scholarly journals Recombinant Measles Viruses Expressing Altered Hemagglutinin (H) Genes: Functional Separation of Mutations Determining H Antibody Escape from Neurovirulence

2001 ◽  
Vol 75 (16) ◽  
pp. 7612-7620 ◽  
Author(s):  
Kerstin Moeller ◽  
Iain Duffy ◽  
Paul Duprex ◽  
Bert Rima ◽  
Rudi Beschorner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Basis ◽  
Measles Virus ◽  
Escape Mutant ◽  
Amino Acid Substitutions ◽  
Structural Alterations ◽  
Recombinant Viruses ◽  
The Brain ◽  
H Protein

ABSTRACT Measles virus (MV) strain CAM/RB, which was adapted to growth in the brain of newborn rodents, is highly neurovirulent. It has been reported earlier that experimentally selected virus variants escaping from the monoclonal antibodies (MAbs) Nc32 and L77 to hemagglutinin (H) preserved their neurovirulence, whereas mutants escaping MAbs K71 and K29 were found to be strongly attenuated (U. G. Liebert et al., J. Virol. 68:1486–1493, 1994). To investigate the molecular basis of these findings, we have generated a panel of recombinant MVs expressing the H protein from CAM/RB and introduced the amino acid substitutions thought to be responsible for antibody escape and/or neurovirulence. Using these recombinant viruses, we identified the amino acid changes conferring escape from the MAbs L77 (377R→Q and 378M→K), Nc32 (388G→S), K71 (492E→K and 550S→P), and K29 (535E→G). When the corresponding recombinant viruses were tested in brains of newborn rodents, we found that the mutations mediating antibody escape did not confer differential neurovirulence. In contrast, however, replacement of two different amino acids, at positions 195G→R and 200S→N, which had been described for the escape mutant set, caused the change in neurovirulence. Thus, antibody escape and neurovirulence appear not to be associated with the same structural alterations of the MV H protein.

scholarly journals Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel

2016 ◽  
Vol 82 (22) ◽  
pp. 6748-6756 ◽  
Author(s):  
Xiaodan Yan ◽  
Jianjun Wang ◽  
Yu Sun ◽  
Junge Zhu ◽  
Sheng Wu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Engineering ◽  
Esterase Activity ◽  
Molecular Basis ◽  
Catalytic Triad ◽  
Amino Acid Substitutions ◽  
Biological Functions ◽  
Catalytic Activities ◽  
Efficient Strategy

ABSTRACTPromiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical α/β fold hydrolase, was previously reported to have both γ-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase fromPseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classicalp-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any γ-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities.IMPORTANCEPromiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of α/β fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies.

scholarly journals Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion, Hemadsorption, Virus Growth, and Penetration Rate

2009 ◽  
Vol 83 (17) ◽  
pp. 8713-8721 ◽  
Author(s):  
Hiromi Okada ◽  
Masae Itoh ◽  
Kyosuke Nagata ◽  
Kaoru Takeuchi
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Measles Virus ◽  
Vero Cells ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Virus Growth ◽  
F Protein ◽  
H Protein ◽  
Cell Cell

ABSTRACT Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.

scholarly journals Two functionally linked amino acids in the stem 2 region of measles virus haemagglutinin determine infectivity and virulence in the rodent central nervous system

2007 ◽  
Vol 88 (11) ◽  
pp. 3112-3120 ◽  
Author(s):  
K. Moeller-Ehrlich ◽  
M. Ludlow ◽  
R. Beschorner ◽  
R. Meyermann ◽  
B. K. Rima ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Measles Virus ◽  
Brain Cell ◽  
Glycosylation Site ◽  
Eukaryotic Expression ◽  
Reading Frame ◽  
Viral Attachment ◽  
Acute Encephalitis ◽  
H Protein

Rodent brain-adapted measles virus (MV) strains, such as CAM/RB and recombinant MVs based on the Edmonston strain containing the haemagglutinin (H) of CAM/RB, cause acute encephalitis after intracerebral infection of newborn rodents. We have demonstrated that rodent neurovirulence is modulated by two mutations at amino acid positions 195 and 200 in the H protein, one of these positions (200) being a potential glycosylation site. In order to analyse the effects of specific amino acids at these positions, we introduced a range of individual and combined mutations into the open reading frame of the H gene to generate a number of eukaryotic expression plasmids. The functionality of the mutant H proteins was assessed in transfected cells and by generating recombinant viruses. Interestingly, viruses caused acute encephalitis only if the amino acid Ser at position 200 was coupled with Gly at position 195, whereas viruses with single or combined mutations at these positions, including glycosylation at position 200, were attenuated. Neurovirulence was associated with virus spread and induction of neuronal apoptosis, whereas attenuated viruses failed to infect brain cells. Similar results were obtained by using primary brain-cell cultures. Our findings indicate that a structural alteration in the stem 2 region of the H protein at position 195 or 200 interferes with infectivity of rodent neurons, and suggest that the interaction of the viral attachment protein with cellular receptors on neurons is affected.

scholarly journals Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480 ◽  
Author(s):  
E Moran ◽  
B Zerler ◽  
T M Harrison ◽  
M B Mathews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Transformation Function ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

scholarly journals Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Michael Herren ◽  
Neeta Shrestha ◽  
Marianne Wyss ◽  
Andreas Zurbriggen ◽  
Philippe Plattet
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Measles Virus ◽  
Cell Entry ◽  
Regulatory Role ◽  
Fusion Activity ◽  
Animal Pathogens

ABSTRACTMorbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerizationper sewas not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCEMeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

PREDICTION OF PATHOGENIC EFFECT FOR AMINO ACID SUBSTITUTIONS IN BRCA1 AND BRCA2 PROTEINS USING MNA DESCRIPTORS AND NAIVE BAYES CLASSIFIER

2020 ◽  
pp. 250-252
Author(s):  
D. Filimonov ◽  
A. Lagunin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Sequence ◽  
Amino Acid Substitutions ◽  
Classification Models ◽  
Bayes Classifier ◽  
Brca1 And Brca2 ◽  
Chemical Structures ◽  
Pathogenic Effect ◽  
Pathogenic Effects

It is advisable to use data peptide's chemical structures with amino acids (AMA) substitution and the corresponding sections of the protein sequence without mutation to construct classification models predicting the pathogenic effects AMA substitutions based on MNA descriptors.

scholarly journals Generation of Recombinant Rotavirus with an Antigenic Mosaic of Cross-Reactive Neutralization Epitopes on VP4

2008 ◽  
Vol 82 (13) ◽  
pp. 6753-6757 ◽  
Author(s):  
Satoshi Komoto ◽  
Masanori Kugita ◽  
Jun Sasaki ◽  
Koki Taniguchi
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Reverse Genetics ◽  
Amino Acid Substitutions ◽  
Neutralization Epitope ◽  
First Report ◽  
P Type

ABSTRACT Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.

scholarly journals Chlorella Virus-Encoded Deoxyuridine Triphosphatases Exhibit Different Temperature Optima

2005 ◽  
Vol 79 (15) ◽  
pp. 9945-9953 ◽  
Author(s):  
Yuanzheng Zhang ◽  
Hideaki Moriyama ◽  
Kohei Homma ◽  
James L. Van Etten
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Recombinant Protein ◽  
Divalent Cations ◽  
Catalytic Efficiency ◽  
Amino Acid Substitutions ◽  
Temperature Optimum ◽  
Docking Simulations ◽  
Chlorella Virus ◽  
Temperature Optima

ABSTRACT A putative deoxyuridine triphosphatase (dUTPase) gene from chlorella virus PBCV-1 was cloned, and the recombinant protein was expressed in Escherichia coli. The recombinant protein has dUTPase activity and requires Mg2+ for optimal activity, while it retains some activity in the presence of other divalent cations. Kinetic studies of the enzyme revealed a Km of 11.7 μM, a turnover k cat of 6.8 s−1, and a catalytic efficiency of k cat/Km = 5.8 × 105 M−1 s−1. dUTPase genes were cloned and expressed from two other chlorella viruses IL-3A and SH-6A. The two dUTPases have similar properties to PBCV-1 dUTPase except that IL-3A dUTPase has a lower temperature optimum (37°C) than PBCV-1 dUTPase (50°C). The IL-3A dUTPase differs from the PBCV-1 enzyme by nine amino acids, including two amino acid substitutions, Glu81→Ser81 and Thr84→Arg84, in the highly conserved motif III of the proteins. To investigate the difference in temperature optima between the two enzymes, homology modeling and docking simulations were conducted. The results of the simulation and comparisons of amino acid sequence suggest that adjacent amino acids are important in the temperature optima. To confirm this suggestion, three site-directed amino acid substitutions were made in the IL-3A enzyme: Thr84→Arg84, Glu81→Ser81, and Glu81→Ser81 plus Thr84→Arg84. The single substitutions affected the optimal temperature for enzyme activity. The temperature optimum increased from 37 to 55°C for the enzyme containing the two amino acid substitutions. We postulate that the change in temperature optimum is due to reduction in charge and balkiness in the active cavity that allows more movement of the ligand and protein before the enzyme and substrate complex is formed.

Apical Transporters for Neutral Amino Acids: Physiology and Pathophysiology

Physiology ◽  
2008 ◽  
Vol 23 (2) ◽  
pp. 95-103 ◽  
Author(s):  
Stefan Bröer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Identification ◽  
Molecular Basis ◽  
Amino Acid Transporters ◽  
Lysinuric Protein Intolerance ◽  
Inherited Disorders ◽  
Lysinuric Protein ◽  
Hartnup Disorder ◽  
Protein Intolerance

Absorption of amino acids in kidney and intestine involves a variety of transporters for different groups of amino acids. This is illustrated by inherited disorders of amino acid absorption, such as Hartnup disorder, cystinuria, iminoglycinuria, dicarboxylic aminoaciduria, and lysinuric protein intolerance, affecting separate groups of amino acids. Recent advances in the molecular identification of apical neutral amino acid transporters has shed a light on the molecular basis of Hartnup disorder and iminoglycinuria.

Sign in / Sign up

Export Citation Format

Share Document