A Novel Sequence Found at the 3′-Terminus of Hepatitis C Virus Genome

Tanaka, Torahiko; Kato, Naoya; Cho, M. J.; Shimotohno, Kunitada

doi:10.1006/bbrc.1995.2526

Cited by 320 publications

(268 citation statements)

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“…Once the highly conserved 3፱ terminus of HCV was discovered and the genome sequence was completed 50,51 , reverse genetics with HCV became possible. Functional complementary DNA clones were assem- lar antiviral pathways may be a major intracellular determinant of HCV tropism.…”

Section: The Replication Era: Reverse Genetic Systems For Hcvmentioning

confidence: 99%

Unravelling hepatitis C virus replication from genome to function

Lindenbach

Rice

2005

Nature

753

561

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Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.virus life cycle: first, as a messenger RNA (mRNA) for translation of the viral proteins; second as a template for RNA replication; and third, as a nascent genome packaged within new virus particles. Virions presumably form by budding into the endoplasmic reticulum (ER) and leave the cell through the secretory pathway.Researchers have followed each aspect of the virus life cycle in turn. Just as infection starts from an HCV genome entering the cytoplasm and progressing through translation, replication and particle production, our understanding has progressed from having a genome sequence to understanding translation and the viral gene products, characterizing RNA replication, and establishing systems to characterize virus particles and infectivity. In this review, we summarize the current understanding of HCV replication with special emphasis on recent developments. As space is limited, we cannot be comprehensive; readers may wish to consult other reviews for detail and breadth 5,13,14. Initial studies: HCV translation and polyprotein processingThe identification of HCV yielded a partial viral genome sequence 5 . Research in the early 1990s focused on dissecting HCV gene expression and characterizing the gene products. Much has been learned about the biochemistry of three key enzymes, and structural information is now available at the atomic level for roughly half of the proteincoding region.Translation of the HCV genome, which lacks a 5፱ cap, depends on an internal ribosome entry site (IRES) within the 5፱-noncoding region (NCR). The HCV IRES binds 40S ribosomal subunits directly and avidly, bypassing the need for pre-initiation factors, and inducing an mRNA-bound conformation in the 40S subunit 15 . The IRES-40S complex then recruits eukaryotic initiation factor (eIF) 3 and the ternary complex of Met-tRNA-eIF2-GTP to form a non-canonical 48S intermediate, before a kinetically slow transition to the translationally active 80S complex 16,17 .Once initiated, translation of the HCV genome produces a large polyprotein that is proteolytically cleaved to produce 10 viral proteins (Fig. 2a). The amino-terminal one-third of the polyprotein encodes the virion structural proteins: the highly basic core (C) protein, and glycoproteins E1 and E2. After the structural region comes a small integral membrane protein, p7, which seems to function as an ion channel 18,19 . The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular process...

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Section: The Replication Era: Reverse Genetic Systems For Hcvmentioning

confidence: 99%

Unravelling hepatitis C virus replication from genome to function

Lindenbach

Rice

2005

Nature

753

561

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“…We compared the 3ЈX sequence of pNIHJ1 clone with those reported previously. 18,19,37 Sequence homology of the 3ЈX of pNIHJ1 against other type 1b clones and different types were 97% and 96%, respectively. These results confirmed that the 3ЈX sequences were highly conserved among different HCV isolates.…”

Section: Determination Of 3јx Sequence In the Plasma Of Donor Andmentioning

confidence: 99%

“…A novel 98-nucleotide (nt) sequence downstream from the poly(U) stretch of the HCV genome was recently reported. 18,19 To construct a complete cDNA clone of the HCV genome, we selected a blood donor sample which was shown to be infectious to both humans and chimpanzees. [20][21][22][23] From that sample we have cloned several fragmental cDNAs and constructed a full-length cDNA containing the 98-base pair (bp)-3ЈX sequence.…”

mentioning

confidence: 99%

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

et al. 1998

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We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3Ј end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.(HEPATOLOGY 1998;27:621-627.)Comparative study of the amino acid sequence of hepatitis C virus (HCV) with those of flaviviruses and pestiviruses and gene expression experiments in bacteria, yeast, and animal cells have revealed that the proteins of HCV are processed by a host-derived signalase and cleaved by virus-coded proteases. [1][2][3][4][5] In vitro culture systems that support partial replication of this virus have been developed from human T-and B-cell lines, 6,7 human fetal hepatocytes, 8 chimpanzee hepatocytes, 9 and human primary hepatocytes. 10 However, fully efficient, long-term viral replication has not yet been established. To study the biology of HCV and its pathogenesis, it is essential to establish an efficient in vitro cell culture system or an infectious complementary DNA (cDNA) clone to support the complete replication of HCV.As a first step to construct an infectious cDNA clone of HCV, it is important to select an adequate sample containing infectious HCV. To date, a considerable number of ''complete'' clones of the HCV genome have been reported. However, it is not certain that those clones have truly originated from infectious HCV, because the materials used usually were pooled plasma samples. Furthermore, the plasma of HCV carriers or patients is generally composed of quasispecies of HCV population. 11 To construct an infectious cDNA clone of RNA viruses, it is crucial to retain both 5Ј and 3Ј ends of the sequence, which are highly conserved and are considered to play essential roles for RNA synthesis, transcription, and translation. 12 Many have reported that the 3Ј untranslated region (UTR) of HCV consisted of poly(U) [13][14][15][16] or poly(A) 17 homopolymer tracts. A novel 98-nucleotide (nt) sequence down...

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“…The fragment containing the vaccinia virus p11 promoter and lacZ gene was excised from pSC10 (Chakrabarti et al, 1985) and inserted into the pFTK5.8 vector yielding pFTKlacZ. A T7HCVLuc expression cassette containing DNA encoding the HCV 5h untranslated region (UTR) (nt 1-341 of the HCV genome), a luciferase gene, the 3h UTR (nt 9354-9523 of HCV) including the 98 bp of 3h extra sequences (Kolykhalov et al, 1996 ;Tanaka et al, 1996), and hepatitis D virus (HDV) ribozyme flanked by the T7 promoter at the 5h end and the T7 terminator at the 3h end was obtained by digestion with HindIII and SpeI from pT7HCVLuc (Y. Aoki, H. Aizaki, T. Shimoike, H. Tani, K. Ishii, I. Saito, Y. Matsuura & T. Miyamura, unpublished results). After being filled in with the Klenow fragment of DNA polymerase and ligated to the BclI linker, the fragment was digested with BclI and subsequently cloned into the BglII site of pFTK5.8 vector yielding pFTKT7HCVLuc (Fig.…”

Section: Methodsmentioning

confidence: 99%

Expression of target genes by coinfection with replication-deficient viral vectors.

Yap

Ishii

Aizaki

et al. 1998

Journal of General Virology

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An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5h untranslated region (UTR), a luciferase gene and the 3h UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase under the control of a mammalian promoter. Only a low level of luciferase expression was ob-

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A Novel Sequence Found at the 3′-Terminus of Hepatitis C Virus Genome

Cited by 320 publications

References 0 publications

Unravelling hepatitis C virus replication from genome to function

Unravelling hepatitis C virus replication from genome to function

Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

Expression of target genes by coinfection with replication-deficient viral vectors.

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