Chien-Kou Shieh - Academia.edu (original) (raw)

Papers by Chien-Kou Shieh

Research paper thumbnail of Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein

Journal of Virology, 1989

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-de... more The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but ...

Research paper thumbnail of Identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus RNA genome

Journal of Virology, 1989

We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) ... more We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) synthesize an additional mRNA species (mRNA 2b, previously called mRNA 2a) with a size intermediate between that of mRNAs 2 and 3, suggesting the presence of an optional transcriptional initiation site. This transcriptional start is dependent on the leader sequence of the virus strains. To study the mechanism of coronavirus transcriptional regulation, we have cloned and sequenced the region of the viral genome corresponding to the 5' unique coding region of mRNA 2 of the JHM strain of MHV. In addition to the open reading frame (ORF) predicted to encode the viral nonstructural protein p30, a second complete ORF, with the potential to encode a 439-amino-acid polypeptide, was discovered. The transcriptional initiation sites of both mRNA 2a (formerly called mRNA 2) and mRNA 2b were determined by primer extension studies and RNA sequencing. The data indicated that transcription of mRNA 2a...

Research paper thumbnail of Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs

Journal of Virology, 1988

Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free l... more Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free leader RNA species. As a result, each virus-specific mRNA contains an identical leader RNA derived from the 5', end of the genomic RNA. In this study, we demonstrate by primer extension studies that the leader-fusion sites on a given species of coronavirus subgenomic mRNA are heterogeneous. The heterogeneity was due to variation in the number of pentanucleotide (UCUAA) repeats present at the leader fusion site. This pentanucleotide repeat region was complementary between the free leader RNA and the transcription start sites on the template RNA. This result suggests that the discontinuous transcription of coronavirus mRNAs occurs within the complementary sequences localized in two different RNA segments and that RNA joining occurs at variable sites.

Research paper thumbnail of Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase

Journal of Virology, 1987

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of t... more A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently t...

Research paper thumbnail of Coronavirus: A Jumping RNA Transcription

Cold Spring Harbor Symposia on Quantitative Biology, 1987

DNA-and RNA-dependent RNA polymerases usually recognize specific promoter sequences and initiate ... more DNA-and RNA-dependent RNA polymerases usually recognize specific promoter sequences and initiate RNA transcription at some distances downstream from the promoter. Initiation of transcription requires only complementary nucleoside triphosphates, although ...

Research paper thumbnail of Leader RNA-primed transcription and RNA recombination of murine coronaviruses

In: Positive-strand RNA, 1987

Research paper thumbnail of Molecular studies of coronavirus RNA

Research paper thumbnail of Sequence andTranslation oftheMurineCoronavirus 5'-End GenomicRNA Reveals theN-Terminal Structure ofthePutative RNA Polymerase

Research paper thumbnail of Murine coronavirus 5'-end genomic RNA sequence reveals mechanism of leader-primed transcription

Advances in experimental medicine and biology, 1987

Research paper thumbnail of Studies into the mechanism of MHV transcription

Advances in experimental medicine and biology, 1987

Research paper thumbnail of Murine Coronavirus Gene 1 Polyprotein Contains an Autoproteolytic Activity

Advances in Experimental Medicine and Biology, 1990

Research paper thumbnail of Primary structure and translation of a defective interfering rna of murine coronavirus

Virology, 1988

An intracellular defective-interfering (DI) RNA, DlssE, of mouse hepatitis virus (MHV) obtained a... more An intracellular defective-interfering (DI) RNA, DlssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DlssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5' end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the S'end of the parental MHV genome. The DlssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DIinfected cells. Sequence comparison of DlssE and the corresponding parts of the parental virus genome revealed that DlssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5' end of DlssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.

Research paper thumbnail of The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase

Research paper thumbnail of Defective-interfering particles of murine coronavirus: Mechanism of synthesis of defective viral RNAs

Virology, 1988

The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interferi... more The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interfering (DI) particles of mouse hepatitis virus was studied. Two DI-specific RNA species, DIssA of genomic size and DIssE of subgenomic size, were detected in DI-infected cells. Purified DI particles, however, were found to contain predominantly DIssA and only a trace amount of DIssE RNA. Despite its negligible amount, the DIssE RNA in virions appears to serve as the template for the synthesis of DIssE RNA in infected cells. This conclusion was supported by two studies. First, the uv target size for DIssE RNA synthesis is significantly smaller than that for DIssA. Second, when purified DIssE RNA was transfected into cells which had been infected with a helper virus, DIssE RNA could replicate itself and became a predominant RNA species in the infected cells. Thus, DIssE RNA was not synthesized from the genomic RNA of DI particles. By studying the relationship between virus dilution and the amount of intracellular viral RNA synthesis, we have further shown that DIssE RNA synthesis requires a helper function, but it does not utilize the leader sequence of the helper virus. In contrast, DIssA synthesis appears to be helper-independent and can replicate itself. Thus DIssA codes for a functional RNA polymerase.

Research paper thumbnail of Analysis of intracellular small RNAs of mouse hepatitis virus: evidence for discontinuous transcription

Virology, 1987

We have previously shown the presence of multiple small leader-containing RNA species in mouse he... more We have previously shown the presence of multiple small leader-containing RNA species in mouse hepatitis virus (MHV)-infected cells. In this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small RNAs. Using cDNA probes specific for leader RNA and genes A, D, and F, we demonstrate that subsets of these small RNAs were derived from the various viral genes. These subsets have discrete and reproducible sizes, varying with the gene from which they are derived. The size of each subset correlates with regions of secondary structure, whose free energy ranges from -1.6 to -77.1 kcal/mol, in each of the mRNAs examined. In addition, identical subsets were detected on the replicative intermediate (RI) RNA, suggesting that they represent functional transcriptional intermediates. The biological significance of these small RNAs is further supported by the detection of leader-containing RNAs of 47, 50, and 57 nucleotides in length, which correspond to the crossover sites in two MHV recombinant viruses. These data, coupled with the high frequency of RNA recombination during MHV infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing RNA intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant RNAs. These data suggest that MHV transcription uses a discontinuous and nonprocessive mechanism in which RNA polymerase allows the partial RNA products to be dissociated from the template temporarily during the process of transcription.

Research paper thumbnail of The 5′-end sequence of the murine coronavirus genome: Implications for multiple fusion sites in leader-primed transcription

Virology, 1987

The coronavirus leader-primed transcription model proposes that free leader RNA species derived f... more The coronavirus leader-primed transcription model proposes that free leader RNA species derived from the 5'-end of the genomic RNA are utilized as a primer for the transcription of subgenomic mRNAs. To elucidate the precise mechanism of leader-priming, we cloned and sequenced the 5'-end of the mouse hepatitis virus genomic RNA. The 5'-terminal sequences are identical to the leader sequences present at the 5'-end of the subgenomic mRNAs. Two possible hairpin loop structures and an AU-rich region around the 3'-end of the leader sequence may provide the termination site for leader RNA synthesis. The comparison of 5'-end genomic sequences and the intergenic start sites for mRNA transcription revealed that there are homologous regions of 7-18 nucleotides at the putative leader/body junction sites. Some intergenic regions contain a mismatching nucleotide within this homologous region. We propose that free leader RNA binds to the intergenic region due to this homology and is cleaved at the mismatching nucleotide before serving as a primer. Thus, the free leader RNA species may be longer than the leader sequences in the subgenomic mRNAs and different mRNAs may have different leader/body junction sites.

Research paper thumbnail of Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus

Virology, 1989

Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the... more Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11 % of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 'H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 1 1-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be:

Research paper thumbnail of Molecular cloning and sequencing of a human hepatitis delta (δ) virus RNA

Nature, 1987

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only... more Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.

Research paper thumbnail of Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus

Virology, 1989

Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the... more Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11 % of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 'H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 1 1-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be:

Research paper thumbnail of Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein

Journal of Virology, 1989

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-de... more The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but ...

Research paper thumbnail of Identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus RNA genome

Journal of Virology, 1989

We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) ... more We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) synthesize an additional mRNA species (mRNA 2b, previously called mRNA 2a) with a size intermediate between that of mRNAs 2 and 3, suggesting the presence of an optional transcriptional initiation site. This transcriptional start is dependent on the leader sequence of the virus strains. To study the mechanism of coronavirus transcriptional regulation, we have cloned and sequenced the region of the viral genome corresponding to the 5' unique coding region of mRNA 2 of the JHM strain of MHV. In addition to the open reading frame (ORF) predicted to encode the viral nonstructural protein p30, a second complete ORF, with the potential to encode a 439-amino-acid polypeptide, was discovered. The transcriptional initiation sites of both mRNA 2a (formerly called mRNA 2) and mRNA 2b were determined by primer extension studies and RNA sequencing. The data indicated that transcription of mRNA 2a...

Research paper thumbnail of Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs

Journal of Virology, 1988

Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free l... more Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free leader RNA species. As a result, each virus-specific mRNA contains an identical leader RNA derived from the 5', end of the genomic RNA. In this study, we demonstrate by primer extension studies that the leader-fusion sites on a given species of coronavirus subgenomic mRNA are heterogeneous. The heterogeneity was due to variation in the number of pentanucleotide (UCUAA) repeats present at the leader fusion site. This pentanucleotide repeat region was complementary between the free leader RNA and the transcription start sites on the template RNA. This result suggests that the discontinuous transcription of coronavirus mRNAs occurs within the complementary sequences localized in two different RNA segments and that RNA joining occurs at variable sites.

Research paper thumbnail of Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase

Journal of Virology, 1987

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of t... more A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently t...

Research paper thumbnail of Coronavirus: A Jumping RNA Transcription

Cold Spring Harbor Symposia on Quantitative Biology, 1987

DNA-and RNA-dependent RNA polymerases usually recognize specific promoter sequences and initiate ... more DNA-and RNA-dependent RNA polymerases usually recognize specific promoter sequences and initiate RNA transcription at some distances downstream from the promoter. Initiation of transcription requires only complementary nucleoside triphosphates, although ...

Research paper thumbnail of Leader RNA-primed transcription and RNA recombination of murine coronaviruses

In: Positive-strand RNA, 1987

Research paper thumbnail of Molecular studies of coronavirus RNA

Research paper thumbnail of Sequence andTranslation oftheMurineCoronavirus 5'-End GenomicRNA Reveals theN-Terminal Structure ofthePutative RNA Polymerase

Research paper thumbnail of Murine coronavirus 5'-end genomic RNA sequence reveals mechanism of leader-primed transcription

Advances in experimental medicine and biology, 1987

Research paper thumbnail of Studies into the mechanism of MHV transcription

Advances in experimental medicine and biology, 1987

Research paper thumbnail of Murine Coronavirus Gene 1 Polyprotein Contains an Autoproteolytic Activity

Advances in Experimental Medicine and Biology, 1990

Research paper thumbnail of Primary structure and translation of a defective interfering rna of murine coronavirus

Virology, 1988

An intracellular defective-interfering (DI) RNA, DlssE, of mouse hepatitis virus (MHV) obtained a... more An intracellular defective-interfering (DI) RNA, DlssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DlssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5' end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the S'end of the parental MHV genome. The DlssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DIinfected cells. Sequence comparison of DlssE and the corresponding parts of the parental virus genome revealed that DlssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5' end of DlssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.

Research paper thumbnail of The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase

Research paper thumbnail of Defective-interfering particles of murine coronavirus: Mechanism of synthesis of defective viral RNAs

Virology, 1988

The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interferi... more The mechanism of synthesis of the defective viral RNAs in cells infected with defective-interfering (DI) particles of mouse hepatitis virus was studied. Two DI-specific RNA species, DIssA of genomic size and DIssE of subgenomic size, were detected in DI-infected cells. Purified DI particles, however, were found to contain predominantly DIssA and only a trace amount of DIssE RNA. Despite its negligible amount, the DIssE RNA in virions appears to serve as the template for the synthesis of DIssE RNA in infected cells. This conclusion was supported by two studies. First, the uv target size for DIssE RNA synthesis is significantly smaller than that for DIssA. Second, when purified DIssE RNA was transfected into cells which had been infected with a helper virus, DIssE RNA could replicate itself and became a predominant RNA species in the infected cells. Thus, DIssE RNA was not synthesized from the genomic RNA of DI particles. By studying the relationship between virus dilution and the amount of intracellular viral RNA synthesis, we have further shown that DIssE RNA synthesis requires a helper function, but it does not utilize the leader sequence of the helper virus. In contrast, DIssA synthesis appears to be helper-independent and can replicate itself. Thus DIssA codes for a functional RNA polymerase.

Research paper thumbnail of Analysis of intracellular small RNAs of mouse hepatitis virus: evidence for discontinuous transcription

Virology, 1987

We have previously shown the presence of multiple small leader-containing RNA species in mouse he... more We have previously shown the presence of multiple small leader-containing RNA species in mouse hepatitis virus (MHV)-infected cells. In this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small RNAs. Using cDNA probes specific for leader RNA and genes A, D, and F, we demonstrate that subsets of these small RNAs were derived from the various viral genes. These subsets have discrete and reproducible sizes, varying with the gene from which they are derived. The size of each subset correlates with regions of secondary structure, whose free energy ranges from -1.6 to -77.1 kcal/mol, in each of the mRNAs examined. In addition, identical subsets were detected on the replicative intermediate (RI) RNA, suggesting that they represent functional transcriptional intermediates. The biological significance of these small RNAs is further supported by the detection of leader-containing RNAs of 47, 50, and 57 nucleotides in length, which correspond to the crossover sites in two MHV recombinant viruses. These data, coupled with the high frequency of RNA recombination during MHV infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing RNA intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant RNAs. These data suggest that MHV transcription uses a discontinuous and nonprocessive mechanism in which RNA polymerase allows the partial RNA products to be dissociated from the template temporarily during the process of transcription.

Research paper thumbnail of The 5′-end sequence of the murine coronavirus genome: Implications for multiple fusion sites in leader-primed transcription

Virology, 1987

The coronavirus leader-primed transcription model proposes that free leader RNA species derived f... more The coronavirus leader-primed transcription model proposes that free leader RNA species derived from the 5'-end of the genomic RNA are utilized as a primer for the transcription of subgenomic mRNAs. To elucidate the precise mechanism of leader-priming, we cloned and sequenced the 5'-end of the mouse hepatitis virus genomic RNA. The 5'-terminal sequences are identical to the leader sequences present at the 5'-end of the subgenomic mRNAs. Two possible hairpin loop structures and an AU-rich region around the 3'-end of the leader sequence may provide the termination site for leader RNA synthesis. The comparison of 5'-end genomic sequences and the intergenic start sites for mRNA transcription revealed that there are homologous regions of 7-18 nucleotides at the putative leader/body junction sites. Some intergenic regions contain a mismatching nucleotide within this homologous region. We propose that free leader RNA binds to the intergenic region due to this homology and is cleaved at the mismatching nucleotide before serving as a primer. Thus, the free leader RNA species may be longer than the leader sequences in the subgenomic mRNAs and different mRNAs may have different leader/body junction sites.

Research paper thumbnail of Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus

Virology, 1989

Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the... more Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11 % of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 'H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 1 1-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be:

Research paper thumbnail of Molecular cloning and sequencing of a human hepatitis delta (δ) virus RNA

Nature, 1987

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only... more Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.

Research paper thumbnail of Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus

Virology, 1989

Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the... more Recent reports have suggested the involvement of arachidonic acid (AA) and its metabolites in the control of body pattern, head and tentacle regeneration and bud formation in Hydra spp. Here we describe for the first time the biosynthesis of hydroxyeicosatetraenoic acids (HETEs) in vitro by hydroid cytosolic extracts. Incubation of both unlabelled and tritiated AA with homogenates of Hydra vulgaris led to the conversion of up to 11 % of the exogenous fatty acid into mainly two metabolites. These were characterized as 11-hydroperoxyeicosatetraenoic acid (11-HPETE) and 11-HETE by means of a combination of chromatographic, chemical, 'H-n.m.r. and electron-impact m.s. techniques. Trace amounts of 9-HETE and 12-HETE were also found. Analysis of 1 1-HETE by chiral-phase h.p.l.c. revealed that this metabolite was composed mainly of the R enantiomer. The production of 11-HPETE and 11-HETE was found to be: