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PubMed1987

Vaccinia recombinant expressing Lassa-virus internal nucleocapsid protein protects guineapigs against Lassa fever.

The Lister strain of vaccinia virus was used to construct a recombinant that expressed the nucleocapsid gene of Lassa virus. Guineapigs immunised with the recombinant virus were protected against challenge with Lassa virus, whereas control animals showed the usual disease course, including pyrexia, anorexia, viraemia, and death. These data indicate not only that protective immunity to Lassa fever can be evoked by an experimental vaccine, but also that this response can be mediated by an internal protein component of the virus.

Animals↗

PubMed1994

trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements.

A region of about 435 bases from the 5' noncoding region of foot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive secondary structure. Precise deletion of five predicted secondary structure features has been performed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of the modified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intact foot-and-mouth disease virus IRES complemented four of these defective elements to an efficiency of up to 80% of wild-type activity. No complementation was observed with the structurally analogous element from encephalomyocarditis virus. The role of RNA-RNA interactions in the function of the picornavirus IRES is discussed.

Amino Acid Sequence↗

PubMed2003

In vivo activity of Rhopalosiphum padi virus internal ribosome entry sites.

The RNA genome of Rhopalosiphum padi virus (RhPV), like other members of the Dicistroviridae, contains two open reading frames that are preceded by internal ribosome entry sites (IRESs). To compare the activities of the two RhPV IRESs in insect cells, a system was established for the in vivo transcription and translation of plasmid templates containing the IRESs. In this system, the two RhPV IRESs directed initiation of translation from bicistronic plasmids with equal efficiency. Competition was observed between the two IRESs when they were in cis in a bicistronic plasmid. A mutation that disrupted the 3'-proximal pseudoknot of the intergenic (IG) IRES reduced translation initiation in vivo. Similarly, mutations in the RhPV IG IRES disrupted its ability to bind 80S particles in vitro. The two IRESs preferentially labelled proteins of different masses in UV cross-linking experiments, illustrating the different translation initiation mechanisms employed by the two elements.

Animals↗

PubMed2000

Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells.

Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5' nontranslated RNA (5'NTR): G(107)-->A, C(204)-->A, and G(243)-->A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). These results suggest that virus with this 5'NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.

5' Untranslated Regions↗

PubMed2005

Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein.

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.

Autoantigens↗

PubMed2000

Cell cycle regulation of hepatitis C virus internal ribosomal entry site-directed translation.

BACKGROUND & AIMS: Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5' nontranslated segment of the viral RNA. This RNA segment is known to form binary complexes with isolated 40S ribosome subunits in vitro, but there is little understanding of how the process of virus translation is regulated in vivo. METHODS: We established 2 stably transformed cell lines from Huh-7 cells that constitutively express dicistronic RNA transcripts containing sequences encoding 2 reporter proteins (Renilla luciferase and firefly luciferase) separated by a functional HCV IRES. The translation of the upstream Renilla luciferase reading frame is initiated in these cells by the usual cellular cap-dependent mechanism, whereas translation of the downstream firefly luciferase reading frame is initiated by the IRES. RESULTS: Compared with cap-dependent translation, the activity of the IRES was greatest in actively growing cells and relatively reduced in resting cells. In synchronized cultures of these stably transformed cells, the IRES activity varied with the cell cycle and was greatest during the mitotic (M) phases and lowest during the quiescent (G(0)) phases. CONCLUSIONS: These findings suggest that HCV translation is regulated by cellular proteins that vary in abundance during the cell cycle and that viral translation may be enhanced by factors that stimulate the regeneration of hepatocytes in patients with chronic hepatitis C.

5' Untranslated Regions↗

PubMedDate not supplied

Cap and polyA tail enhance translation initiation at the hepatitis C virus internal ribosome entry site by a discontinuous scanning, or shunting, mechanism.

OBJECTIVES: Hepatitis C virus (HCV) does not replicate in vitro, suggesting that cultured cells may lack factors that are essential for efficient use of HCV messenger RNAs (mRNAs). Here, we have studied the efficiency of HCV mRNA translation compared with translation of capped and polyadenylated mRNAs in human cells. STUDY DESIGN/METHODS: We have generated noninfectious minivirus mRNAs from an infectious HCV genome. These mRNAs were transfected into human cells, and the translation efficiency was determined. RESULTS: Hepatitis C virus mRNAs under control of the HCV internal ribosome entry site (IRES) were inefficiently translated compared with capped and polyadenylated mRNAs. Addition of a cap and a polyA tail on the HCV mRNAs revealed that these structures interacted with the hepatitis C IRES in a synergistic manner to load ribosomes onto the HCV mRNAs, thereby strongly enhancing translation. The positive effect of the cap and the polyA tail on initiation of translation at the initiator AUG embedded in the HCV IRES was the result of a discontinuous scanning, or shunting, mechanism. CONCLUSIONS: The results demonstrated that recruitment of ribosomes to the HCV mRNAs was inefficient in dividing cultured cells. Factors that are necessary for efficient translation of the HCV mRNAs in hepatocytes may be absent or inactive in cultured cells. This may be one reason for the inefficient replication of the HCV in vitro.

5' Untranslated Regions↗

PubMed1994

Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions.

Translational initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' nontranslated region of the EMCV mRNA, rather than by ribosomal scanning. Internal ribosomal binding requires a cis-acting element termed the internal ribosomal entry site (IRES). IRES elements have been proposed to be involved in the translation of picornavirus mRNAs and some cellular mRNAs. Internal ribosome binding likely requires the interaction of trans-acting factors that recognize both the mRNA and the ribosomal complex. Five cellular proteins (p52, p57, p70, p72, and p100) cross-link the EMCV IRES or fragments of the IRES. For one of these proteins, p57, binding to the IRES correlates with translation. Recently, p57 was identified to be very similar, if not identical, to polypyrimidine tract-binding protein. On the basis of cross-linking results with 21 different EMCV IRES fragments and cytoplasmic HeLa extract or rabbit reticulocyte lysate as the source of polypeptides, consensus binding sites for p52, p57, p70, and p100 are proposed. It is suggested that each of these proteins recognizes primarily a structural feature of the RNA rather than a specific sequence.

Base Sequence↗

PubMed1985

Indication and study of antigenic properties of influenza virus internal proteins by means of monoclonal antibodies.

Influenza A and B viruses isolated from animals and man were studied by solid phase radioimmunoassay (SP RIA) using monoclonal antibodies to the A/Dunedin/4/73 strain nucleoprotein (NP). Only influenza A viruses isolated before 1980 interacted with the monoclonal antibody set used, while strains isolated between 1980 and 1983 failed to do so. It was shown that RIA employing monoclonal antibodies was useful for rapid identification of influenza virus NP in the allantoic fluid containing fresh influenza A virus isolates.

Animals↗

PubMed2003

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human La autoantigen.

The human La autoantigen has been shown to interact with the internal ribosome entry site (IRES) of hepatitis C virus (HCV) in vitro. Using a yeast three-hybrid system, we demonstrated that, in addition to full-length La protein, both N- and C-terminal halves were able to interact with HCV IRES in vivo. The exogenous addition of purified full-length and truncated La proteins in rabbit reticulocyte lysate showed dose-dependent stimulation of HCV IRES-mediated translation. However, an additive effect was achieved adding the terminal halves together in the reaction, suggesting that both might play critical roles in achieving full stimulatory activity of the full-length La protein. Using computational analysis, three-dimensional structures of the RNA recognition motifs (RRM) of the La protein were independently modeled. Of the three putative RRMs, RRM2 was predicted to have a good binding pocket for the interaction with the HCV IRES around the GCAC motif near the initiator AUG and RRM3 binds perhaps in a different location. This observation was further investigated by the filter-binding and toe-printing assays. The results presented here strongly suggest that both the N- and C-terminal halves can interact independently with the HCV IRES and are involved in stimulating internal initiation of translation.

Amino Acid Sequence↗

PubMed2006

Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2alpha under oxidative stress.

Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2alpha phosphorylation. A phosphomimetic of eIF2alpha, which mimics the structure of the phosphorylated eIF2alpha, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2alpha. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2alpha-independent mechanism, whereas the simultaneous phosphorylation of eIF2alpha repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2alpha-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.

Cell Line↗

PubMed1996

Requirements for mouse mammary tumour virus internalization in mouse mammary epithelial cells.

Methylamine, a lysosomotropic alkalinizing agent, blocked mouse mammary tumour virus (MMTV) infection in normal mouse mammary epithelium, suggesting that internalization and acidification are necessary for cell penetration. This mechanism was further supported by the fact that intact MMTV induced the translocation of its cellular binding protein from the plasmalemma to the microsomes; however, isolated gp52, the MMTV envelope protein that binds this receptor, did not redistribute the binding protein. These data suggest that either another viral component, in addition to gp52, is needed for cell entry or that internalization requires receptor aggregation, which only the multivalent viral envelope can induce.

Animals↗

PubMed2001

Hepatitis C virus internal ribosome entry site RNA contains a tertiary structural element in a functional domain of stem-loop II.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5'-terminal sequence, most of it in the uncapped 5'-untranslated region (5'-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem-loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53-68 and 103-117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem-loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem-loop II element is directly involved in IRES action.

5' Untranslated Regions↗

PubMed1999

[Democratic Republic of the Congo: between civil war and the Marburg virus. International Committee of Technical and Scientific Coordination of the Durba Epidemic].

Because the epidemiology of viral hemorrhagic fever is unclear, each outbreak is a spectacular event that focuses the attention of the international scientific community. When an epidemic of Marburg virus disease occurred in the Durba region located in the northeastern part of the People's Republic of the Congo, 23 scientists were sent from 12 different countries. Sixty of the 73 people infected died. The first case was observed in December 1998 and the last in May 1999. Because of political unrest in the country, the outbreak was not reported immediately and most data was collected by observers retrospectively. However Marburg virus infection was confirmed in 5 of 16 patients in whom testing was performed and person-to-person transmission was demonstrated. Thus the Durba outbreak was the first epidemic of Marburg virus disease not involving laboratory contamination. Initial epidemiologic findings suggest that the first cases involved miners who were probably infected by contact with an animal reservoir such as bats. Further studies to determine seroprevalence in the general population and virologic testing on animals captured in the zone should provide answers to these questions.

Adolescent↗

PubMed2000

Human La antigen is required for the hepatitis C virus internal ribosome entry site-mediated translation.

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

5' Untranslated Regions↗

PubMed1993

Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection.

Mice were vaccinated with recombinant vaccinia virus (rVac) expressing the glycoprotein (G), nucleoprotein (N), phosphoprotein (NS) or matrix protein (M) of rabies virus and their resistance to peripheral lethal infection with street rabies virus was examined. Mice vaccinated with rVac-G or rVac-N developed strong antibody responses to the corresponding proteins and essentially all mice survived challenge infection. Mice vaccinated with rVac-NS or rVac-M developed only a slight antibody response, however, a significant protection (59%) was observed in the rVac-NS-vaccinated mice, whereas rVac-M-vaccinated mice were not protected. No anti-G antibodies were detected in the sera of mice which has been vaccinated with rVac-N or rVac-NS and survived challenge infection. Passive transfer of anti-N monoclonal antibodies (MAbs) recognizing an epitope located on amino acids 1-224 of the protein prior to challenge resulted in significant protection, although the protection was not complete even with a high amount of antibodies. In contrast, none of the mice given MAbs recognizing an epitope of amino acids 247-415 or F(ab')2 fragments from a protective MAb IgG were protected. Administration of anti-CD 8 MAb to rVac-N-vaccinated mice showed no significant effect on protection. Our observations suggest that a considerable part of the protection achieved by the vaccination with rVac-N can be ascribed to the intact anti-N antibodies recognizing an epitope located on amino acids 1-224 of the protein.

Animals↗

PubMed2004

La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro.

Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

5' Untranslated Regions↗

PubMed2002

Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.

5' Untranslated Regions↗