Use of an internal ribosome entry site for bicistronic expression of Cre recombinase or rtTA transactivator.
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Conditional gene targeting depends on tissue and time specificity of recombination events. Endogenous promoters are often used to drive various transgenic constructs. To avoid the problems associated with reconstituting a specific expression pattern in transgenic animals by this method, we tested the internal ribosome entry site of the encephalomyocarditis virus, to enable linkage of the Cre recombinase or rtTA trans-activator to 3' untranslated ends of endogenous genes. Here we report that these constructs function effectively in COS cells. The data suggest that these cassettes will be appropriate for 3' targeting of mouse genes. (+info)
Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus.
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A cDNA encoding the human guanylate binding protein-1 (hGBP-1) was expressed in HeLa cells using a constitutive expression vector. Stably transfected clones expressing hGBP-1 exhibited resistance to the cytopathic effect mediated by both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) and produced less viral progeny than control cells following infection with these viruses. To study the role hGBP-1 plays in the IFN-mediated antiviral effect, cells were stably transfected with a construct expressing antisense RNA for hGBP-1. VSV infection of IFN-alpha-treated antisense RNA-expressing cells produced an amount of virus comparable to that produced in the parental cell line, while EMCV infection of the IFN-alpha-treated transfected cells and VSV and EMCV infection of the IFN-gamma-treated transfected cells produced far more virus than was produced in the parental cell line. These results demonstrate that GBP-1 mediates an antiviral effect against VSV and EMCV and plays a role in the IFN-mediated antiviral response against these viruses. (+info)
Identification and characterization of a protein destruction signal in the encephalomyocarditis virus 3C protease.
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The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3alpha, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease. (+info)
Targeted overexpression of elafin protects mice against cardiac dysfunction and mortality following viral myocarditis.
(4/642)
Serine elastases degrade elastin, stimulate vascular smooth muscle cell migration and proliferation, and are associated with myocardial damage. To evaluate the impact of elastase inhibition on cardiovascular development and disease, transgenic mice were created in which the mouse preproendothelin-1 promoter was used to target elafin overexpression to the cardiovascular system. To distinguish the transgene from endogenous elafin, constructs were made incorporating a FLAG sequence; the COOH-terminus FLAG-tagged elafin construct produced a stable, functionally active gene product and was used to create transgenic mice. Consistent with endothelin expression, abundant elafin mRNA was observed in transgenic F1 embryos (embryonic day 13.5) and in adult transgenic mice heart, trachea, aorta, kidney, lung, and skin, but not in liver, spleen, and intestine. Functional activity of the transgene was confirmed by heightened myocardial elastase inhibitory activity. No tissue abnormalities were detected by light microscopy or elastin content. However, injection of 10 plaque-forming units (PFU) of encephalomyocarditis virus resulted in death within 11 days in 10 out of 12 nontransgenic mice compared with one out of nine transgenic littermates. This reduced mortality was associated with better cardiac function and less myocardial inflammatory damage. Thus, elafin expression may confer a protective advantage in myocarditis and other inflammatory diseases. (+info)
Encephalomyocarditis (EMC) virus infection in PC12 and C6 cells.
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PC12 cells derived rom rat pheochromocytoma and C6 cells derived from rat glioma were infected with 0.3 plaque forming units (PFU)/cell of the D variant of encephalomyocarditis virus (EMC-D), after pretreatment with or without nerve growth factor (NGF). The virus titres in medium and cells were investigated at 6, 12, 24, 48 and 72 h post infection (HPI), and histopathology and viral antigens in cells were examined at 24 and 48 HPI, respectively. As a result, neither viral replication nor light and electron microscopic changes were observed in PC12 cell cultures without NGF-pretreatment. On the contrary, in PC12 cell cultures with NGF-pretreatment, the virus titre prominently increased at 12 HPI, and peaked at 48 HPI. In addition, distinct histological and ultrastructural changes with viral antigens in cells were observed. C6 cells showed similar morphology and susceptibility to EMC-D-infection irrespective of NGF-pretreatment. Namely, the virus titres in C6 cell cultures increased slightly and viral antigens were found in a small number of C6 cells, but there were no evident histological and ultrastructural changes. These results suggest that PC12 cells pretreated with NGF and C6 cells are susceptible to EMC-D infection in vitro. (+info)
Interstitial fibrin-fibronectin deposition with T cell infiltrates precedes fibrosis in murine viral myocarditis.
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This study was carried out to investigate interstitial fibrin and fibronectin deposition and subsequent myocardial connective tissue abnormalities in BALB/c-nu/+ (euthymic and normal T cell function) and BALB/c-nu/nu (athymic and T cell-deficient) mice. Both types of mice were inoculated with encephalomyocarditis virus and sacrificed periodically. Sections of the hearts were stained with haematoxylin-eosin, trichrome, lymphocyte subsets, silver impregnation, and fibrin or fibronectin. In addition, myocardial collagen concentration was measured. Interstitial fibrin and fibronectin appeared in parallel with inflammatory T lymphocytes and myocardial necrosis in the BALB/c-nu/+ mice. The changes increased until 14 days, subsequently decreasing with time. Interstitial fibrosis and abnormal reticulin fibres were absent until 7 days postinfection, and then increased with time until 60 days. In BALB/c-nu/nu mice, in contrast, although myocardial necrosis and fibrin-fibronectin deposition associated with immature T lymphocytes were evident on days 7 and 14, subsequent myocardial fibrosis and reticulin fibre abnormalities were minimal on days 30 and 60. In BALB/c-nu/+ mice, myocardial collagen concentration increased on day 30, but it did not in BALB/c-nu/nu mice. Thus, interstitial fibrin-fibronectin deposition resulting from virus-induced and T lymphocyte-mediated myocyte necrosis precedes the subsequent development of interstitial fibrosis and abnormal reticulin architectures in this model of murine myocarditis. (+info)
Interferon action in triply deficient mice reveals the existence of alternative antiviral pathways.
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Antiviral proteins encoded by the interferon (IFN)-stimulated genes provide a front-line defense against viral infections. In particular, PKR, RNase L, and Mx are considered to be the principal proteins through which IFNs mount an antiviral state. To determine whether alternative antiviral pathways exist, RNase L-/- mice and PKR-/- mice were crossed onto an Mx1(-/-) background to generate a strain of triply deficient (TD) mice. After infections with encephalomyocarditis virus, the TD mice died 3-4 days earlier than infected, wild-type mice. However, there was an IFN dose-dependent increase in survival times after encephalomyocarditis virus infections for both the TD and wild-type mice. Mice that were deficient for PKR or RNase L showed intermediate survival times between those of the TD and wild-type mice. Surprisingly, cultured embryonic fibroblasts lacking RNase L, PKR, or both proteins were still able to mount a substantial residual antiviral response against encephalomyocarditis virus or vesicular stomatitis virus after IFN-alpha treatments. These results confirm the antiviral functions of RNase L and PKR in vivo but also provide unequivocal evidence for the existence of novel, innate immune pathways against viruses. (+info)
The properties of chimeric picornavirus IRESes show that discrimination between internal translation initiation sites is influenced by the identity of the IRES and not just the context of the AUG codon.
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The internal ribosome entry segment (IRES) of picornaviruses consists of approximately 450 nt of 5'-untranslated region, terminating at the 3' end with an approximately 25 nt element consisting of an absolutely conserved UUUC motif followed by a more variable pyrimidine-rich tract and G-poor spacer, and finally an AUG triplet, which is considered to be the actual ribosome entry site. Events following entry at this site differ among picornaviruses: in encephalomyocarditis virus (EMCV) virtually all ribosomes initiate translation at this site (AUG-11); in foot-and-mouth-disease virus (FMDV), one-third of the ribosomes initiate at this AUG (the Lab site), and the rest at the next AUG 84 nt downstream (Lb site); and in poliovirus (PV), the AUG at the 3' end of the IRES (at nt 586 in PV type 1) is considered to be a silent entry site, with all ribosomes initiating translation at the next AUG downstream (nt 743). To investigate what determines this different behavior, chimeras were constructed with a crossover at the conserved UUUC motif: the body of the IRES, the sequences upstream of this UUUC motif, was derived from one species, and the downstream sequences from another. When the body of the FMDV or PV IRESes was replaced by that of EMCV, there was a marked increase in the absolute and relative frequency of initiation at the upstream AUG, the Lab site of FMDV and 586AUG of PV, respectively. In contrast, when the body of the EMCV IRES was replaced by that of PV, initiation occurred with no preference at three AUGs: the normal site (AUG-11), AUG-10 situated 8 nt upstream, and AUG-12, which is 12 nt downstream. Thus although the context of the AUG at the 3' end of the IRES may influence initiation frequency at this site, as was shown by improving the context of 586AUG of PV, the behavior of the ribosome is also highly dependent on the nature of the upstream IRES. Delivery of the ribosome to this AUG in an initiation-competent manner is particularly efficient and accurate with the EMCV IRES. (+info)