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At least 91 records · Page 5Linked to original sources
PubMed1986

Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes.

It has generally been assumed that most if not all CTL specific for vesicular stomatitis virus (VSV)-infected cells recognize the viral glycoprotein (G), an integral membrane protein abundantly expressed on infected cell surfaces. Using recombinant vaccinia viruses containing copies of cloned VSV genes to examine CTL recognition of VSV, we have confirmed that G is recognized by VSV-specific CTL. More interestingly, however, we have also found that nucleocapsid protein (N), an internal virion protein, can be detected on infected cell surfaces using mAb, and serves as a major target antigen for VSV-specific CTL. In contrast to the highly serotype-specific recognition of G, N is recognized by a major population of CTL able to lyse cells infected with either the Indiana or New Jersey VSV serotypes. Using target cells expressing a cloned MHC class I gene, we could directly show that CTL recognition of N occurs in the context of the MHC Ld molecule.

Animals↗
PubMed1998

A small yeast RNA blocks hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation and inhibits replication of a chimeric poliovirus under translational control of the HCV IRES element.

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

Autoantigens↗
PubMed2001

Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation.

Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3'X) targeting the HCV minus-strand replication intermediate at position 40 within the 3'X tail. Surprisingly, Rz3'X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3'X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3'X tail sequences, the results indicate that Rz3'X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome alpha-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3'X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

Antiviral Agents↗
PubMed2004

Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication.

Polypyrimidine tract-binding protein (PTB) has previously been shown to affect Hepatitis C virus (HCV) IRES-mediated translation. In the present study we investigated the functional role of PTB for HCV translation, replication and chronic HCV infection. Bicistronic HCV IRES reporter plasmids and two different subgenomic replicons (bicistronic: pHCVrep1bBB7 (s1179I); monocistronic: pFK1-389/hyg-ubi/NS3-3'/5.1) were used to analyze the effects of PTB. Following transfection of plasmids expressing PTB RNA in sense or antisense orientation, translational activity and HCV RNA were analyzed by luciferase assay, quantitative real-time RT-PCR and northern blot analysis. Additionally, in liver tissue (n = 53) intrahepatic PTB RNA levels were determined by quantitative real-time RT-PCR. Significant inhibition of HCV IRES activity up to 42.6% was observed upon PTB sense RNA expression for HCV IRES reporter plasmids, while translational activity was enhanced up to 63.8% for PTB antisense RNA expression. In the HCV replicons PTB did not affect replication and no correlation was found between intrahepatic PTB mRNA levels and serum HCV RNA or histological changes in liver tissue of HCV infected patients. Although PTB inhibits HCV IRES-mediated translation from bicistronic reporter constructs, data obtained from two subgenomic HCV replicons and liver tissue do not indicate a significant role of PTB for HCV replication and chronic HCV infection.

5' Untranslated Regions↗
PubMed1983

Localization with the electron microscope of p27 Rous sarcoma virus internal protein in chicken embryo fibroblasts. A time study at different stages after infection.

Protein 76 (Pr76), the precursor of the so-called 'gag' protein of avian leukosarcoma viruses, has been localized in the light and electron microscope by using the immunoperoxidase technique in Rous sarcoma virus-infected chick embryo fibroblasts. The kinetics of the progression of the protein through the cell was studied at different times after infection. The protein was first found (after 8 h) on ribosomes located near the nucleus and then proceeded rapidly to the periphery of the cell. After 5-6 days, intracytoplasmic staining faded away and only the ribosomes close to the plasma membrane were stained. A possible association of a protein with the cytoskeleton is suggested.

Animals↗
PubMed2001

Functional interaction of translation initiation factor eIF4G with the foot-and-mouth disease virus internal ribosome entry site.

In the life-cycle of picornaviruses, the synthesis of the viral polyprotein is initiated cap-independently at the internal ribosome entry site (IRES) far downstream from the 5' end of the viral plus-strand RNA. The cis-acting IRES RNA elements serve as binding sites for translation initiation factors that guide the ribosomes to an internal site of the viral RNA. In this study, we show that the eukaryotic translation initiation factor eIF4G interacts directly with the IRES of foot-and-mouth disease virus (FMDV). eIF4G binds mainly to the large Y-shaped stem-loop 4 RNA structure in the 3' region of the FMDV IRES element, whereas stem-loop 5 contributes only slightly to eIF4G binding. Two subdomains of stem-loop 4 are absolutely essential for eIF4G binding, whereas another subdomain contributes to a lesser extent to binding of eIF4G. At the functional level, the translational activity of stem-loop 4 subdomain mutants correlates with the efficiency of binding of eIF4G in the UV cross-link assay. This indicates that the interaction of eIF4G with the IRES is crucial for the initiation of FMDV translation. A model for the interaction of initiation factors with the IRES element is discussed.

Aphthovirus↗
PubMed1996

Revertant analysis of J-K mutations in the encephalomyocarditis virus internal ribosomal entry site detects an altered leader protein.

The internal ribosomal entry site (IRES) of picornaviruses consists of various sequence and structural elements that collectively impart translational function to the genome. By engineering substitution and deletion mutations into the J-K elements of the encephalomyocarditis virus IRES, translationally defective viruses with small-plaque phenotypes were generated. From these, 60 larger-plaque revertant viruses were isolated and characterized, and their sequences were compared with a structural model of the IRES. The data provide confirming evidence for the existence of helix J3 within stem J but suggest that helix J1 is 3 bp longer than previously estimated. They also suggest that previously modeled stems L and M should be replaced by an alternative structure. One reversion mutation was mapped to the leader protein coding region. This change of leader amino acid 20 from Pro to Ser increased the viral plaque size dramatically but did not alter the cell-free translational activity of the mutated, parental IRES.

Animals↗
PubMed2002

High translation efficiency is mediated by the encephalomyocarditis virus internal ribosomal entry sites if the natural sequence surrounding the eleventh AUG is retained.

Internal ribosomal entry sites (IRES) allow cap-independent translation and are sequences that are often used in gene therapy for strategies in which several genes need to be expressed. In this study, two different sequences of encephalomyocarditis virus (EMCV) IRES were compared for their translation efficiency in the context of retroviral vectors. When the sequence surrounding the 11th AUG of the IRES was conserved (IRESg), the translation efficiency was significantly higher than if the AUG of the downstream gene started with the 11th AUG of the IRES (IRESb). The translation efficiency with IRESg was influenced by the cell type and also by the nature of the transgene.

Animals↗
PubMed1998

Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT)

BACKGROUND: Only 15-20% of patients with chronic hepatitis C achieve a sustained virological response with interferon therapy. The aim of this study was to compare the efficacy and safety of interferon alpha2b in combination with oral ribavirin with interferon alone, for treatment of chronic infection with hepatitis C virus (HCV). METHODS: 832 patients aged 18 years or more with chronic HCV who had not been treated with interferon or ribavirin, were enrolled and randomly allocated one of three regimens: 3 mega units (MU) interferon alpha2b three times a week plus 1000-1200 mg ribavirin per day for 48 weeks; 3 MU interferon alpha2b three times a week plus 1000-1200 mg ribavirin per day for 24 weeks; or 3 MU interferon alpha2b three times a week and placebo for 48 weeks. All patients were assessed for safety, tolerance, and efficacy at the end of weeks 1, 2, 4, 6, and 8, and every 4 weeks during treatment. After treatment was completed patients were followed up on weeks 4, 8, 12, and 24. The primary endpoint was loss of detectable HCV-RNA (serum HCV-RNA <100 copies/mL) at week 24 after treatment. FINDINGS: Sustained virological response at 24 weeks after treatment, was found in 119 (43%) of the 277 patients treated for 48 weeks with the combination regimen, 97 (35%) of the 277 patients treated for 24 weeks with the combination regimen (p=O.055), and 53 (19%) of the 278 patients treated for 48 weeks with interferon alone (p<0.001 vs both combination regimens, intention-to-treat analysis). Logistic regression identified five independent factors significantly associated with response: genotype 2 or 3, viral load less than 2 million copies/mL, age 40 years or less, minimal fibrosis stage, and female sex. Among patients with fewer than three of these factors the odds ratio of sustained response was 2.6 (95% Cl 1.4-4.8; p=0.002) for the 48 week combination regimen compared with 24 weeks of the combination regimen. Discontinuation of therapy for adverse events was more frequent with combination (19%) and monotherapy (13%) given for 48 weeks than combination therapy given for 24 weeks (8%). INTERPRETATION: An interferon alpha2b plus ribavirin combination is more effective than 48 weeks of interferon alpha2b monotherapy and has an acceptable safety profile. Patients with few favourable factors benefit more from extending the duration of combination therapy to 48 weeks.

Administration, Oral↗
PubMed1999

La protein is required for efficient translation driven by encephalomyocarditis virus internal ribosomal entry site.

Translation of internal ribosomal entry site (IRES)-dependent mRNAs is mediated by RNA-binding proteins as well as canonical translation factors. In order to elucidate the roles of RNA-binding proteins in IRES-dependent translation, the role of polypyrimidine tract-binding protein (PTB) and La protein in encephalomyocarditis virus (EMCV) IRES-dependent translation was investigated. PTB was required for efficient EMCV IRES-driven translation but, intriguingly, an excess of PTB suppressed it. Such a translational suppression by surplus PTB was relieved by addition of La protein. A possible role for La protein in IRES-dependent translation is discussed.

Autoantigens↗
PubMed2003

Cell cycle regulation of hepatitis C and encephalomyocarditis virus internal ribosome entry site-mediated translation in human embryonic kidney 293 cells.

We have established stably transformed human embryonic kidney cell lines (HEK293) containing bicistronic constructs to study regulation of viral internal ribosome entry site (IRES)-mediated translation in vivo. These cells produce Renilla luciferase (Rluc) in a cap-dependent manner, while Firefly luciferase (Luc) synthesis is mediated by IRES elements. Using these cell lines, we demonstrate here that IRES-mediated translation directed by both hepatitis C (HCV) and encephalomyocarditis (EMCV) virus varies with the cell cycle. Experiments involving arrest of the cell lines at different phases of the cell cycle, release of synchronized cells from cell cycle arrest, as well as direct sorting of the cells based on position in the cell cycle have shown that the activity of the HCV and EMCV IRES elements is lowest during the G2/M phase in HEK293 cells. These results suggest that cellular trans-acting factors either stimulate viral IRES-mediated translation during G1 and S phases or repress translation during the G2/M phase in HEK293 cells.

5' Untranslated Regions↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1995

The human foamy virus internal promoter is required for efficient gene expression and infectivity.

The human foamy or spumaretrovirus (HFV) is a complex retrovirus that codes for the three retroviral genes gag, pol, and env and the regulatory and accessory bel genes. A particular feature of HFV gene expression was recently described: not only does the HFV provirus contain the classical retroviral long terminal repeat promoter, a second functionally active promoter is present in the env gene upstream of the bel genes (M. Löchelt, W. Muranyi, and R. M. Flügel, 1993, Proc. Natl. Acad. Sci. USA 90, 7317-7321). Both, the HFV long terminal repeat promoter I and internal promoter II depend upon the HFV transcriptional transactivator Bel 1 for efficient gene expression. The internal promoter directs the synthesis of functionally active Bel 1 transactivator and Bet proteins that are expressed early after HFV infection. In this report, it is shown that mutation of the promoter II TATA box resulted in HFV proviral clones with a reduction in infectivity by a factor of approximately 100. Gene expression by promoter II TATA box mutant HFV proviruses was reduced. HFV proviruses with the mutated promoter II TATA box used cryptic start sites of transcription upstream of the original promoter II TATA box, resulting in an inefficient and less accurate transcriptional initiation. The reduced HFV structural gene expression by the mutated HFV proviruses was relieved by providing Bel 1 protein in trans. This demonstrates that HFV promoter II-directed Bel 1 expression is important for producing the high levels of Bel 1 that increases virus replication.

Animals↗
PubMed2006

Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site.

The hepatitis C viral mRNA initiates translation using an internal ribosome entry site (IRES) located in the 5' noncoding region of the viral genome. At physiological magnesium ion concentrations, the HCV IRES forms a binary complex with the 40S ribosomal subunit, recruits initiation factor eIF3 and the ternary eIF2/GTP/Met-tRNA(i)Met complex, and joins 60S subunits to assemble translation-competent 80S ribosomes. Here we show that in the presence of 5 mM MgCl2, the HCV IRES can initiate translation by an alternative mechanism that does not require known initiation factors. Specifically, the HCV IRES was shown to initiate translation in a reconstituted system consisting only of purified 40S and 60S subunits, elongation factors, and aminoacylated tRNAs at high magnesium concentration. Analyses of assembled complexes supported a mechanism by which preformed 80S ribosomes can assemble directly on the HCV IRES at high cation concentrations. This mechanism is reminiscent of that employed by the divergent IRES elements in the Dicistroviridae, exemplified by the cricket paralysis virus, which mediates initiation of protein synthesis without initiator tRNA.

Aminoacylation↗
PubMed2005

SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.

A new class of small molecules that bind the HCV RNA IRES IIA subdomain with sub-micromolar affinity is reported. The benzimidazole 'hit' 1 with a KD approximately 100 microM to a 29-mer RNA model of Domain IIA was identified from a 180000-member library using mass spectrometry-based screening methods. Further MS-assisted SAR (structure-activity relationships) studies afforded benzimidazole derivatives with sub-micromolar binding affinity for the IIA RNA construct. The optimized benzimidazoles demonstrated activity in a cellular replicon assay at concentrations comparable to their KD for the RNA target.

Base Sequence↗
PubMed2005

Amantadine inhibits hepatitis A virus internal ribosomal entry site-mediated translation in human hepatoma cells.

The effect of six drugs (amantadine, glycyrrhizin, ribavirin, ursodeoxycholic acid, alcohol, and IFN) on HAV RNA translation from the HAV internal ribosomal entry site (IRES) was investigated using a bicistronic reporter construct containing HAV IRES as intragenic spacer. Huh-7 cells and derivatives were transfected with in vitro transcripts, and the reporter gene activity was determined. IFN suppressed both cap-dependent and HAV IRES-dependent translation, while amantadine specifically inhibited HAV IRES-dependent translation. In contrast to IFN, by reporter assay, amantadine did not activate the interferon-stimulated response element (ISRE) or interferon gamma-activated sequence (GAS)-associated pathways. Immunoblot analysis revealed that amantadine had no effect on PKR and on IFN-regulatory factor-1 (IRF-1) expression. These findings demonstrated a novel antiviral effect of amantadine against HAV with or without HCV infection.

Amantadine↗