The complete nucleotide sequence of cell fusing agent (CFA): homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses.

Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.     

Use of insertion sequence element IS1126 in a genotyping and transmission study of Porphyromonas gingivalis

Porphyromonas gingivalis is strongly associated with periodontal diseases and is regarded as one of the risk factors for periodontitis. Insertion sequence element IS1126-based PCR was used to investigate the genetic heterogeneity of P. gingivalis from periodontitis patients and to examine the frequency of the parent-child and spouse-spouse transmission. Two sets of IS1126-specific primers were used for the PCR. The inward primer set (PI1 and PI2), which amplifies the IS1126 fragment of approximately 690 bp, was used to identify P. gingivalis. The outward primer set (PI1RC and PI2RC), which is reverse complementary to PI1 and PI2, respectively, and amplifies the gene fragments between the adjacent IS1126 elements was used to characterize the genotypes of the P. gingivalis strains. PCR of P. gingivalis with PI1RC and PI2RC resulted in the production of two to seven amplicons, which showed a unique electrophoretic pattern in each strain (4 laboratory strains and 37 clinical isolates cultured from 12 patients with aggressive periodontitis). The usefulness of the method for transmission study was confirmed by detecting identical genotypes between the isolates and the plaque samples from which the isolates were cultured and between the plaque samples from different tooth sites in the same patient. Thirty probands with periodontal diseases and their thirty immediate family members were included in the transmission study. In 11 of 14 parent-child pairs (78.6%), P. gingivalis revealed an identical or similar band pattern, whereas 5 of 16 spouse pairs (31.25%) had this similarity. These results show that IS1126-based PCR for genotyping P. gingivalis has a highly discriminating potential with reproducible data and is a simple and reliable method for a transmission study.     

Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis

An insertion sequence-like element, IS6110, was isolated from a Mycobacterium tuberculosis cosmid library as a repetitive sequence. IS6110 shows similarities with elements of the IS3 family. This insertion sequence was found to be specific to mycobacteria belonging to the M. tuberculosis complex. For detection and identification of M. tuberculosis bacilli in uncultured specimens, oligonucleotides derived from the IS6110 sequence were used as primers and probes in polymerase chain reaction studies. The results obtained were consistent with results of classical identification procedures, bacteriological data, and clinical criteria.     

Biologically distinct subtypes of Mycobacterium avium differ in possession of insertion sequence IS901.

Mycobacterium avium causes disease, principally tuberculosis in immunocompromised individuals. It is the most frequent cause of disseminated infections in AIDS patients in the West. The pathogen is also associated with disease in animals, chiefly birds and livestock, and may be isolated from environmental samples such as soil and water. Analysis of strains of M. avium isolated from clinical, veterinary, and environmental sources for the presence of the mycobacterial insertion sequences IS900 and IS901 demonstrates the specific association of IS901 to animal pathogenic M. avium strains. In contrast, most clinical M. avium strains and all AIDS-derived strains examined so far lacked IS901. Significant differences in the plasmid contents and serotypes of strains with and without IS901 were also found. We therefore suggest that the presence of IS901 divides M. avium into two clearly distinct subtypes with differing host range, virulence, plasmid possession, and serotyping antigens. By using DNA sequence data from IS901 and M. avium DNA, a set of polymerase chain reactions were developed for the specific detection and differentiation of these subtypes.     

Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins

The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined.     

Inhibition of cellular protein secretion by picornaviral 3A proteins

During poliovirus infection, anterograde traffic between the endoplasmic reticulum and the Golgi is inhibited due to the action of 3A, an 87 amino acid viral protein. The ability of poliovirus protein 3A to inhibit ER-to-Golgi traffic is not required for virus growth. Instead, we have suggested that the inhibition of host protein secretion, shown to reduce the secretion of interferon-beta, IL-6, and IL-8 and the expression of both newly synthesized MHC class I and TNF receptor in the plasma membrane of infected cells, affects growth in host organisms. To determine whether the ability of poliovirus 3A to inhibit ER-to-Golgi traffic is conserved, the ability of 3A proteins from several picornaviruses, including human rhinovirus 14, foot-and-mouth disease virus, enterovirus 71, hepatitis A, and Theiler's virus, was tested. Only the 3A proteins from another poliovirus, Sabin 3, and closely related coxsackievirus B3 inhibited ER-to-Golgi traffic as effectively as the 3A protein from poliovirus Mahoney type 1. Site-directed mutagenesis based on these findings and the three-dimensional structure of the amino-terminal domain of poliovirus 3A protein revealed that residues in the unstructured amino terminus of 3A are critical for the inhibition of host protein secretion.     

Immunodetection and subcellular localization of the proteins encoded by ORF 3 of grapevine viruses A and B

Summary.  Polyclonal antisera were raised to Escherichia coli-expressed ORF3 products (putative movement proteins) of Grapevine virus A (GVA) and Grapevine virus B (GVB) (genus Vitivirus), and used for their immunodetection in infected plants. Western blot analysis of subfractionated cellular compartments showed that the distribution of both proteins was comparable to that of plant virus movement proteins, as they were transiently present in a crude membrane fraction and accumulated in a cell wall-enriched fraction. The GVA ORF3-encoded protein, but not the comparable GVB protein, was also present in large amount in a cytoplasmic soluble fraction. Intracellular immunogold labelling localized these proteins in the cell wall and plasmodesmata of infected cells and, especially for GVA, in association with cytoplasmic virus aggregates. Received February 9, 2000 Accepted March 23, 2000     

DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110.

Strains of Mycobacterium bovis, the causative organism of bovine tuberculosis, can be clearly distinguished from each other by restriction fragment analysis. This method of DNA fingerprinting has been used for many epidemiological studies in New Zealand, but the technique presents practical difficulties that hinder its widespread use. The insertion element IS6110 is being widely used as a DNA probe for distinguishing restriction fragment polymorphisms among strains of Mycobacterium tuberculosis. Both this element and another recently sequenced element, IS1081, are also present in M. bovis. We assessed the usefulness of these two elements for distinguishing between 160 strains of M. bovis. These strains, most of which were isolated in New Zealand, were selected to be representative of the 95 different types that were identified among 530 strains that were previously typed by restriction fragment analysis. Fifteen IS6110 types were identified, but more than half of the strains representing 46 restriction types had the same IS6110 type. Virtually all M. bovis strains as well as strains of M. tuberculosis and Mycobacterium africanum had the same IS1081 type. The results indicate that for M. bovis, IS1081 cannot be used to type strains, IS6110 can be used to distinguish strains into broad groups, but only restriction fragment analysis is sufficiently sensitive for detailed epidemiological studies. An investigation of the host range of IS1081 revealed that, apart from its presence in species of the tuberculosis complex, it is also present in a strain of Mycobacterium xenopi.     

Inhibition of mouse TAP by immune evasion molecules encoded by non-murine herpesviruses

Herpesviruses escape elimination by cytotoxic T lymphocytes through specific interference with the antigen-presenting function of MHC class I (MHC I) molecules. The transporter associated with antigen processing (TAP) forms a bottleneck in the MHC I antigen presentation pathway. The fact that multiple viruses, especially herpesviruses, encode molecules blocking TAP function is a case in point. The action of these viral immuno evasins is usually potent and very specific, making these proteins valuable tools for studying the cell biology of antigen presentation, including alternative antigen processing pathways. Yet, no dedicated TAP inhibitor has been described for any of the mouse herpesviruses. To permit the use of immuno evasins derived from non-mouse herpesviruses in mouse models, we assessed the cross-species activity of four TAP inhibitors and one tapasin inhibitor in the context of three different mouse haplotypes, H-2(b), H-2(d), and H-2(k). Two of the four TAP inhibitors, the bovine herpesvirus 1-encoded UL49.5 and the human cytomegalovirus (HCMV)-encoded US6 protein, potently inhibited mouse TAP. ICP47 and BNLF2a, encoded by herpes simplexvirus 1 and Epstein-Barr virus, respectively, failed to inhibit TAP in all mouse cells tested. Previous work, however, demonstrated that US6 did not cross the mouse species barrier. We now show that substitution of the cysteine residue at position 108 was responsible for this lack of activity. The HCMV-encoded tapasin inhibitor US3 efficiently downregulated H-2(d) molecules on 3T3 cells, but not in other cell lines tested. Finally, we show that synthetic peptides comprising the functional domain of US6 can be exploited as a versatile TAP inhibitor. In conclusion, a complete overview is presented of the applicability of herpesvirus-encoded TAP and tapasin inhibitors in mouse cells of different genetic background.     

Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.     

Identification of IS1356, a new insertion sequence, and its association with IS402 in epidemic strains of Burkholderia cepacia infecting cystic fibrosis patients.

Burkholderia cepacia is now recognized as an important opportunistic pathogen in cystic fibrosis (CF) and other compromised patients. Epidemicity among CF patients has been attributed to at least one particularly infectious strain (strain ET12), and both genetic evidence and anecdotal evidence suggest that this strain, currently endemic in Ontario, and those causing an epidemic in the United Kingdom, are indeed the same. Our study was conducted to determine whether there was any association between the presence of various insertion sequence (IS) elements, the cable pilin subunit gene (cblA), electrophoretic type (ET), and ribotype (RT) in a collection of 97 clinical and 2 environmental isolates of B. cepacia. No apparent linkage was found for IS elements IS401, IS402, IS406, IS407, and IS408 with ET or RT. The cblA target, said to be a marker for high infectivity, was detected in 100% (38 of 38) of strains of B. cepacia ET12 and in a single strain of ET13 that differed in a single enzyme allele. A new IS, IS1356, identified during the investigation, was present in 71.7% of all isolates, and 50.7% of these isolates harbored IS1356 as a hybrid IS element inserted into IS402. IS1356 is 1,353 bp in length, and when it is inserted into IS402 it results in a 10-bp duplication at the site of insertion. IS1356 contains one major open reading frame of 1,260 bp coding for a putative transposase which has significant homology to ISRm3 in Rhizobium meliloti (59%) and to an undesignated IS element in Corynebacterium diphtheriae (49%). The IS402-IS1356 element was found exclusively in the epidemic strains from Ontario and the United Kingdom, being detected in 94.7% (36 of 38 isolates) of B. cepacia ET12 isolates. Of the two ET12 isolates found to be devoid of the IS402-IS1356 element, both contained IS1356 unassociated with IS402, one was temporally unrelated to the epidemic, and the other was from a CF patient in a geographic area remote from Ontario and the United Kingdom. It is evident that the IS402-IS1356 hybrid element, the cblA pilin subunit gene, and the allelic suite represented by multilocus enzyme electrophoretic type ET12 may provide useful markers for the epidemic, highly transmissible transatlantic strain isolated in Ontario and the United Kingdom.     

Silent information regulator 2 proteins encoded by Cryptosporidium parasites

Screening in a database has revealed that Cryptosporidium hominis encodes a silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase. Cellular localization of the protein, ChSir2, was analyzed by the use of the social amoeba Dictyostelium discoideum as a model system. Fluorescent microscopic analysis showed that ChSir2 fused with green fluorescent protein was localized in the D. discoideum nucleus. D. discoideum expressing ChSir2 grew faster and reached higher cell density than did D. discoideum harboring a control vector. These results suggest that ChSir2 is a nucleus-localizing protein that plays an important role in the growth of C. hominis. We cloned and sequenced the genes for Sir2 orthologs encoded by three isolates of C. hominis, two isolates of Cryptosporidium parvum and one isolate of Cryptosporidium meleagridis. The orthologs conserve critical catalytic or NAD-binding residues but do not have similarity with human Sir2 proteins (SIRTs). Cryptosporidium Sir2 orthologs would therefore be attractive therapeutic targets. The Cryptosporidium orthologs were classified into four variants based on their nucleotide sequences. Each of the four variants produces its own unique restriction fragment length polymorphism pattern by digestion with TfiI.     

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.     

Structural insight into the membrane insertion of tail‐anchored proteins by Get3

Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C‐terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP‐bound and nucleotide‐free forms at 3.0 Å and 2.8 Å resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn²⁺‐mediated homodimer in a head‐to‐head orientation, representing an open dimer conformation. Our cross‐link experiments indicated the closed dimer‐stimulating ATP hydrolysis, which might be coupled with TA‐protein release. Further, our coexpression‐based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA‐protein insertion mediated by the conserved DTAPTGH motif.     

Dramatically increased rearrangement and peripheral representation of Vbeta14 driven by the 3'Dbeta1 recombination signal sequence

V(D)J recombination is targeted by short recombination signal (RS) sequences that are relatively conserved but exhibit natural sequence variations. To evaluate the potential of RS sequence variations to determine the primary and peripheral TCRbeta repertoire, we generated mice containing specific replacement of the endogenous Vbeta14 RS with the 3'Dbeta1 RS (Vbeta14/3'DbetaRS). These mice exhibited a dramatic increase in Vbeta14(+) thymocyte numbers at the expense of thymocytes expressing other Vbetas. In addition, the percentage of peripheral Vbeta14(+) alphabeta T lymphocytes was similarly increased. Strikingly, this altered Vbeta repertoire resulted predominantly from a higher relative level of primary Vbeta14/3'DbetaRS rearrangement to DbetaJbeta complexes, despite the ability of the 3'Dbeta1 RS to break B12/23 restriction and allow direct rearrangement of Vbeta14/3'DbetaRS to Jbeta segments.     

Comparative analysis of the Epstein-Barr virus encoded nuclear proteins of EBNA-3 family

It is known that the EBNA-3 family proteins (EBNA-3, -4 and -6, alternative nomenclature EBNA-3A, B and C correspondingly) show a limited sequence similarity.We have analyzed EBNA-3 proteins both at the primary sequence and secondary structure levels. EBNA-3 and EBNA-4 were structurally more similar compared to other combinations with EBNA-6. We found “Stonin Homology Domain” profile in EBNA-4 and “Proline Rich Domain” in all EBNA-3 family of proteins. We have also found positive and negative charge clusters in all three proteins and mixed charge clusters in EBNA-3. Charged clusters are believed to play an important role in interactions with DNA or signaling proteins. Additionally, unique primary sequence repeats were found in all three proteins.     

Development of a rapid PCR method using the insertion sequence IS1203 for genotyping Shiga toxin-producing Escherichia coli O157

We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.