The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37-to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.The cap-independent initiation of translation on the plussense genomic RNAs of picornaviruses and some flaviviruses requires binding of the small ribosome subunit to the RNA at a site located hundreds of nucleotides downstream of its 5Ј terminus (34,54,75). This interaction is controlled by highly structured, cis-acting RNA elements located within the 5Ј nontranslated region (5ЈNTR), the internal ribosome entry site (IRES). IRES elements were first identified within viral RNAs but have been increasingly recognized within the lengthy 5ЈNTRs of cellular mRNAs encoding certain critical mammalian growth factors, including fibroblast growth factor 2 (76), human insulin-like growth factor II (74), and platelet-derived growth factor B (4), as well as eukaryotic translation initiation factor 4G (eIF4G) (19). In directing the internal initiation of translation, these viral and cellular IRES elements function in concert with both canonical and possibly noncanonical transacting cellular translation initiation factors. Factors influencing the efficiency of internal ribosome entry directed by IRESs are likely to play important roles in determining the cellular tropisms of some viruses or the posttranscriptional regulation of proteins translated under the...
Progress in understanding the pathogenesis of hepatitis C virus (HCV) has been slowed by the absence of tractable small animal models. Whereas GB virus B (GBV-B, an unclassified flavivirus) shares a phylogenetic relationship and several biologic attributes with HCV, including hepatotropism, it is not known to cause persistent infection, a hallmark of HCV. Here, we document persistent GBV-B infection in one of two healthy tamarins (Saguinus oedipus) inoculated intrahepatically with infectious synthetic RNA. High-titer viremia (10 8 to 10 9 genome equivalents per ml) and transiently elevated serum alanine transaminase activities were present from weeks 4 to 12 postinoculation in both animals. However, whereas GBV-B was eliminated from one animal by 20 weeks, the second animal remained viremic (10 3 to 10 7 genome equivalents per ml) for >2 years, with alanine transaminase levels becoming elevated again before spontaneous resolution of the infection. A liver biopsy taken late in the course of infection demonstrated hepatitis with periportal mononuclear infiltrates, hepatocellular microvesicular changes, cytoplasmic lipid droplets, and disordered mitochondrial ultrastructure, findings remarkably similar to chronic hepatitis C. GBV-B-infected hepatocytes contained numerous small vesicular membranous structures resembling those associated with expression of HCV nonstructural proteins, and sequencing of GBV-B RNA demonstrated a rate of molecular evolution comparable to that of HCV. We conclude that GBV-B is capable of establishing persistent infections in healthy tamarins, a feature that substantially enhances its value as a model for HCV. Mitochondrial structural changes and altered lipid metabolism leading to steatosis are conserved features of the pathogenesis of chronic hepatitis caused by these genetically distinct flaviviruses.
We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a thirdgeneration enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera. (HEPATOLOGY 1999;30:316-324.)Unlike other human hepatitis viruses, the majority of immunocompetent persons who become infected with hepatitis C virus (HCV) are unable to clear the virus and go on to develop persistent infection. 1,2 Such infections are frequently associated with active necroinflammatory liver disease, and eventually result in cirrhosis in 20% to 30% of individuals. 2 These individuals are also at risk for the development of hepatocellular carcinoma. 2 Thus, it is not surprising that HCV is the most important infectious cause of chronic liver disease in the United States and in many other countries. Despite the fact that chronic hepatitis C is increasingly recognized as an important disease, treatment options remain very limited. Even the most efficacious treatment, the combination of interferon with ribavirin, 3 results in a lack of sustained response in at least one half of all persons receiving therapy. There is an urgent need for better therapeutics. However, antiviral discovery efforts are severely restricted by the absence of a cell culture system that supports the efficient replication of HCV, as well as the lack of a small-animal model. Overcoming these limitations to hepatitis C research efforts will likely require a greater understanding of the molecular mechanisms involved in HCV replication. While it is pos...
Translation of the foot and mouth disease virus genome in vitro and in vivo indicated that all seven serotypes initiate protein synthesis at two separate AUGs. Sequence analysis of the region surrounding these AUGs has shown that the efficiency with which the initiating AUG is recognized is dependent on the flanking nucleotides. However, in vitro, the major factor determining which AUG is used is the concentration of Mg2+.
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