Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as,well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Transcriptome analysis of Taenia solium cysticerci using Open Reading Frame ESTs (ORESTES)

Background: Human infection by the pork tapeworm Taenia solium affects more than 50 million people worldwide, particularly in underdeveloped and developing countries. Cysticercosis which arises from larval encystation can be life threatening and difficult to treat. Here, we investigate for the first time the transcriptome of the clinically relevant cysticerci larval form. Results: Using Expressed Sequence Tags (ESTs) produced by the ORESTES method, a total of 1,520 high quality ESTs were generated from 20 ORESTES cDNA mini-libraries and its analysis revealed fragments of genes with promising applications including 51 ESTs matching antigens previously described in other species, as well as 113 sequences representing proteins with potential extracellular localization, with obvious applications for immune-diagnosis or vaccine development. Conclusion: The set of sequences described here will contribute to deciphering the expression profile of this important parasite and will be informative for the genome assembly and annotation, as well as for studies of intra- and inter-specific sequence variability. Genes of interest for developing new diagnostic and therapeutic tools are described and discussed.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

Abstract Background The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. Results Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. Conclusions The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Investigation of a unique short open reading frame within the 3' untranslated region of the canine distemper virus matrix messenger RNA

Increasing evidence suggest that the long "untranslated" region (UTR) between the matrix (M) and the fusion (F) proteins of morbilliviruses has a functional role. In canine distemper virus (CDV), the F 5' UTR was recently shown to code for a long F signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with the long Fsp were synergistically regulating the F mRNA and protein expression, thereby modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has been identified within the wild-type CDV-M 3' UTR (termed M2). Here, we investigated whether M2 was expressed from the genome of the virulent and demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-RFP was successfully recovered from cDNA (termed recA75/17(green)-HA-M2-RFP). M2 expression in cells transfected or infected with these mutants was studied by immunoprecipitation, immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence emission in several recA75/17(green)-HA-M2-RFP-infected cell types suggested lack of M2 biosynthesis, which was confirmed by the other techniques. Consistent with these data, no functional role of the short polypeptide was revealed by infecting various cell types with HA-M2-RFP over-expressing or M2-knockout recombinant viruses. Thus, in sharp contrast to the CDV-F 5' UTR reported to translate a long Fsp, our data provided evidence that the CDV-M 3' UTR does not express any polypeptides.

The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.

OBJECTIVES This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). BACKGROUND Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. METHODS Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 +/- 15 years, mean QTc 428 +/- 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). RESULTS Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. CONCLUSIONS Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that approximately 65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.

Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons.

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Hydrophobic cluster analysis predicts an amino-terminal domain of varicella-zoster virus open reading frame 10 required for transcriptional activation.

Varicella-zoster virus open reading frame 10 (ORF10) protein, the homolog of the herpes simplex virus protein VP16, can transactivate immediate-early promoters from both viruses. A protein sequence comparison procedure termed hydrophobic cluster analysis was used to identify a motif centered at Phe-28, near the amino terminus of ORF10, that strongly resembles the sequence of the activating domain surrounding Phe-442 of VP16. With a series of GAL4-ORF10 fusion proteins, we mapped the ORF10 transcriptional-activation domain to the amino-terminal region (aa 5-79). Extensive mutagenesis of Phe-28 in GAL4-ORF10 fusion proteins demonstrated the importance of an aromatic or bulky hydrophobic amino acid at this position, as shown previously for Phe-442 of VP16. Transactivation by the native ORF10 protein was abolished when Phe-28 was replaced by Ala. Similar amino-terminal domains were identified in the VP16 homologs of other alphaherpesviruses. Hydrophobic cluster analysis correctly predicted activation domains of ORF10 and VP16 that share critical characteristics of a distinctive subclass of acidic activation domains.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.

Distributional profiles of homologous open reading frames among bacterial phyla: implications for vertical and lateral transmission

If open reading frames (ORFs) have been transmitted primarily by vertical descent, the distributional profile of orthologues of each ORF should be congruent with the organismal tree or a subtree thereof. Distributional patterns not reconciled parsimoniously with tree-like descent and loss are prima facie evidence of lateral gene transfer. Herein, a rigorous criterion for recognizing ORF distributions is described and implemented; it does not require the inference of phylogenetic trees, nor does it assume any specific tree. Because lineage-specific differences in rates of sequence change can also generate unexpected distributional patterns, rate artefacts, were controlled for by requiring pairwise matches between ORFs to exceed a rigorous inclusion threshold, but absence of a match was assessed against a more-permissive exclusion threshold. Applying this dual-threshold criterion to cross-domain and cross-phylum distributional patterns for ORFs in 23 bacterial genomes, a relative abundance of ORFs was observed that find a match in exactly seven other bacterial phyla; 94-99% of these ORFs also find matches among the Archaea and/or Eukarya. In the larger (and some smaller) bacterial genomes, ORFs that find matches in exactly one other bacterial phylum are also relatively abundant, but fewer of these have non-bacterial homologues; most of their matches within the Bacteria are to the Proteobacteria and/or Firmicutes, which cannot be sister lineages to all bacteria. ORFs that are neither distributed universally among the Bacteria, nor necessarily shared with topologically adjacent lineages, are preferentially enriched in large bacterial genomes.

Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the b-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.