expression using transcriptome (2008) and quantitative polymerase chain reaction (qPCR) (2008/09) analyses at V8 were compared among the stresses and with nonstressed corn. N stress did not affect vegetative parameters, although grain yield was reduced by 40% compared with nonstressed plants. Shade, present until V2, reduced biomass and leaf area &gt; 50% at V2, and recovering plants remained smaller than nonstressed plants at V12. However, grain yields of shade-stressed and nonstressed plants were similar, unless shade remained until V8. Weed stress reduced corn growth and yield in 2008 when weeds remained until V6. In 2009, weed stress until V2 reduced corn vegetative growth, but yield reductions occurred only if weed stress remained until V6 or later. Principle component analysis of differentially expressed genes indicated that shade and weed stress had more similar gene expression patterns to each other than they did to nonstressed or N-stressed tissues. However, corn grown in N-stressed conditions shared 252 differentially expressed genes with weed-stressed plants. Ontologies associated with light/photosynthesis, energy conversion, and signaling were down-regulated in response to all three stresses. Shade and weed stress clustered most tightly together, based on gene expression, but shared only three ontologies, O-METHYLTRANSFERASE activity (lignification processes), POLY(U)-BINDING activity (posttranscriptional gene regulation), and stomatal movement. Based on morphologic and genomic observations, weed stress to corn was not explained by individual effects of N or light stress. Therefore, we hypothesize that these stresses share limited signaling mechanisms.