Human cytomegalovirus escapes a naturally occurring neutralizing antibody by incorporating it into assembling virions

Human cytomegalovirus (CMV) is a common but difficult to treat infection of immunocompromised patients. MSL-109 is a human monoclonal IgG isolated from a CMV seropositive individual that recognizes the viral glycoprotein H (gH) surface antigen complexes that mediate entry. Although MSL-109 blocks CMV infection in?vitro, it lacked sufficient efficacy in human trials, and CMV isolated from treated patients suggested the evolution of MSL-109 resistance. To understand how CMV escapes MSL-109, we characterized a MSL-109-resistant CMV strain. Our results elucidate a nongenetic escape mechanism in which the antibody is selectively taken up by infected cells and incorporated into assembling virions in a dose-dependent manner. The resistant virus then utilizes the Fc domain of the incorporated antibody to infect naive nonimmune cells. This resistance mechanism may explain the clinical failure of MSL-109, illustrate a general mechanism of viral antibody escape, and inform antiviral vaccine and therapeutic development.     

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (K_D < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.     

In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (K_D < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.     

Isolation and characterization of nine microsatellite loci from the pike-perch, Sander lucioperca (Linnaeus, 1758)

In order to facilitate studies on the genetic structure of wild populations as well as to monitor genetic changes in cultured stocks, nine polymorphic microsatellite loci were isolated from pike-perch (Sander lucioperca). Single loci allele numbers varied between two (loci MSL-3 and MSL-7) and six (loci MSL-1 and MSL-2), and observed heterozygosities ranged from 0.36 (locus MSL-2) to 1.00 (locus MSL-9) in a test panel of 25 individuals. Only one pair of loci (MSL-5 and MSL-8) displayed significant linkage disequilibrium after sequential Bonferroni corrections. Hardy-Weinberg tests revealed significant excesses of heterozygotes at three loci (MSL-1, MSL-7, and MSL-9).     

Purification and characterization of three galactose specific lectins from mulberry seeds (Morus sp.).

Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion-exchange chromatography on DEAE and CM-cellulose. The lectins were found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular masses of the lectins as determined by gel filtration were 175 000 for MSL-1, 120 000 for MSL-2 and 89 500 for MSL-3. MSL-1 is dimer in nature, with the two monomers held together by disulfide bond(s), while MSL-2 and MSL-3 contain four nonidentical subunits that are held together by nonionic hydrophobic interactions. The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl-alpha-d-galactopyranoside, methyl-beta-d-galactopyranoside, lactose and raffinose. The lectins MSL-1, MSL-2 and MSL-3 contained 5.7, 5.4 and 4.5% neutral sugars, respectively, and the sugar composition of the lectins was glucose and mannose for MSL-1 and galactose for both MSL-2 and MSL-3. The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay.     

Drosophila Male-Specific Lethal-2 Protein: Structure/Function Analysis and Dependence on Msl-1 for Chromosome Association

MSL-2 is required for the male-specific assembly of a dosage compensation regulatory complex on the X chromosome of Drosophila melanogaster. We found that MSL-2 binds in a reproducible, partial pattern to the male X chromosome in the absence of MLE or MSL-3, or when ectopically expressed at a low level in females. Moreover, the pattern of MSL-2 binding corresponds precisely in each case to that of MSL-1, suggesting that the two proteins function together to associate with the X. Consistent with this hypothesis, we isolated EMS-induced loss of function msl-1 and msl-2 alleles in a screen for suppressors of the toxic effects of MSL-2 expression in females. We also used site-directed mutagenesis to determine the importance of the MSL-2 RING finger domain and second cysteine-rich motif. The mutations, including those in conserved zinc coordinating cysteines, confirm that the RING finger is essential for MSL-2 function, while suggesting a less stringent requirement for an intact second motif.     

<Emphasis Type="Italic">Nocardioides islandiensis</Emphasis> sp. nov., isolated from soil in Bigeum Island Korea

A novel actinobacterium designated as MSL-26^T was isolated from soil in Bigeum Island Korea. A polyphasic study was undertaken to establish the taxonomic position of isolate MSL-26^T. Strain MSL-26^T was found to have chemical and morphological characteristics similar to Nocardioides. The strain grew optimally at pH 7·5 and 28°C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MSL-26^T forms a distinct line of descent within the radiation enclosed by the genus Nocardioides. The cell wall of strain MSL-26^T contained LL-2, 6-diaminopimelic acid. The principal menaquinone was MK-8 (H₄). The phospholipids detected were diphosphatidylglycerol, phosphatidylglycerol and some unidentified lipids. C_18:1 w7c (50.38%) was the major fatty acid. The DNA G + C content of strain MSL-26^T was 71.4 mol%. The 16S rRNA gene sequence of strain MSL-26^T shares the highest sequence similarity with Nocardioides kribbensis KCTC 19038^T (95.78%) and Nocardioides aquaticus DSM 11439^T (95.52%). Based on the morphological, physiological, biochemical and chemotaxonomical data presented in this study, strain MSL-26^T should be classified as a novel species, for which the name Nocardioides islandiensis sp. nov. is proposed. The type strain is MSL-26^T (=KCTC 19275^T =DSM 19321^T)     

Nocardioides tritolerans sp. nov., Isolated from soil in Bigeum Island, Korea

A Gram-positive strain designated as MSL-14T isolated from a soil sample collected from Bigeum Island, Korea, was subjected to polyphasic taxonomy. The isolate was strictly aerobic. Cells were short rods and motile. Optimum growth temperature and pH was 28 degrees and 7.0, respectively. It was characterized chemotaxonomically as having a cell-wall peptidoglycan type based on LL-2,6-diaminopimelic acid and MK-8(H4) as the predominant menaquinone. The major fatty acids were iso-C16:0, C17:1 omega8c, and C18:1 omega9c. The G+C content was 67.6 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain MSL-14T is affiliated to the genus Nocardioides and formed a distinct lineage within the genus. MSL-14T showed highest sequence similarity to Nocardioides aestuarii JCM 12125T, having a similarity of 96.5%. Based on the 16S rRNA gene sequence divergence and phenotypic characteristics, it is proposed that strain MSL-14T should be classified as representing a novel member of the genus Nocardioides, for which we propose the name Nocardioides tritolerans sp. nov. The type strain is strain MSL-14T (=KCTC 19289T= DSM 19320T).     

Nocardioides halotolerans sp. nov., isolated from soil on Bigeum Island, Korea

A strictly aerobic, Gram-positive, motile, coccoid-shaped, halotolerant actinobacterium (10% NaCl, w/v), designated MSL-23^T, was isolated from a soil sample on Bigeum Island, Korea. Results of 16S rRNA gene sequence analysis indicated that the isolate belonged to the genus Nocardioides, with the highest sequence similarity (95.63%) being to Nocardioides kribbensis KCTC 19038^T. The major menaquinone was MK-8(H₄), and the predominant cellular fatty acids were i-C_16:0, ai-C_17:0, C_18:1 ω9c and 10-methyl-C_16:0. The DNA G+C content was 69.7 mol%. The 16S rRNA gene sequence of strain MSL 23^T and its chemotaxonomic properties showed it to be unique in the genus Nocardioides. Phenotypic characteristics distinguished strain MSL-23^T from other Nocardioides species. On the basis of the phenotypic, chemotaxonomic and phylogenetic data strain MSL-23^T represents a novel species, for which the name Nocardioides halotolerans sp. nov. is proposed, with MSL-23^T (=KCTC 19274^T=DSM 19273^T) as the type strain.     

<Emphasis Type="Italic">Nocardioides dilutes</Emphasis> sp. nov. Isolated from Soil in Bigeum Island,Korea

A short coccoid-rod-shaped, nonmotile actinobacteria strain MSL-11^T was isolated from soil in Bigeum Island, Korea. A polyphasic study was undertaken to establish the taxonomic position of this strain. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain MSL-11^T forms an evolutionary lineage within the radiation of the genus Nocardioides. The cell wall peptidoglycan of strain MSL-11^T contained ll-diaminopimelic acid, indicating wall chemotype I. The predominant menaquinone was MK-8(H₄). Strain MSL-11^T had a cellular fatty acid profile containing straight-chain, branched, unsaturated, and 10-methyl fatty acids, with iso-C_16:0 as a major fatty acid component detected. The DNA G + C content of the strain was 71.8 mol%. Comparative 16S rRNA gene sequencing revealed that the strains constituted a distinct subclade within the genus Nocardioides, displaying a 16S rRNA gene sequence similarity of about 95.68% with Nocardioides jensenii DSM 20641^T. On the basis of both phenotypic and phylogenetic evidence, the strain is separated from previously described Nocardioides species and should be assigned to represent a novel species of the genus Nocardioides, for which the name Nocardioides dilutes sp. nov. is proposed. The type strain is strain MSL-11^T (= KCTC 19288^T = DSM 19318^T).     

Evolution of Chromatin-Remodeling Complexes: Comparative Genomics Reveals the Ancient Origin of “Novel” Compensasome Genes

Dosage compensation in Drosophila is mediated by a complex, called compensasome, composed of at least five proteins and two noncoding RNAs. Genes encoding compensasome proteins have been collectively named male-specific lethals or msls. Recent work showed that three of the Drosophila msls (msl-3, mof, and mle) have an ancient origin. In this study, I describe likely orthologues of the two remaining msls, msl-1 and msl-2, in several invertebrates and vertebrates. The MSL-2 protein is the only one found in Drosophila and vertebrate genomes that contains both a RING finger and a peculiar type of CXC domain, related to the one present in Enhancer of Zeste proteins. MSL-1 also contains two evolutionarily conserved domains: a leucine zipper and a second characteristic region, described here for the first time, which I have called the PEHE domain. These two domains are present in the likely orthologues of MSL-1 as well as in other genes in several invertebrate and vertebrate species. Although it cannot be excluded that the compensasome complex is a recent evolutionary novelty, these results shows that all msls are found in mammals, suggesting that protein complexes related to the compensasome may be present in mammalian species. Metazoans that lack several of the msls, such as Caenorhabditis elegans, cannot contain compensasomes. The evolutionary relationships of the compensasome and the NuA4 complex, another chromatin-remodeling complex that contains related subunits, are discussed.     

Tissue specificity of alternative splicing products of mouse mRNA encoding new protein hampin homologous to the Drosophila MSL-1 protein

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.     

X Chromosome Sites Autonomously Recruit the Dosage Compensation Complex in Drosophila Males

It has been proposed that dosage compensation in Drosophila males occurs by binding of two core proteins, MSL-1 and MSL-2, to a set of 35–40 X chromosome “entry sites” that serve to nucleate mature complexes, termed compensasomes, which then spread to neighboring sequences to double expression of most X-linked genes. Here we show that any piece of the X chromosome with which compensasomes are associated in wild-type displays a normal pattern of compensasome binding when inserted into an autosome, independently of the presence of an entry site. Furthermore, in chromosomal rearrangements in which a piece of X chromosome is inserted into an autosome, or a piece of autosome is translocated to the X chromosome, we do not observe spreading of compensasomes to regions of autosomes that have been juxtaposed to X chromosomal material. Taken together these results suggest that spreading is not involved in dosage compensation and that nothing distinguishes an entry site from the other X chromosome sites occupied by compensasomes beyond their relative affinities for compensasomes. We propose a new model in which the distribution of compensasomes along the X chromosome is achieved according to the hierarchical affinities of individual binding sites.     

X chromosome sites autonomously recruit the dosage compensation complex in Drosophila males

It has been proposed that dosage compensation in Drosophila males occurs by binding of two core proteins, MSL-1 and MSL-2, to a set of 35–40 X chromosome “entry sites” that serve to nucleate mature complexes, termed compensasomes, which then spread to neighboring sequences to double expression of most X-linked genes. Here we show that any piece of the X chromosome with which compensasomes are associated in wild-type displays a normal pattern of compensasome binding when inserted into an autosome, independently of the presence of an entry site. Furthermore, in chromosomal rearrangements in which a piece of X chromosome is inserted into an autosome, or a piece of autosome is translocated to the X chromosome, we do not observe spreading of compensasomes to regions of autosomes that have been juxtaposed to X chromosomal material. Taken together these results suggest that spreading is not involved in dosage compensation and that nothing distinguishes an entry site from the other X chromosome sites occupied by compensasomes beyond their relative affinities for compensasomes. We propose a new model in which the distribution of compensasomes along the X chromosome is achieved according to the hierarchical affinities of individual binding sites.     

Tissue Specificity of Alternative Splicing of Transcripts Encoding Hampin,a New Mouse Protein Homologous to the <Emphasis Type="Italic">Drosophila</Emphasis> MSL-1 Protein

A number of mammalian genomes have one gene copy encoding the protein that we named hampin. A search in a number of databases revealed a distant homologue, the well-known Drosophila protein MSL-1 (male-specific lethal 1). An alternative splicing of mRNA led to a significant diversity of structural hampin variants with different domain compositions. We analyzed the tissue-specific expression of five mouse hampin variants using RT-PCR. Two variants encoding hampin proteins with truncated N termini were shown to have a restricted tissue specificity: they are exclusively expressed in the testes. The mRNAs of other hampin variants were detected in all the tested tissues at comparable levels. We obtained polyclonal antibodies to the recombinant hampin and used them to demonstrate that at least one of the variants is predominantly localized in the nucleus. The specific features of the hampin primary structure and its possible functions as a member of the hampin/MSL-1 family of proteins are discussed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 363–371.Original Russian Text Copyright © 2005 by Dmitriev, Pestov, Korneenko, Gerasimova, Zhao, Modyanov, Kostina, Shakhparonov.The article was translated by the authors.     

Potentiation of invasive activity of hepatoma cells by reactive oxygen species is mediated by autocrine/paracrine loop of hepatocyte growth factor

We have already reported that reactive oxygen species (ROS) promote rat ascites hepatoma cell invasion beneath mesentery-derived mesothelial cell monolayer. To investigate the mechanism for this, we examined the involvement of motility factors, particularly hepatocyte growth factor (HGF). Rat ascites hepatoma cell line of AH109A expressed HGF and c-Met mRNAs. Treatment with ROS augmented amounts of HGF mRNA in AH109A and HGF concentration in the medium. ROS also induced HGF gene expression in mesothelial cells. Exogenously added HGF enhanced invasive activity of AH109A cells, but exerted no effect on proliferation. AH109A cells pretreated with ROS showed an increased invasive activity, which was cancelled by simultaneous pretreatment with anti-HGF antibody. These results suggest that the invasive activity of AH109A is mediated by the autocrine and paracrine pathways of HGF, and ROS potentiate invasive activity by inducing gene expression of HGF in AH109A and mesothelial cells.