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

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.75.16.7612-7620.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7612-7620 ◽

Cited By ~ 29

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.

Download Full-text

Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽

10.1139/g91-098 ◽

1991 ◽

Vol 34 (4) ◽

pp. 644-651 ◽

Cited By ~ 16

Author(s):

Kenneth Koo ◽

W. Dorsey Stuart

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurospora Crassa ◽

Rna Binding ◽

Signal Sequence ◽

Average Length ◽

Open Reading Frame ◽

Mrna Transcript ◽

S1 Nuclease ◽

Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

Download Full-text

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1711-1721.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1711-1721

Author(s):

E M McIntosh ◽

R H Haynes

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequences ◽

Open Reading Frame ◽

Northern Analysis ◽

Hindiii Site ◽

Reading Frame ◽

Hindiii Restriction Site ◽

Cycle Dependent

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.00679-18 ◽

2018 ◽

Vol 92 (18) ◽

Cited By ~ 7

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.

Download Full-text

Nucleotide and Amino Acid Sequences of the Metallo-β-Lactamase, ImiS, from Aeromonas veronii bv. sobria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.2.436 ◽

1998 ◽

Vol 42 (2) ◽

pp. 436-439 ◽

Cited By ~ 22

Author(s):

T. R. Walsh ◽

W. A. Neville ◽

M. H. Haran ◽

D. Tolson ◽

D. J. Payne ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Active Site ◽

Amino Acid Sequences ◽

Open Reading Frame ◽

Reading Frame ◽

Aeromonas Veronii ◽

Putative Active Site ◽

Lactamase Gene ◽

Active Site Sequence

ABSTRACT The Aeromonas veronii bv. sobria metallo-β-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-β-lactamases.

Download Full-text

Molecular characterization of MHC class II DRA cDNA in Deoni cattle

Indian Journal of Animal Research ◽

10.18805/ijar.b-3592 ◽

2018 ◽

Author(s):

K. Swathi ◽

M. Gnana Prakash ◽

D. Sakaram ◽

T. Raghunandan ◽

A. Sarat Chandra ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Mhc Class Ii ◽

Signal Peptide ◽

Class Ii ◽

Glycosylation Site ◽

Amino Acid Sequences ◽

Conserved Residues ◽

A2 Domain

The present study was undertaken to clone and characterize DRA gene in Deoni cattle. The cDNA for the DRA gene was amplified by using specific primers designed based on available cattle sequences and purified products were cloned in competent E.coli (DH5á) strain. The full length 1013bp product of cDNA of DRA contained a single ORF of 762 nucleotides that coded for 253 amino acids translated product. Twenty four amino acids formed signal peptide while 229 constituted mature peptide. The deduced amino acid sequences resembled those of class II molecules of other species for all the conserved residues having critical functional role. But a single N-linked glycosylation site in á1 was observed in cattle and buffalo when compared to human and swine which contain a second site in á2 domain. The signal peptide was found more variable among the species compared. Comparison of nucleotide and amino acid sequences among related species and dendrogram constructed revealed that the cattle sequences are more similar to buffalo sequences.

Download Full-text

Glycine cleavage enzyme complex: Rabbit H-protein cDNA sequence analysis and comparison to human, cow, and chicken

Biochemistry and Cell Biology ◽

10.1139/o00-081 ◽

2000 ◽

Vol 78 (6) ◽

pp. 725-730 ◽

Cited By ~ 1

Author(s):

Francis Choy ◽

Lisa Sharp ◽

Derek A Applegarth

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Matrix Protein ◽

Protein Gene ◽

Mitochondrial Targeting ◽

Reading Frame ◽

Cleavage Enzyme ◽

Glycine Cleavage ◽

H Protein ◽

Sequence Similarities

The H-protein is one of the four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex, the major degradative pathway of glycine. We have isolated the full-length cDNA of the H-protein gene from the rabbit (Oryctolagus caniculus) by reverse transcription of liver poly-A mRNA and determined its nucleotide sequence (GenBank Acc. No. BankIt 318281 AF 231451). Similar to that in human, the rabbit H-protein gene possesses a 519-bp open reading frame that translates a 173-amino-acid (aa) protein. This reading frame is comprised of a 48-aa mitochondrial targeting sequence and a 125-aa residue that constitutes the mature mitochondrial matrix protein. In the mature protein region, there is a 95.5% nucleotide and 98.4% amino-acid sequence similarity to human. This conservation was also noted in the mature protein of the cow (Bos taurus) and chicken (Gallus domesticus), where there are a 94.1% and 85.3% nucleotide similarities, and 95.2% and 85.6% amino-acid sequence similarities, respectively. However, the targeting region is not as well conserved. Comparison of the rabbit targeting sequence to that in human, cow, and chicken reveals 84.0%, 79.2%, and 72.9% nucleotide, and 72.9%, 75.0%, and 54.2% amino-acid sequence similarities, respectively. These findings suggest that within the H-protein gene, the regions encoding the mitochondrial targeting and matrix protein may have evolved differently. Gene diversification in the former may reflect the species specificity in targeting proteins destined for the mitochondria, whereas homology in the latter suggests a very similar structure-function of the mature H-protein among these species. This homology in structure-function likely accounts for the observation that non-human H-protein can replace the human protein in the activity assay of the glycine cleavage enzyme system. This includes the biochemical diagnosis of non-ketotic hyperglycinemia (NKH) resulting from defects other than the H-protein, e.g., mutation(s) in the P-protein.Key words: glycine cleavage enzyme, H-protein, sequence comparison, non-ketotic hyperglycinemia.

Download Full-text

Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver

Biochemical Journal ◽

10.1042/bj3150807 ◽

1996 ◽

Vol 315 (3) ◽

pp. 807-814 ◽

Cited By ~ 16

Author(s):

Said MODARESSI ◽

Bruno CHRIST ◽

Jutta BRATKE ◽

Stefan ZAHN ◽

Tilman HEISE ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Human Liver ◽

Phosphoenolpyruvate Carboxykinase ◽

Amino Acid Level ◽

Cdna Probe ◽

Rat Hepatocytes ◽

Eukaryotic Expression ◽

Amino Acid Sequences ◽

Sequence Identity

In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) is about equally distributed between cytosol and mitochondria in contrast with rat liver in which it is essentially a cytosolic enzyme. Recently, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlain, Keith, Falls and Meisler (1993) Genomics 16, 698–706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mol. Genet. 2, 1–4]. It was the goal of this investigation to isolate the cDNA of the human mitochondrial form of hepatic PCK. A human liver cDNA library was screened with a rat cytosolic PCK cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68% DNA sequence and a 70% deduced amino acid sequence identity with the human cytosolic PCK cDNA. Without the flanking 270 bases (=90 amino acids) each at the 5´ and 3´ end, the sequence identity was 73% on the DNA and 78% on the amino acid level. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cytosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic expression vector pcDNAI and transfected into human embryonal kidney cells HEK293; PCK activity was increased by 3-fold in the mitochondria, which normally contain 70% of total PCK activity, but not in the cytosol. The isolated cDNA was also transfected into cultured rat hepatocytes; again, PCK activity was enhanced by about 40-fold in the mitochondria, which normally possess only 10% of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glucagon. Comparison of the amino acid sequences deduced from the isolated cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses revealed the human mitochondrial PCK mRNA to be 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension experiments showed that the 5´-untranslated region of mitochondrial PCK mRNA was 134 nucleotides in length.

Download Full-text

Sequence of the Structural Gene for Xanthine Dehydrogenase (rosy Locus) in Drosophila melanogaster

Genetics ◽

10.1093/genetics/116.1.67 ◽

1987 ◽

Vol 116 (1) ◽

pp. 67-73

Author(s):

Tim P Keith ◽

Margaret A Riley ◽

Martin Kreitman ◽

R C Lewontin ◽

Daniel Curtis ◽

...

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Drosophila Melanogaster ◽

Amino Acid ◽

Nucleotide Sequence ◽

Structural Gene ◽

Xanthine Dehydrogenase ◽

Open Reading Frame ◽

Reading Frame ◽

Eco Ri

ABSTRACT We determined the nucleotide sequence of a 4.6-kb Eco RI fragment containing 70% of the rosy locus. In combination with information on the 5′ sequence, the gene has been sequenced in entirety. rosy cDNAs have been isolated and intron/exon boundaries have been determined. We find an open reading frame which spans four exons and would encode a protein of 1335 amino acids. The molecular weight of the encoded protein (xanthine dehydrogenase), based on the amino acid translation, is 146,898 daltons which agrees well with earlier biophysical estimates. Characteristics of the protein are discussed.

Download Full-text

Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe Butyrivibrio fibrisolvensOR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.2128-2135.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 2128-2135 ◽

Cited By ~ 32

Author(s):

M. L. Kalmokoff ◽

D. Lu ◽

M. F. Whitford ◽

R. M. Teather

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gel Filtration ◽

Culture Fluid ◽

Northern Blotting ◽

Coding Region ◽

Gene Encoding ◽

Butyrivibrio Fibrisolvens ◽

Reading Frame ◽

Significant Homology

ABSTRACT The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79Awas a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.

Download Full-text