Yellow fever virus (YFV), a member of the Flavivirus genus, has a plus-sense RNA genome encoding a single polyprotein. Viral protein NS3 includes a protease and a helicase that are essential to virus replication and to RNA capping. The 1.8-Å crystal structure of the helicase region of the YFV NS3 protein includes residues 187 to 623. Two familiar helicase domains bind nucleotide in a triphosphate pocket without base recognition, providing a site for nonspecific hydrolysis of nucleoside triphosphates and RNA triphosphate. The third, C-terminal domain has a unique structure and is proposed to function in RNA and protein recognition. The organization of the three domains indicates that cleavage of the viral polyprotein NS3-NS4A junction occurs in trans.Flaviviruses are arthropod-borne pathogens that cause a number of serious human diseases throughout the world. Despite their importance as human pathogens, the molecular mechanisms of flavivirus replication are minimally understood, hindering the development of effective antiviral therapies and vaccines. Yellow fever virus (YFV) is the prototype member of the Flavivirus genus. Like many other flaviviruses, YFV is transmitted by mosquitoes. Symptoms of YFV infection include kidney failure, internal bleeding, high fever, and hepatitis, which leads to the yellow coloring of the skin for which the disease is named. Although vaccination using a live attenuated strain has been successful for decades, yellow fever still causes over 30,000 deaths per year, mostly in Africa and South America.In addition to YFV, the Flavivirus genus also includes West Nile virus, four serotypes of dengue virus, and Japanese encephalitis virus. Flavivirus is the largest genus in the Flaviviradae family, whose other genera are Hepacivirus and Pestivirus. The only characterized hepacivirus is hepatitis C virus (HCV), which is well studied due to its importance in chronic liver disease and in liver failure leading to transplant. Although many molecular details differ between HCV and the flaviviruses and their protein sequences are less than 20% identical, there are sufficient similarities in replication strategy to allow comparisons between the viral proteins.The flavivirus genome is a Ϸ11-kb plus-sense RNA containing a 5Ј cap (m 7 G5Јppp5ЈA) but lacking a 3Ј poly(A) tail (16, 38). The genome encodes a 370-kDa polyprotein precursor, which is inserted into the membrane of the endoplasmic reticulum and processed to yield three structural proteins (C, M, and E) and seven replication proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (16). Host proteases process the polyprotein at sites in the endoplasmic reticulum lumen and a viral protease cleaves at specific sites on the cytoplasmic side of the endoplasmic reticulum membrane. The viral serine protease is formed by the N-terminal Ϸ175 residues of the NS3 protein and a cofactor peptide within the NS2B protein (2,15,16,34,54).The C-terminal 440 amino acids of the NS3 protein constitute a helicase region, based on sequence analysis (30). The precise ...

Flaviviruses are serious human pathogens for which treatments are generally lacking. The proteolytic maturation of the 375-kDa viral polyprotein is one target for antiviral development. The flavivirus serine protease consists of the N-terminal domain of the multifunctional nonstructural protein 3 (NS3) and an essential 40-residue cofactor (NS2B 40 ) within viral protein NS2B. The NS2B-NS3 protease is responsible for all cytoplasmic cleavage events in viral polyprotein maturation. This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3. Recombinant proteases were created by fusion of West Nile virus (WNV) NS2B 40 to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg 459 2Gly 460 within the C-terminal helicase region of NS3 was cleaved more slowly. Autolytic cleavage reactions also occurred in NS2B-NS3 recombinant proteins from yellow fever virus, dengue virus types 2 and 4, and Japanese encephalitis virus. Cis and trans cleavages were distinguished using a noncleavable WNV protease variant and two types of substrates as follows: an inactive variant of recombinant WNV NS2B-NS3, and cyan and yellow fluorescent proteins fused by a dodecamer peptide encompassing a natural cleavage site. With these materials, the autolytic cleavages were found to be intramolecular only. Autolytic cleavage of the helicase site was insensitive to protein dilution, confirming that autolysis is intramolecular. Formation of an active protease was found to require neither cleavage of NS2B from NS3 nor a free NS3 N terminus. Evidence was also obtained for product inhibition of the protease by the cleaved C terminus of NS2B. Most flaviviruses, including West Nile virus (WNV),2 yellow fever virus (YFV), dengue viruses, and Japanese encephalitis virus (JEV), cause severe human diseases. The plus-sense RNA genome of flaviviruses is a single open reading frame encoding a polyprotein precursor of ϳ3400 amino acids, consisting of three structural proteins (C, prM, and E) and seven nonstructural replication proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Fig. 1A) (1-3). Signal sequences direct the polyprotein into the host endoplasmic reticulum (ER) membrane so that NS1 and the exogenous domains of prM and E are in the lumen; C protein, NS3 and NS5 are cytoplasmic; and proteins NS2A, NS2B, NS4A, and NS4B are predominantly trans-membrane. Post-translational processing of the polyprotein, which is required for virus replication, is performed by a viral NS3 protease (4, 5) in the cytoplasm and by host proteases in the ER lumen. NS3 protease activity is dependent upon association with an NS2B cofactor (NS2B 40 ), a central 40-amino acid hydrophilic domain within the largely hydrophobic NS2B protein (6 -8). The viral NS2B-NS3 protease cleaves the viral polyprotein precursor at the NS2A/NS2B, NS2B/NS3, NS3/ NS4A, and NS4B/NS5 junctions (Fig. 1A), as well as at internal sites within C, NS2A, N...

BackgroundSundarban is the world's largest coastal sediment comprising of mangrove forest which covers about one million hectares in the south-eastern parts of India and southern parts of Bangladesh. The microbial diversity in this sediment is largely unknown till date. In the present study an attempt has been made to understand the microbial diversity in this sediment using a cultivation-independent molecular approach.ResultsTwo 16 S rRNA gene libraries were constructed and partial sequencing of the selected clones was carried out to identify bacterial strains present in the sediment. Phylogenetic analysis of partially sequenced 16 S rRNA gene sequences revealed the diversity of bacterial strains in the Sundarban sediment. At least 8 different bacterial phyla were detected. The major divisions of detected bacterial phyla were Proteobacteria (alpha, beta, gamma, and delta), Flexibacteria (CFB group), Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, Planctomycetes and Gammatimonadates.ConclusionThe gammaproteobacteria were found to be the most abundant bacterial group in Sundarban sediment. Many clones showed similarity with previously reported bacterial lineages recovered from various marine sediments. The present study indicates a probable hydrocarbon and oil contamination in this sediment. In the present study, a number of clones were identified that have shown similarity with bacterial clones or isolates responsible for the maintenance of the S-cycle in the saline environment.

Ribosomcs from it number of prokaryotic and eukaryolic sources (e.g., Ewhwichirr c d i , wheat germ and rat livcr) can refold a number of enzymes which iirc denatured with guanidine/HCl prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured liictate dehydrogenase from rabbit muscle and glucose-&phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver, The protein-folding acivity of E. d i ribosomes was found to be prcsent in 50s particles arid in 23s rRNA. The 30s particle 01-16s rKNA did not show any protein-folding activity. The protein-folding activity of 23s rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of ElITA, inhibited the protein-folding activily of 23s rRNA. This low concentration of EDTA had no cffecl on folding of thc denatured enzymes by themselves.Kp.ywords: protein folding; ribosomes; 23s rRNA ; lactate dehydrogenase; glucose-6-phosphate dehydrogenase.The process by which a genetic message is converted from a linear urray of nucleotides to a linear polypeptide chain has been worked out in great detail id bacterial and eukaryotic cells. However, the mechanism of folding of linear polypeptides into three-dimensional structures has not been explained. Whereas some proteins can fold spontaneously from their denatured stales (Anfinsen, 1971), the folding of many proteins into active conformations, which occurs rapidly during and after synthesis of lhc polypeptide, has been shown by means of genetic and biochemical experiments to be assisted by molecular chaperones (Cething and Sambrook, 1992; Hartle and Martin, 1992).A nutnbcr of examples of i~r v i m protein synthesis by cell extracts have been reported (Zubay ct al., 1970; decrornbrugghe e l al., 1971 ; Zubay, 1973) in which the synthesized polypeptide could fold into biologically active forms. We asked whether ribosomes, the site of protein synthesis, could also fold thc newly synthesized polypeptide chain into its active form. Although the structure and function of some ribosomal proteins and rRNA species are known in great detail, new findings are emerging on the role of olhcr proteins and rRNA in protein synthesis and in a number of other cellular processes. Resistance mutations against a number of antibiotics have been located in rRNA (reviewed by Noller, 1991), arid 236 rRNA has been reportcd to possess pepticiyl transferase activity (Noller et al., 1992). Kccent reports on the initiation of folding of the /{-subunit of tryptophan synthiise during its translation on ribosomes (Fedorov et : 11. , 1992) In this report, we show that the protein-folding activity is a general activity since ribosomes from E. cwli (prokaryote), wheat germ and rat liver (eukaryok) could efficiently rcfold denatured lactate dehydrogenase (LDH) from rabbit muscle (a tetrnmeric enLyme of I40 kDdtctramer) and glucosz-6-phosphate dehydrogenasc (GlcCiP-DH) from baker's yeast (a ditneric enzyme of 102 kDa/ditner)...

Globally, it is established that the partial lockdown system assists to improve the health of the total environment due to inadequate anthropogenic actions in different economic sectors. The ample research on fitness of environment has been proved that the strict imposition of lockdown was the blessings of environment. The river Damodar has historical significance and lifeline for huge population of Jharkhand and West Bengal state of India but in the recent years the water quality has been deteriorated due to untreated industrial effluents and urban sewage. The main objective of this study is to examine the water quality of river Damodar during and prelockdown phase for domestic use and restoration of river ecosystem. A total of eleven (11) effluent discharge sites were selected in prelockdown and during lockdown phase. A new approach of water quality assessment, i.e., water pollution index (WPI) has been applied in this study. WPI is weightage free, unbiased method to analysis of water quality. The result shows that the physical, chemical and heavy elements were found beyond the standard limit in prelockdown period. The cation and anion were arranged in an order of Na 2+ > K + > Ca 2+ > Mg 2+ and Cl − > So 4 − > No 3 − > F − in both the sessions. WPI of prelockdown showed that about 100% water samples are of highly polluted. WPI of lockdown period showed that around 90.90% samples improved to 'good quality' and 9.10% of samples are of 'moderately polluted.' Hypothesis testing by 't' test proved that there was a significant difference (ρ = 0.05%) in values of each parameter between two periods. Null hypothesis was rejected and indicated the improvement of river water quality statistically. Spatial mapping using Arc GIS 10.4 interpolation (IDW) helps to understand spatial intensity of pollution load in two periods. This research study should be helpful for further management and spatial diagnosis of water resource of river Damodar.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalyzes the first reaction of de novo purine nucleotide synthesis in two steps at two sites. Glutamine is hydrolyzed to glutamate plus NH 3 at an N-terminal glutaminase site, and NH 3 is transferred through a 20-Å hydrophobic channel to a distal PRPP site for synthesis of phosphoribosylamine. Binding of PRPP is required to activate the glutaminase site (termed interdomain signaling) to prevent the wasteful hydrolysis of glutamine in the absence of phosphoribosylamine synthesis. Mutations were constructed to analyze the function of the NH 3 channel. In the wild type enzyme, NH 3 derived from glutamine hydrolysis was transferred to the PRPP site, and little or none was released. Replacement of Leu-415 at the PRPP end of the channel with an alanine resulted in a leaky channel and release of NH 3 to the solvent. Mutations in five amino acids that line the channel and two other residues required for the reorganization of phosphoribosyltransferase domain "flexible loop" that leads to formation of the channel perturbed channel function as well as interdomain signaling. The data emphasize the role of the NH 3 channel in coupling interdomain signaling and NH 3 transfer. Glutamine PRPP1 amidotransferase catalyzes the first reaction in the pathway for de novo purine nucleotide synthesis and is the key regulatory enzyme in the pathway. The enzyme is a member of an Ntn, N-terminal nucleophile family of glutamine amidotransferases (1). The overall reaction, shown by Equation 1, takes place in two steps at active sites in two domains.An N-terminal glutaminase domain is responsible for hydrolysis of glutamine (Equation 2). NH 3 derived from glutamine hydrolysis reacts with PRPP at a site in the C-terminal PRTase domain (Equation 3). Crystal structures have been determined for a ligand-free (2) and an enzyme-substrate analog ternary complex (3). In the ligand-free enzyme, denoted state I, the two active sites are separated by 16 Å, and neither of the sites is properly organized for catalysis. The PRPP site is open to the solvent such that bound substrate would be susceptible to hydrolysis. The glutamine site in the ligand-free enzyme is in a closed conformation, unfavorable for entry of glutamine, and Arg-73, a residue required for glutamine binding, is extensively hydrogen-bonded and unavailable for interaction with the glutamine ␣-carboxyl group. In the crystal structure of the ternary complex, designated state III, conformational changes have optimized the two sites for catalysis, and a 20-Å hydrophobic tunnel has formed to channel NH 3 derived from glutamine hydrolysis to the PRPP site. There are thus several functional consequences resulting from PRPP binding. First, binding of PRPP activates the glutamine site by lowering the K m for glutamine by 100-fold and increasing k cat by 3-fold (4). This interdomain signaling prevents the wasteful hydrolysis of glutamine in the absence of PRA synthesis. Formation of the NH 3 channel is a second consequence of PRPP bind...

A single helicase amino acid substitution, NS3-T249P, has been shown to increase viremia magnitude/mortality in American crows (AMCRs) following West Nile virus (WNV) infection. Lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of WNV isolates associated with recent outbreaks reported worldwide have a proline at the NS3-249 residue. In order to evaluate the impact of NS3-249 variants on avian and mammalian virulence, multiple amino acid substitutions were engineered into a WNV infectious cDNA (NY99; NS3-249P) and the resulting viruses inoculated into AMCRs, house sparrows (HOSPs) and mice. Differential viremia profiles were observed between mutant viruses in the two bird species; however, the NS3-249P virus produced the highest mean peak viral loads in both avian models. In contrast, this avian modulating virulence determinant had no effect on LD50 or the neurovirulence phenotype in the murine model. Recombinant helicase proteins demonstrated variable helicase and ATPase activities; however, differences did not correlate with avian or murine viremia phenotypes. These in vitro and in vivo data indicate that avian-specific phenotypes are modulated by critical viral-host protein interactions involving the NS3-249 residue that directly influence transmission efficiency and therefore the magnitude of WNV epizootics in nature.

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD(+)-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.