Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles

Coronavirus replication is facilitated by a number of highly conserved viral proteins. The viruses also encode accessory genes, which are virus group specific and believed to play roles in virus replication and pathogenesis in vivo. Of the eight putative accessory proteins encoded by the severe acute respiratory distress syndrome associated coronavirus (SARS-CoV), only two-open reading frame 3a (ORF3a) and ORF7a-have been identified in virus-infected cells to date. The ORF7b protein is a putative viral accessory protein encoded on subgenomic (sg) RNA 7. The ORF7b initiation codon overlaps the ORF7a stop codon in a -1 shifted ORF. We demonstrate that the ORF7b protein is expressed in virus-infected cell lysates and from a cDNA encoding the gene 7 coding region, indicating that the sgRNA7 is bicistronic. The translation of ORF7b appears to be mediated by ribosome leaky scanning, and the protein has biochemical properties consistent with that of an integral membrane protein. ORF7b localizes to the Golgi compartment and is incorporated into SARS-CoV particles. We therefore conclude that the ORF7b protein is not only an accessory protein but a structural component of the SARS-CoV virion.     

The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments.

Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.     

Characterization of the I-Spom I endonuclease from fission yeast: insights into the evolution of a group I intron-encoded homing endonuclease

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli. Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo, the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro. Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes.     

A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA

Dihydrouridine modification of tRNA is widely observed in prokaryotes and eukaryotes, as well as in some archaea. In Saccharomyces cerevisiae every sequenced tRNA has at least one such modification, and all but one have two or more. We have used a biochemical genomics approach to identify the gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w), using yeast pre-tRNA(Phe) as a substrate. Dus1 is a member of a widespread family of conserved proteins, three other members of which are found in yeast: YNR015w, YLR405w, and YLR401c. We show that one of these proteins, Dus2, encoded by ORF YNR015w, has activity with two other substrates: yeast pre-tRNA(Tyr) and pre-tRNA(Leu). Both Dus1 and Dus2 are active as a single subunit protein expressed and purified from Escherichia coli, and the activity of both is stimulated in the presence of flavin adenine dinucleotide. Dus1 modifies yeast pre-tRNA(Phe) in vitro at U17, one of the two positions that are known to bear this modification in vivo. Yeast extract from a dus1-A strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-delta and dus2-delta strains is significantly depleted in dihydrouridine content.     

Two alternative translation mechanisms are responsible for the expression of the HCV ARFP/F/core+1 coding open reading frame

HCV-1 produces a novel protein, known as ARFP, F, or core+1. This protein is encoded by an open reading frame (ORF) that overlaps the core gene in the +1 frame (core+1 ORF). In vitro this protein is produced by a ribosomal frameshift mechanism. However, similar studies failed to detect the ARFP/F/core+1 protein in the HCV-1a (H) isolate. To clarify this issue and to elucidate the functions of this protein, we examined the expression of the core+1 ORF by the HCV-1 and HCV-1a (H) isolates in vivo, in transfected cells. For this purpose, we carried out luciferase (LUC) tagging experiments combined with site-directed mutagenesis studies. Our results showed that the core+1-LUC chimeric protein was efficiently produced in vivo by both isolates. More importantly, neither changes in the specific 10-A residue region of HCV-1 (codons 8-11), the proposed frameshift site for the production of the ARFP/F/core+1 protein in vitro, nor the alteration of the ATG start site of the HCV polyprotein to a stop codon significantly affected the in vivo expression of the core+1 ORF. Furthermore, we showed that efficient translation initiation of the core+1 ORF is mediated by internal initiation codon(s) within the core/core+1-coding sequence, located between nucleotides 583 and 606. Collectively, our data suggest the existence of an alternative translation initiation mechanism that may result in the synthesis of a shorter form of the core+1 protein in transfected cells.     

First open reading frame protein (ORF1p) of the Blattella germanica R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains

The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was islated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established by us for R1 ORF1p of B. germanica.     

GST pull-down结合质谱筛选猪圆环病毒2型ORF4潜在互作蛋白

猪圆环病毒2型编码的ORF4蛋白是近年来发现的新蛋白。迄今为止,人们对ORF4所参与的细胞生物学过程知之甚少。本研究首先构建了带双标签的真核表达载体pCMV-N-Flag-GST,再将ORF4基因插入该载体中,形成pCMV-N-Flag-GST-ORF4。将质粒转染293T细胞表达ORF4后,通过GSTPull-down试验捕获细胞内潜在与ORF4互作的蛋白库。经SDS-PAGE分离及银染后,对所得的特异性条带进行质谱鉴定,筛选出5个与ORF4潜在互作的蛋白,包括丝氨酸/苏氨酸蛋白磷酸酶6催化亚基、α心肌蛋白、β肌动蛋白、SEC-14样蛋白5和肌球蛋白myosin 9。上述研究结果为深入揭示ORF4在病毒感染细胞过程中发挥的作用提供新的思路与方向。     

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides.

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.     

Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitro translation studies.

The 7.5-kb plus-stranded genomic RNA of rabbit hemorrhagic disease virus contains two open reading frames of 7 kb (ORF1) and 351 nucleotides (ORF2) that cover nearly 99% of the genome. The aim of the present study was to identify the proteins encoded in these open reading frames. To this end, a panel of region-specific antisera was generated by immunization of rabbits with bacterially expressed fusion proteins that encompass in total 95% of the ORF1 polyprotein and almost the complete ORF2 polypeptide. The antisera were used to analyze the in vitro translation products of purified virion RNA of rabbit hemorrhagic disease virus. Our studies show that the N-terminal half of the ORF1 polyprotein is proteolytically cleaved to yield three nonstructural proteins of 16, 23, and 37 kDa (p16, p23, and p37, respectively). In addition, a cleavage product of 41 kDa which is composed of VPg and a putative nonstructural protein of approximately 30 kDa was identified. Together with the results of previous studies which identified a trypsin-like cysteine protease (TCP) of 15 kDa, a putative RNA polymerase (pol) of 58 kDa, and the major capsid protein VP60, our data establish the following gene order in ORF1: NH2-p16-p23-p37 (helicase)-p30-VPg-TCP-pol-VP60-COOH. Immunoblot analyses showed that a minor structural protein of 10 kDa is encoded in ORF2. The data provide the first complete genetic map of a calicivirus. The map reveals a remarkable similarity between caliciviruses and picornaviruses with regard to the number and order of the genes that encode the nonstructural proteins.     

First open reading frame protein (ORF1p) of the <Emphasis Type="Italic">Blattella germanica</Emphasis> R1 retroposon and phylogenetically close GAG-like proteins of insects and fungi contain RRM domains

The rDNA locus of insects and other arthropods contains non-LTR retrotransposons (retroposons) that are specifically inserted into 28S rRNA genes. The most frequent retroposons are R1 and R2, but the mechanism of insertion and the functions of these mobile elements have not been studied in detail. A clone containing a full-length R1 retroposon copy was isolated from the cosmid library of Blattella germanica genes and sequenced. The amino acid sequences encoded by ORF1 of the R1 retroposon were subjected to bioinformatic analysis. It was found that ORF1 of this mobile element encodes a protein (ORF1p) belonging to the superfamily of zinc finger (CCHC) retroviral nucleocapsid proteins and contains two conserved RRM domains (RNA-recognizing motifs) identified on the basis of analysis of the secondary structure of this protein. The discovery of RRM domains in ORF1p of R1 retroposons can contribute to the understanding of the mechanisms of their retrotransposition. We revealed a coiled-coil motif in the N-terminal region of R1 ORF1p, which is similar to the coiled-coil domain involved in homo- or heteromultimerization of proteins and in protein-protein interactions. The domain organization of homologous Gag-like proteins of retroposons in some insects and fungi was found to be similar to the structure established for R1 ORF1p of B. germanica.     

Identification,cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.