Aloke Kumar Bera

Structure of the Flavivirus Helicase: Implications for Catalytic Activity, Protein Interactions, and Proteolytic Processing

ABSTRACT 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.

Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease *□

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 (NS2B40) 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) NS2B40 to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg459↓Gly460 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.

A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation

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.

Establishment of l-arabinose fermentation in glucose/xylose co-fermenting recombinant Saccharomyces cerevisiae 424A(LNH-ST) by genetic engineering

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and l-arabinose. Wild-type strains of Saccharomyces cerevisiae used in industrial ethanol production cannot ferment d-xylose and l-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of l-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi l-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from l-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock.

Interdomain Signaling in Glutamine Phosphoribosylpyrophosphate Amidotransferase

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp83 in the glutamine loop (residues 73–84) and Trp482 in the C-terminal helix (residues 471–492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile335 and Tyr74 mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased theK d for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile335 in the phosphoribosyltransferase domain and Tyr74 in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404–420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.

Host Competence and Helicase Activity Differences Exhibited by West Nile Viral Variants Expressing NS3-249 Amino Acid Polymorphisms

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.

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90 degrees C, with half-lives of 65 min for GPAT and 60 h for GARS at 80 degrees C. GPAT activity is known to depend upon channeling of NH(3) from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH(3) for synthesis of PRA was found to increase from 34% at 37 degrees C to a maximum of 84% at 80 degrees C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90 degrees C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50 degrees C, and remained relatively constant up to 90 degrees C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.