Detection of genomic RNA of the hepatitis A virus in clinical samples using the blot hybridization method

The synthetic oligonucleotide sequence 3'CTCCTCGTACTTTA-5' complementing hepatitis A virus RNA was compared with cDNA probes in identification of viral genomic RNA. The clinical materials from patients in the 1-2 weeks of jaundice were screened. High specificity of the technique was demonstrated. Possibility of clinical using of the blot hybridization technique is discussed.     

Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus.

Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera. The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169. A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11. Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment. The gene was transcribed into a late 1.3-kilobase RNA. The nucleotide sequence of the coding region was determined. Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase. In Western blots these proteins were recognized by human sera. Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots. In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.     

Synthesis of a novel hepatitis C virus protein by ribosomal frameshift.

Hepatitis C virus (HCV) is an important human pathogen that affects approximately 100 million people worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. We report here the discovery of a novel HCV protein synthesized by ribosomal frameshift. This protein, which we named the F protein, is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frameshift into the -2/+1 reading frame. This ribosomal frameshift requires only codons 8-14 of the core protein-coding sequence, and the shift junction is located at or near codon 11. An F protein analog synthesized in vitro reacted with the sera of HCV patients but not with the sera of hepatitis B patients, indicating the expression of the F protein during natural HCV infection. This unexpected finding may open new avenues for the development of anti-HCV drugs.     

Nucleotide sequence and tissue distribution of the human mitochondrial aspartate aminotransferase mRNA

The cDNA of human mitochondrial aspartate aminotransferase (E.C.2.6.1.1.) was isolated from a human liver cDNA library using a rat mitochondrial aspartate aminotransferase cDNA as probe. The sequence of this cDNA gives a predicted aminoacid sequence for the human presequence and for the human mature protein exhibiting respectively 93% and 95% homology with rat sequences. A Northern blot of total RNA, isolated from various human tissues and hybridized with this cDNA, revealed a single 2.4 Kb RNA band. Mitochondrial aspartate aminotransferase RNA was clearly detected in human kidney, placenta, stomach and spleen as well as in both fetal and adult liver.     

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.     

Molecular cloning of human plasma glutathione peroxidase gene and its expression in the kidney.

A genomic clone containing the human plasma glutathione peroxidase (GSH-Px) gene has been isolated using a rat plasma GSH-Px cDNA as a probe. The partial nucleotide sequence of the clone completely matched the sequence of the human plasma GSH-Px cDNA. The results of Southern blot hybridization indicate that the human plasma GSH-Px gene consists of at least 4 exons and 3 introns, and spans about 12 kb. RNA blot analysis demonstrated that the human plasma GSH-Px gene is expressed in the kidney.     

Non-A, non-B hepatitis-related hepatocellular carcinoma in a chimpanzee

Epidemiology has indicated the possible association of non-A, non-B hepatitis (NANBH) with hepatocellular carcinoma (HCC) in man, but there are no means for confirmation. Chimpanzees are recognized models for studying hepatitis B and NANBH, and may become carriers of both. The first case of HCC to be reported in chimpanzees was found after longitudinal study of a hepatitis B-free chimpanzee 7 years after inoculation with human plasma from a patient reported to have chronic NANBH.     

Isolation and characterization of a complementary DNA expressing human U1 small nuclear ribonucleoprotein C polypeptide

A cloned complementary DNA, termed pS2, was isolated from a human fibroblast cDNA library in the bacteriophage expression vector lambda gt11 after screening with a patient's serum containing a high titer of anti-ribonucleoprotein (RNP) antibodies. A reasonable amount of cro-beta-galactosidase fusion protein (pS2EX) was obtained through subcloning of the pS2 insert into a plasmid expression vector pEX-2. Antibody against pS2EX (anti-pS2EX) was purified from this patient's serum by Sepharose 4B conjugated with pS2EX. Immunofluorescent staining of HeLa cells with anti-pS2EX antibody exhibited a typical speckled pattern in the interphase nuclei. In the immunoblot analysis, the anti-pS2EX antibody recognized the 22 kDa protein. Using immunoprecipitation of cell lysate and subsequent RNA analysis, anti-pS2EX antibody was shown to precipitate U1 RNP only. The reactivities of various anti-RNP sera to pS2EX correlated well with the positive reaction to C polypeptide in the immunoblot. These findings indicate that pS2 is a cDNA for C polypeptide of U1 snRNP. In the Northern blot using human RNA and radiolabeled pS2, a single band about 800 base was observed. The nucleotide sequence of pS2 showed no significant homologies to known proteins.     

The organisation and interviral homologies of genes at the 3' end of tobacco rattle virus RNA1

The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3'-terminal region. The sequence contains three long open reading frames. One of these starts 5' of the cDNA and probably corresponds to the carboxy-terminal sequence of a 170-K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29-K polypeptide, was shown by Northern blot analysis to coincide with a 1.6-kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co-migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29-K polypeptide showed homology with two regions in the 30-K protein of TMV. This homology includes positions in the TMV 30-K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16-K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7-kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.     

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.     

cDNA cloning and amino acid sequence of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase

A cDNA of 1762 base pairs was obtained from a cDNA library of human brain by immunoscreening, and the nucleotide sequence of the cDNA was determined. The complete amino acid sequence of human 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced from the nucleotide sequence of the cDNA. Human enzyme was found to contain 401 amino acids including initiation methionine and have a molecular weight of 45,098. RNA blot hybridization revealed a single mRNA band at the position of about 3000 bases. DNA blot hybridization suggested that a single-copy 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene exists per haploid genome.     

Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previousl     

Identification of a novel human member of the DEAD box protein family

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.     

我国非肠道传播非甲非乙型肝炎(丙型)病毒NS3蛋白的部份cDNA克隆及序列分析

用15ml非肠道传播非甲非乙型肝炎病人的混合血清提取病毒RNA,经逆转录和聚合酶链反应(PCR)扩增,获得583个核苷酸的非肠道传播非甲非乙型肝炎病毒(HCV)NS3蛋白的cDNA片段.该片段与美国报道的同片段HCV cDNA原型比较,核酸序列同源性为80.9％,氨基酸序列同源性为93.2％。与日本报道的同片段J1 HCV cDNA相比较,核酸序列和氨基酸序列的同源性分别为92.6％和95.2％。用α-~(32)P同位素标记该片段,与HCV病人血清出现杂交反应。     

A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan.

Recently, a cDNA from the hepatitis C virus (HCV) RNA genome has been isolated in the USA from a chronically infected chimpanzee. In order to isolate HCV cDNA derived from human material, RNA was extracted from plasma of a Japanese blood donor implicated in post-transfusion non-A, non-B hepatitis and HCV cDNA was synthesized and amplified by the PCR method using HCV-specific oligonucleotide primers. The cDNA fragment, 583 nucleotides long, showed 79.8% homology at the nucleotide level and 92.2% homology at the amino acid level compared with the prototype HCV cDNA. These results provides further evidence to show that HCV is closely associated with the development of post transfusion non-A, non-B hepatitis.