The Experts below are selected from a list of 129 Experts worldwide ranked by ideXlab platform
Michael C Lawrence - One of the best experts on this subject based on the ideXlab platform.
-
sequence and structure alignment of paramyxoVirus hemagglutinin neuraminidase with influenza Virus neuraminidase
Journal of Virology, 1993Co-Authors: Peter M. Colman, Peter A Hoyne, Michael C LawrenceAbstract:A model is proposed for the three-dimensional structure of the paramyxoVirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza Virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the Enzyme-active center of influenza Virus neuraminidase. The influenza Virus Enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences. Images
-
Sequence and structure alignment of paramyxoVirus hemagglutinin-neuraminidase with influenza Virus neuraminidase.
Journal of virology, 1993Co-Authors: Peter M. Colman, Peter A Hoyne, Michael C LawrenceAbstract:A model is proposed for the three-dimensional structure of the paramyxoVirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza Virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the Enzyme-active center of influenza Virus neuraminidase. The influenza Virus Enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.
Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
-
A Yeast-Based Genetic System for Functional Analysis of Viral mRNA Capping Enzymes
Journal of Virology, 2000Co-Authors: Alexandra Martins, Stewart ShumanAbstract:Eukaryotic Viruses have evolved diverse strategies to acquire a 5′ cap structure for their mRNAs (4). RNA Viruses that encode RNA-dependent RNA polymerases to synthesize their mRNAs either steal the caps from cellular transcripts, encode their own Enzymes that cap and methylate the plus-strand transcripts, or circumvent the capping problem by including cis-acting elements in the plus strand that promote cap-independent translation. The mRNAs of most DNA Viruses are synthesized by cellular RNA polymerase II (pol II) and are therefore capped by the cellular capping and methylating Enzymes. However, vaccinia Virus and other poxViruses, which replicate entirely in the cytoplasm, encode and encapsidate their own DNA-dependent RNA polymerase and mRNA capping apparatus (12, 16). African swine fever Virus, which has a cytoplasmic replication phase, also encodes and encapsidates its own RNA polymerase and capping Enzyme (39). BaculoViruses, which replicate in the nucleus, use pol II to transcribe early genes and then switch at late times to a Virus-encoded transcription system that includes RNA polymerase and capping activities (14, 15, 26). Chlorella Virus PBCV-1 encodes a capping Enzyme (25) but appears not to encode its own RNA polymerase. Cap formation by the Enzymes of DNA Viruses, double-stranded RNA Viruses, and eukaryotic cells occurs via three sequential reactions: (i) the 5′ triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA 5′ triphosphatase, (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by S-adenosylmethonine:RNA (guanine-N7) methyltransferase (48). This “conventional” pathway of cap synthesis was defined using soluble Enzymes purified from vaccinia Virus particles (12, 54). The mechanisms and structures of cellular and DNA Virus capping Enzymes have since been delineated through mutagenesis and crystallography (10, 11, 17, 21, 28, 30, 33, 37, 42, 56, 57, 60–62). Several single-stranded RNA Viruses have evolved alternative cap synthetic pathways, which entail unconventional chemistry and are less well understood with respect to Enzyme structure and mechanisms (1–3, 47). The genetic and physical organizations of the known Virus-encoded mRNA capping Enzymes are significantly different from those of metazoan host cells (48). Hence, the viral cap-forming Enzymes are potential targets for antiviral drugs that would interfere with capping of pathogen mRNAs but spare the host capping Enzymes. For any given Virus that provides its own caps, there are a number of questions that need to be addressed before capping can be validated as a target, such as (i) whether the viral gene encoding the capping Enzyme is essential for Virus replication and (ii) whether the capping activity of the viral gene product is essential for Virus replication. These questions have not been answered fully, even where the biochemistry of viral cap formation is well understood. For example, vaccinia Virus capping Enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities (45, 54). The Enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia Virus D1 and D12 genes, respectively. The vaccinia Virus D1 and D12 genes are essential for Virus replication, insofar as mutations that elicit temperature-sensitive Virus growth phenotypes have been mapped to the two capping Enzyme subunits (6, 18). However, the genetic landscape is complicated, because vaccinia Virus capping Enzyme plays a larger role in viral gene expression; it serves as a transcription termination factor during the synthesis of viral early mRNAs (29, 50) and as an initiation factor during the transcription of intermediate genes (55). Amazingly, the D1 and D12 temperature-sensitive mutant Viruses display no gross defect in viral gene expression at the restrictive temperature but are instead defective in resolving concatemeric DNA replication intermediates into the hairpin telomeres of the mature viral genome (6, 18). This mysterious phenotype has no obvious connection to the known mRNA-processing or transcription functions of the D1 and D12 proteins. Similar problems arise in interpreting the essentiality of the baculoVirus LEF-4 capping Enzyme, which is an intrinsic subunit of baculoVirus RNA polymerase, because it is not clear if the conditional phenotype of a lef-4 mutant Virus is a consequence of failure to transcribe or failure to cap viral mRNAs (15, 26). Genetic, and ultimately pharmacologic, analysis of viral capping Enzymes would be facilitated by the development of in vivo assays in which the functional readout is clearly and exclusively dependent on the capacity of the viral gene product to catalyze cap synthesis. Here we explore the possibility of using the budding yeast Saccharomyces cerevisiae as a genetic model for the study of viral capping Enzymes. S. cerevisiae encodes a three-component capping system consisting of separate triphosphatase (Cet1p), guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products (22, 31, 43, 53). All three genes are essential for yeast cell growth. Mutational analyses of Cet1p, Ceg1p, and Abd1p have resulted in the delineation of minimal catalytic domains for each protein and the identification of catalytically important amino acid side chains that comprise the triphosphatase, guanylyltransferase, and methyltransferase active sites (21, 27, 30, 37, 56, 57). By correlating mutational effects on catalysis in vitro with effects on function in vivo, we have shown that the triphosphatase, guanylyltransferase, and methyltransferase activities are essential for yeast cell growth. The feasibility of using yeast growth as a readout of the function of capping Enzymes from heterologous sources is underscored by the demonstration that the entire three-component yeast capping apparatus can be replaced in vivo by the two-component mammalian capping system, consisting of a bifunctional triphosphatase-guanylyltransferase protein (Mce1p) and a separate cap methyltransferase (Hcm1p) (41). This result is remarkable because the structure and catalytic mechanism of the mammalian RNA triphosphatase are completely different from those of the yeast RNA triphosphatase (48). The salient question here is whether a viral capping Enzyme can function in place of one or more of the yeast capping Enzymes. To address this issue, we tested the ability of the N-terminal triphosphatase-guanylyltransferase domain of the vaccinia Virus D1 polypeptide [vD1(1-545)p] to complement the growth of yeast cet1Δ or ceg1Δ cells. We report that the vaccinia Virus Enzyme is active in vivo, provided that it is targeted to the pol II transcription complex by fusion to a cellular protein that binds to pol II. Mutations of vD1(1-545)p that abrogate triphosphatase activity in vitro abolish complementation of cet1Δ. Additional mutational analysis of vD1(1-545)p defines the triphosphatase active site and suggests a mechanism of metal-dependent catalysis common to DNA viral and fungal capping Enzymes. Isogenic strains bearing vaccinia Virus and human capping Enzymes can be used to screen for cytotoxic compounds that specifically inhibit the poxVirus capping apparatus.
-
Characterization of a DNA Topoisomerase Encoded byAmsacta mooreiEntomopoxVirus
Virology, 1997Co-Authors: Birgitte Ø. Petersen, Richard W. Moyer, Richard L. Hall, Stewart ShumanAbstract:Abstract We have identified an Amsacta moorei entomopoxVirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxViruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant Enzyme was partially purified. AmEPV topoisomerase is a monomeric Enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma Virus, and Orf Virus Enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase ( k obs = 0.08 sec −1 ; K cl = 0.22) are similar to those of the vaccinia Virus Enzyme.
-
Characterization of a DNA Topoisomerase Encoded byAmsacta mooreiEntomopoxVirus
Virology, 1997Co-Authors: Birgitte Ø. Petersen, Richard W. Moyer, Richard L. Hall, Stewart ShumanAbstract:Abstract We have identified an Amsacta moorei entomopoxVirus (AmEPV) gene encoding a DNA topoisomerase. The 333-amino acid AmEPV topoisomerase displays instructive sequence similarities to the previously identified topoisomerases encoded by five genera of vertebrate poxViruses. One hundred nine amino acids are identical or conserved among the six proteins. The gene encoding AmEPV topoisomerase was expressed in bacteria and the recombinant Enzyme was partially purified. AmEPV topoisomerase is a monomeric Enzyme that catalyzes the relaxation of supercoiled DNA. Like the vaccinia, Shope fibroma Virus, and Orf Virus Enzymes, the AmEPV topoisomerase forms a covalent adduct with duplex DNA at the target sequence CCCTT↓. The kinetic and equilibrium parameters of the DNA cleavage reaction of AmEPV topoisomerase ( k obs = 0.08 sec −1 ; K cl = 0.22) are similar to those of the vaccinia Virus Enzyme.
Peter M. Colman - One of the best experts on this subject based on the ideXlab platform.
-
sequence and structure alignment of paramyxoVirus hemagglutinin neuraminidase with influenza Virus neuraminidase
Journal of Virology, 1993Co-Authors: Peter M. Colman, Peter A Hoyne, Michael C LawrenceAbstract:A model is proposed for the three-dimensional structure of the paramyxoVirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza Virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the Enzyme-active center of influenza Virus neuraminidase. The influenza Virus Enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences. Images
-
Sequence and structure alignment of paramyxoVirus hemagglutinin-neuraminidase with influenza Virus neuraminidase.
Journal of virology, 1993Co-Authors: Peter M. Colman, Peter A Hoyne, Michael C LawrenceAbstract:A model is proposed for the three-dimensional structure of the paramyxoVirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza Virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the Enzyme-active center of influenza Virus neuraminidase. The influenza Virus Enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.
Alba Guarné - One of the best experts on this subject based on the ideXlab platform.
-
A structural model of picornaVirus leader proteinases based on papain and bleomycin hydrolase.
Journal of General Virology, 1998Co-Authors: Tim Skern, Ignacio Fita, Alba GuarnéAbstract:The leader (L) proteinases of aphthoViruses (foot-and-mouth disease Viruses) and equine rhinoVirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease Virus Enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analysis have been used to classify the L proteinases as papain-like cysteine proteinases. However, sequence identity within the L proteinases themselves is low (between 18% and 32%) and only 14% between the L proteinases and papain. Secondary structure predictions, sequence alignments that take into account the positions of the essential catalytic residues, and structural considerations have been used in this study to investigate more closely the relationships between the L proteinases and papain. In spite of the low sequence identities, the analyses strongly suggest that the L proteinases of foot-and-mouth disease Virus and of equine rhinoVirus 1 have a similar overall fold to that of papain. Regions in the L proteinases corresponding to all five alpha-helices and seven beta-sheets of papain could be identified. Further comparisons with the proteinase bleomycin hydrolase, which also displays a papain topology in spite of important differences in size and amino acid sequence, support these conclusions and suggest how a C-terminal extension, present in all three L proteinases, and predicted to be an alpha-helix, might enable C-terminal self-processing to occur.
Peter A Hoyne - One of the best experts on this subject based on the ideXlab platform.
-
sequence and structure alignment of paramyxoVirus hemagglutinin neuraminidase with influenza Virus neuraminidase
Journal of Virology, 1993Co-Authors: Peter M. Colman, Peter A Hoyne, Michael C LawrenceAbstract:A model is proposed for the three-dimensional structure of the paramyxoVirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza Virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the Enzyme-active center of influenza Virus neuraminidase. The influenza Virus Enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences. Images
-
Sequence and structure alignment of paramyxoVirus hemagglutinin-neuraminidase with influenza Virus neuraminidase.
Journal of virology, 1993Co-Authors: Peter M. Colman, Peter A Hoyne, Michael C LawrenceAbstract:A model is proposed for the three-dimensional structure of the paramyxoVirus hemagglutinin-neuraminidase (HN) protein. The model is broadly similar to the structure of the influenza Virus neuraminidase and is based on the identification of invariant amino acids among HN sequences which have counterparts in the Enzyme-active center of influenza Virus neuraminidase. The influenza Virus Enzyme-active site is constructed from strain-invariant functional and framework residues, but in this model of HN, it is primarily the functional residues, i.e., those that make direct contact with the substrate sialic acid, which have identical counterparts in neuraminidase. The framework residues of the active site are different in HN and in neuraminidase and appear to be less strictly conserved within HN sequences than within neuraminidase sequences.
