Glycoprotein H-related complexes of human cytomegalovirus: identification of a third protein in the gCIII complex.

Previous studies have described three disulfide-bonded glycoprotein complexes within the envelope of human cytomegalovirus (HCMV). These have been designated gCI, gCII, and gCIII. Although gCI has been identified as homodimeric glycoprotein B (gB, gpUL55), the compositions of gCII and gCIII remain incompletely defined. Earlier studies suggested that gCIII was composed of glycoprotein H (gH, gpUL75) complexed with a second glycoprotein, the gL homolog of HCMV. We characterized the gCIII complex of HCMV using recombinant vaccinia virus-expressed gH and gL. Our results indicated that authentic gCIII was not reconstituted by coexpression of gH and gL. The presence of a third, structurally and antigenically unique glycoprotein with an estimated molecular mass of 125,000 Da in virion-derived gCIII complexes suggested that at least three proteins were necessary for formation of this envelope glycoprotein complex. This third glycoprotein, gp125, contained both simple and complex N-linked carbohydrates and had an estimated deglycosylated mass of 64,000 Da. Furthermore, we demonstrated that mature gH existed as both a covalently complexed and noncovalently associated component of the gCIII complex within the envelope of infectious extracellular virions. These findings provide further evidence for the structural complexity of the envelope of HCMV and emphasize the uncertainties associated with the previous assignment of specific functions to envelope proteins of HCMV.     

Intracellular formation and processing of the heterotrimeric gH-gL-gO (gCIII) glycoprotein envelope complex of human cytomegalovirus

The human cytomegalovirus (HCMV) gCIII complex contains glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and glycoprotein O (gO; gpUL74). To examine how gH, gL, and gO interact within HCMV-infected cells to assemble the tripartite complex, pulse-chase experiments were performed. These analyses demonstrated that gH and gL associate by the end of the pulse period to form a disulfide dependent gH-gL complex. Subsequently, the gH-gL complex interacts with a 100-kDa precursor form of gO to form a 220-kDa precursor of the mature gH-gL-gO complex that contains a 125-kDa form of gO. The 220-kDa precursor complex (pgCIII) was sensitive to treatment with endoglycosidase H (endo H), while the mature gCIII complex was essentially resistant to digestion with this enzyme, suggesting that formation of pgCIII complex occurs in the endoplasmic reticulum (ER) and is processed to mature gH-gL-gO (gCIII) in a post-ER compartment. While the N-linked glycans on the 100-kDa form of gO were modified to endo H-resistant states as the 125-kDa gO formed, additional posttranslational modifications were detected on gO. These processing alterations were non-N-linked oligosaccharide modifications that could not be accounted for by phosphorylation or by O-glycosylation of the type sensitive to O-glycanase. Of gH, gL, gO, and the various complexes that they form, only the mature form of the complex was detectable at the infected cell membrane, as judged by surface biotinylation studies.     

UL146 variability among clinical isolates of human cytomegalovirus from Japan

Human cytomegalovirus (HCMV) is a herpesvirus associated with serious diseases in immunocompromised subjects. The region between ORF UL133 and UL151 from HCMV, named ULb' is frequently deleted in attenuated AD169 and in highly passaged laboratory strains. However, this region is conserved in low-passaged and more virulent HCMV, like the Toledo strain. The UL146 gene, which is located in the ULb' region, encodes a CXC-chemokine analogue. The diversity of UL146 gene was evaluated among fifty-six clinical isolates of HCMV from Japan. Results show that UL146 gene was successfully amplified by the polymerase chain reaction (PCR) in only 17/56 strains (30%), while the success rate for UL145/UL147 gene was 18/56 strains (32%). After DNA sequencing, the 35 amplified strains were classified into 8 groups. When compared, variability of UL146 ranged from 25.1% to 52.9% at the DNA level and from 34.5% to 67% at the amino acid level. Seven groups had the interleukin-8 (IL-8) motif ERL (Glu-Leu-Arg) CXC and one group had only the CXC motif, suggesting the absence of the IL-8 function of UL146. In conclusion, we found that UL146 gene of HCMV is hyper-variable in clinical strains from Japan suggesting the possibility of a different function in each sequence group.     

The chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types

The chicken Ig-like receptors (CHIR) have been described as two Ig domain molecules with long cytoplasmic tails containing inhibitory motifs. In this study, we demonstrate that CHIR form a large family, with multiple members showing great sequence variability among members as well as a great diversity in domain organization and properties of the transmembrane and cytoplasmic segments. We characterize various novel receptor types with motifs indicative of inhibitory, activating, or both functions. In addition to the inhibitory receptors with two ITIM, receptors with a single immunoreceptor tyrosine-based switch motif or receptors lacking a cytoplasmic domain were isolated. Activating receptors with a short cytoplasmic domain and a transmembrane arginine assembled with the newly identified chicken FcepsilonRIgamma chain. Three bifunctional receptor types were characterized composed of one or two C2-type Ig-like domains, a transmembrane region with a positively charged residue and combinations of cytoplasmic motifs such as ITIM, immunoreceptor tyrosine-based switch motif, and YXXM. RT-PCR revealed distinct expression patterns of individual CHIR. All receptor types shared a conserved genomic architecture, and in single Ig domain receptors a pseudoexon replaced the second Ig exon. Southern blot analyses with probes specific for the Ig1 domain were indicative of a large multigene family. Of 103 sequences from the Ig1 domain of a single animal, 41 unique sequences were obtained that displayed extensive variability within restricted Ig regions. Fluorescence in situ hybridization localized the CHIR gene cluster to microchromosome 31 and identified this region as orthologous to the human leukocyte receptor complex.     

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains.

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.     

Five genes encoding surface-exposed LPXTG proteins are enriched in hospital-adapted Enterococcus faecium clonal complex 17 isolates

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.     

Recent clinical isolates of cytomegalovirus suppress human cytomegalovirus-specific human leukocyte antigen-restricted cytotoxic T-lymphocyte activity.

Peripheral blood mononuclear cells harvested from healthy adults seropositive for human cytomegalovirus (HCMV) and cultured with laboratory strain AD-169 demonstrated human leukocyte antigen-restricted and HCMV-specific killing on target cells infected with either HCMV laboratory strain AD-169 or recent low-passage HCMV isolates. These results indicated that the determinants recognized by cytotoxic T lymphocytes (CTLs) are shared among different strains of HCMV. However, when low-passage isolates, rather than high-passage AD-169 virions, were used to stimulate CTL activity, the lytic response was significantly lower against all targets. Mixing of AD-169 and low-passage HCMV isolates induced low CTL activity. Collectively, the findings suggest that low-passage HCMV isolates have dual effects--antigenic stimulation and immunosuppression--whereas laboratory strain AD-169 is primarily immunogenic. The study of several recent isolates indicated that they varied in their ratio of immunostimulation to suppression, that infectious virus was necessary to produce suppression, and that suppressive isolates did not have to be present at the initiation of culture to exert their suppressive effects.     

Alternative splice variants encoding unstable protein domains exist in the human brain

Alternative splicing has been recognized as a major mechanism by which protein diversity is increased without significantly increasing genome size in animals and has crucial medical implications, as many alternative splice variants are known to cause diseases. Despite the importance of knowing what structural changes alternative splicing introduces to the encoded proteins for the consideration of its significance, the problem has not been adequately explored. Therefore, we systematically examined the structures of the proteins encoded by the alternative splice variants in the HUGE protein database derived from long (>4 kb) human brain cDNAs. Limiting our analyses to reliable alternative splice junctions, we found alternative splice junctions to have a slight tendency to avoid the interior of SCOP domains and a strong statistically significant tendency to coincide with SCOP domain boundaries. These findings reflect the occurrence of some alternative splicing events that utilize protein structural units as a cassette. However, 50 cases were identified in which SCOP domains are disrupted in the middle by alternative splicing. In six of the cases, insertions are introduced at the molecular surface, presumably affecting protein functions, while in 11 of the cases alternatively spliced variants were found to encode pairs of stable and unstable proteins. The mRNAs encoding such unstable proteins are much less abundant than those encoding stable proteins and tend not to have corresponding mRNAs in non-primate species. We propose that most unstable proteins encoded by alternative splice variants lack normal functions and are an evolutionary dead-end.     

Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.     

Sequence analysis of human cytomegalovirus US28 gene in low-passage clinical isolates from children and AIDS patients

Human cytomegalovirus (HCMV) is often a dangerous opportunistic pathogen that causes significant morbidity and mortality in newborn children and immunocompromised patients. The different symptoms and tissue tropisms of HCMV infection may result from genetic polymorphism. This study investigated the sequence variability of the HCMV US28 ORF, which shows sequence homology to the G protein-coupled receptor. HCMV isolated from suspected pediatric cases and isolates from AIDS patients were compared in order to examine the possible associations between polymorphisms and pathogenesis. Seventy children with suspected congenital HCMV infection, who suffered from jaundice (47), megacolon (10), and microcephaly (13), and 17 AIDS patients, were studied. Mutation was prevalent among the sequences of US28, with a focus on the two ends of US28. The important functional groups of US28 are highly conserved. An unrooted tree showed that all sequences from suspected congenitally infected infants and AIDS patients were divided into three groups. Comparison showed that most of the sequences (12/17) from pediatric patients were included in the first group (G1), whereas most of the sequences (11/17) from AIDS patients were included in the third group (G3). The specific high mutation sites in US28 from children were located at the C terminus of the protein, whereas those from AIDS patients were located at the N terminus. We demonstrated the existence of polymorphisms among the US28 genes of clinical isolates of HCMV from infants with suspected congenital infection. Comparison of US28 sequences from AIDS patients with those from children showed that both sequences have their own specific high mutation points.     

Mapping genes of complex diseases in genetic isolates of Dagestan

Original results of the analysis of genetic linkage between some genomic markers and two complex clinical phenotypes, schizophrenia and mental retardation, in pedigrees from Dagestan genetic isolates are described. Interpopulation differences in the epidemiology of the complex phenotypes were studied and in their genetic linkage was demonstrated. These differences are evidently related to the genetic structure of the isolates determined by their genetic history. The MR epidemiological index characterizing the lifetime morbid risk of schizophrenia varies in the Dagestan isolates studied from 0 to 4.95%, which is almost five times higher than the average worldwide population rate, 1%. Comparative genetic mapping permitted determination of the most probable genetic linkages and associations of loci from chromosomal regions 17p11.1-12, 3q13.3, and a locus from 22q with schizophrenia and locus 12q23 with mental retardation. There is evidence that this approach is effective for detailed study of the relationship between the genetic (allele and locus) and clinical heterogeneity of complex diseases, which favors successful identification of the genes determining them. The study of linkage disequilibrium (LD) in genetic isolates of Daghestan populations (which have a common genetic background) may be an effective methodological approach for revealing the numerous contradictory results of mapping of the same genes of complex disease performed by different researchers in different regions of the world.     

Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene

Among the mitochondrial disorders, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, in the majority of the complex I-deficient patients mutations of nuclear genes are expected. This review attempts to summarize genetic defects affecting nuclear encoded subunits of complex I reported to date focusing on those found in the NDUFS4 gene. NDUFS4 product is 18 kDa protein which appears to have a dual role in complex I, at least: cAMP-dependent phosphorylation activates the complex; non-sense mutation of NDUFS4 prevents normal assembly of a functional complex in the inner mitochondrial membrane.     

Identity and conservation of mating type genes in geographically diverse isolates of Phaeosphaeria nodorum

Mating type idiomorphs (MAT1-1 and MAT1-2) were identified from the heterothallic loculoascomycete Phaeosphaeria nodorum (wheat biotype) using DNA from a pair of isolates from Poland and Georgia, USA that are known to mate. MAT predicted proteins of P. nodorum are similar in sequence and in phylogenetic relationship to those described for other loculoascomycetes such as Cochliobolus spp., Alternaria alternata, and Didymella zeae-maydis. The organization of the MAT locus of the P. nodorum differs from these species in that its idiomorph begins within an adjacent upstream conserved ORF of unknown function. MAT-specific primers were used to identify isolates of both mating types in field populations, demonstrating that an absence of either mating type is not the reason that the teleomorph has not been found in New York. Portions of MAT1-1 and MAT1-2 were sequenced from geographically diverse isolates, including those from regions where the teleomorph has been reported. MAT was highly conserved and no significant differences in sequence were found.     

The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type

Clostridium difficile causes diarrhoeal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis. Endospore production plays a vital role in transmission of infection, and in order to cause disease these spores must then germinate and return to vegetative cell growth. Type BI/NAP1/027 strains of C. difficile have recently become highly represented among clinical isolates and are associated with increased disease severity. It has also been suggested that these 'epidemic' types generally sporulate more prolifically than 'non-epidemic' strains, although the few existing reports are inconclusive and encompass only a small number of isolates. In order to better understand any differences in sporulation rates between epidemic and non-epidemic C. difficile types, we analysed these characteristics using 14 C. difficile clinical isolates of a variety of types. Sporulation rates varied greatly between individual BI/NAP1/027 isolates, but this variation did not appear to be type-associated. Furthermore, a number of BI/NAP1/027 spores appeared to form colonies with a lower frequency than specific non-BI/NAP1/027 strains. The data suggest that (i) careful experimental design is required in order to accurately quantify sporulation; and (ii) current evidence cannot link differences in sporulation rates with the disease severity of the BI/NAP1/027 type.     

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.     

Exons encoding the highly conserved part of human glutaminyl-tRNA synthetase

Summary Aminoacyl-tRNA synthetases are important components of the genetic apparatus. In spite of common catalytic properties, synthetases with different amino acid specificities are widely diverse in their primary structures, subunit sizes, and subunit composition. However, synthetases with given amino acid specificities are well conserved throughout evolution. We have been studying the human glutaminyl-tRNA synthetase possessing a sequence of about 400 amino acid residues (the core region) that is very similar to sequences in the corresponding enzymes from bacteria and yeast. The conserved sequence appears to be essential for the basic function of the enzyme, the charging of tRNA with glutamine. As a first step to a better understanding of the evolution of this enzyme, we determined the coding region for the conserved part of the human glutaminyl-tRNA synthetase. The coding region is composed of eight exons. It appears that individual exons encode defined secondary structural elements as parts of functionally important domains of the enzyme. Evolution of the gene by assembly of individual exons seems to be a viable hypothesis; alternative pathways are discussed. Offprint requests to: R. Knippers