2'-5'-oligoadenylate synthetase gene expression in normal and murine sarcoma virus-transformed NIH 3T3 cells.
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Mouse fibroblasts transformed by murine sarcoma virus (MSV) are highly sensitive to the antiproliferative effect of interferon (IFN) (M. Bakhanashvili, D. H. Wreschner, and S. Salzberg, Cancer Res. 43:1289-1294, 1983). To elucidate the mechanism leading to this IFN sensitivity, the expression of the 2'-5'-oligoadenylate synthetase (2-5A synthetase) gene, the presence of the 2-5A synthetase protein, and the level of its enzymatic activity were determined in IFN-treated and untreated cultures. NIH 3T3 mouse fibroblasts were compared with two different NIH 3T3 clones transformed by MSV. Cultures were treated with 300 IU of beta IFN (IFN-beta) per ml for 16 to 24 h. While no detectable 2-5A synthetase-derived transcripts were seen in untreated NIH 3T3 cells, two size classes of RNA transcripts, i.e., 1.7 and 4.2 kilobases, were detected in IFN-treated cultures. Surprisingly, a similar amount of transcripts were present in untreated transformed cells. However, following IFN treatment, an eightfold increase in the level of RNA was readily detected in these cells, with no change in the size classes. Similar results were obtained with the 2-5A synthetase protein, for which three size classes of 42, 71, and 102 kilodaltons were demonstrated by immunoblotting, and with the enzymatic activity, for which again, the highest level was seen in IFN-treated MSV-transformed cultures. The basal level of 2-5A synthetase gene expression in the transformed cells has biological significance since these cells were more resistant to mengovirus infection than NIH 3T3 mouse fibroblasts. Medium collected from transformed cultures failed to induce 2-5A synthetase activity in NIH 3T3 cells. Furthermore, antibodies directed against mouse IFN-beta failed to inhibit 2-5A synthetase activity detected in transformed cultures. These results suggest that at least IFN-beta secretion is not involved in the elevated level of 2-5A synthetase gene expression in these cells. (+info)
Cloning and synthesis of infectious cardiovirus RNAs containing short, discrete poly(C) tracts.
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Mengovirus RNA transcripts with 5' noncoding poly(C) tracts of C8, C12, and C13UC10 have been synthesized in vitro from cDNA clones and shown to be infectious to HeLa cells. A chimeric clone has also been constructed which links the 5' end from one mengovirus clone (299 nucleotides, containing C13UC10) to a 7,424-base fragment derived from the 3' end of encephalomyocarditis (EMC) virus. Progeny virus isolated after transfection with the clone-derived RNAs had the same poly(C) tracts, mengovirus-specific sequences, or EMC virus-specific sequences as the transcript from which it was derived. Although the cloned poly(C) tracts were considerably shorter than those found in viral RNA from mengovirus (C50UC10) or EMC virus (C115UCUC3UC10), the growth characteristics of the progeny viruses in HeLa cells were indistinguishable from those of the parental viruses, indicating the length of this tract does not play a significant restrictive role for cardiovirus infectivity in tissue culture. (+info)
A theoretical study of the acidification of the rhinovirus capsid.
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Electrostatic calculations for human rhinovirus 14 indicate that histidine-base residue pairs in the region of a beta-strand interaction between pentamers may be involved in a pH-induced process that leads to the release of viral RNA. Other picornavirus sequences are examined for these residue pairs, a subset of which is present in enteroviruses. Foot and mouth disease virus possesses one of the residue pairs, and cardioviruses, which undergo a separate pH and halide ion-induced capsid dissociation, possess none. (+info)
Selection and characterization of interferon response mutants from mouse L929 fibroblast cells.
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Interferon (IFN) response mutants were selected from mouse L929 fibroblast cells and their specific resistance to is-1, an IFN-sensitive mutant of mengovirus, was studied. The standard L cell subline used in our laboratory (G3), is resistant to is-1 infection after pretreatment with low levels of IFN. Two clonal sublines that support the growth of is-1 in the presence of IFN (AS-4 and TA-6) were isolated from it, and two revertant lines (AS-4R1 and TA-6R1) were subsequently selected from AS-4 and TA-6. The kinetics of is-1 growth in the presence of IFN were found to vary in each of these sublines. Specific resistance to is-1 cannot be accounted for by enhanced induction of IFN, ability to bind IFN, or increased 2'-5'-oligo(A)-dependent endonuclease activity. AS-4 and TA-6 appear to have arisen through loss of one or more whole chromosomes. The origin of TA-6R1 is unclear. (+info)
Biological activities of human recombinant interferon alpha/beta targeted by anti-Epstein-Barr virus monoclonal antibodies.
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The requirement of high doses of interferon (IFN) during therapy severely restrict its application. Thus a model using an Epstein-Barr virus (EBV) membrane antigen (MA) specific monoclonal antibody (MAb) was developed to assess the feasibility of coupling minimal amounts of IFN to a MAb and specifically delivering the IFN to the target cells. Coupled IFN was first shown to retain fully both its anti-viral and anti-proliferative properties when tested on human tumor cell lines QIMR-WIL (EBV-MA+) and the U-266 (EBV-MA-). A series of in vitro pulsing experiments demonstrated the specific targeting of both the anti-viral and anti-proliferative properties of IFN to the EBV-MA+ QIMR-WIL cells and not EBV-MA- cell lines. (+info)
Evidence that IFN-alph/beta induces two antiviral states active against different viruses.
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IFN-alpha/beta has been suggested to require only one round of mRNA and protein synthesis to induce an antiviral state. We have examined the mechanism of induction of the antiviral states shown against three types of viruses: mengovirus (plus strand, sense RNA), vesicular stomatitis virus (VSV, minus strand RNA), and vaccinia virus (DNA). Mouse L cells were treated with IFN-alpha/beta and cycloheximide and then with actinomycin D on a schedule which allowed only one round of mRNA and protein synthesis. The cells were challenged with virus under single cycle growth conditions to determine the amount of antiviral activity against the particular challenge virus employed. These studies confirmed that most of the antiviral effect directed against VSV is achieved with one round of macromolecular synthesis. However, most of the antiviral effect directed against mengovirus and vaccinia virus seemed to require more than one round. These results suggest that IFN-alpha/beta induces two different antiviral states: one requiring one round of synthesis which is primarily responsible for the inhibition observed for VSV; and another requiring more than one round of synthesis which is primarily responsible for the inhibition observed for mengovirus and vaccinia virus. (+info)
Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.
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After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation. (+info)
Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs.
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The RNA genome of poliovirus hybridizes to 28S and 18S rRNAs of higher eukaryotes under stringent conditions. The hybridization detected by Northern blot analyses is specific since little or no signal was detected for yeast or prokaryotic rRNAs or other major cellular RNAs. Southern blot analysis of DNA clones of mouse rRNA genes leads us to conclude that several regions of 28S rRNA, and at least one region in 18S rRNA, are involved in the hybridization to polio RNA, and that G/C regions are not responsible for this phenomenon. We have precisely mapped one of these hybridizing regions in both molecules. Computer analysis confirms that extensive intermolecular base-pairing (81 out of 104 contiguous bases in the rRNA strand) could be responsible for this one particular site of interaction (polio genome, bases 5075-5250; 28S rRNA, bases 1097-1200). We discuss the possible functional and/or evolutionary significance of this novel type of interaction. (+info)