Interferon-stimulated response element (ISRE)-binding protein complex DRAF1 is activated in Sindbis virus (HR)-infected cells.

To elucidate the host cell defense mechanisms in response to Sindbis viral infection, we have started to characterize interferon (IFN)-stimulated response element (ISRE)-binding proteins activated in infected cells that are involved in the transcriptional induction of IFN type I-inducible genes. Using electromobility shift assays (EMSA), we detected several protein complexes with a human IFN-stimulated gene 15 (ISG15) ISRE in extracts from virus-infected L929 cells that were absent in extracts from uninfected cells. Comigration with Newcastle disease virus-activated ISRE-binding complexes, ISRE-binding specificity, supershift experiments, and conditions of formation indicate that the complexes activated by Sindbis viral infection in L929 cells correspond to DRAF1 and ISG factor 3 (ISGF3). Transfection of L929 cells with poly rI:rC induced only ISGF3. DRAF1 could be detected in Sindbis virus-infected mouse embryo fibroblasts derived from IFNR type I and type II KO mice. Viral RNA synthesis is required for activation of DRAF1.

Sequence comparison of avian interferon regulatory factors and identification of the avian CEC-32 cell as a quail cell line.

Interferon (IFN) regulatory factor-1 (IRF-1) is a well-characterized member of the IRF family. Previously, we have cloned cDNA of several members of the chicken IRF (ChIRF) family and studied the function of ChIRF-1 in the avian cell line CEC-32. The IRF-1 proteins from primary chicken embryo fibroblasts (CEF) and CEC-32 cells differed in their electrophoretic mobility. To characterize the different forms of IRF-1 in avian cells, we compared the sequences of IRF-1 cDNA from CEC-32 cells, primary CEF, and quail fibroblasts (QEF). The deduced amino acid sequences of IRF-1 cDNA from chicken and quail show high similarity. Comparison of genomic sequences of IRF-1 and IFN consensus sequence binding protein (ICSBP) also confirm the relatedness of the members of the IRF family in quail and chicken. Based on these data, it is concluded that the avian fibroblast cell line CEC-32 is derived from quail. This conclusion is further supported by deoxynucleotide sequence comparison of a DNA fragment in an avian MHC class II gene and by fluorescence in situ hybridization (FISH) using the vertebrate telomeric (TTAGGG) repeat. Chromosome morphology and the lack of interstitial hybridization signals in macrochromosomes suggest that the CEC-32 cell line has probably been derived from Japanese quail.

Allosteric properties of cyanobacterial cytochrome c oxidase: inhibition of the coupled enzyme by ATP and stimulation by ADP.

Thorough analysis of the cta operon of Synechocystis sp. PCC6803 (grown in high-concentration salt medium to enhance the expression of respiratory proteins) showed that, apart from ctaCDE and Fb genes potentially encoding subunits I, II, III, and a small pseudo-bacteria-like subunit-IV of unknown function, a large mitochondria-like cta-Fm gene and a pronounced terminator structure are additional components of the operon. The deduced cta Fm gene product shows approximately 50% and 20% sequence identity to the Saccharomyces cerevisiae and beef heart mitochondrial COIV proteins, respectively. It also shows amino acid regions (near the N terminus, on the cytosolic side) with conspicuous sequence similarities to adenylate-binding proteins such as ATP synthase beta subunit Walker A and B consensus regions or to adenylate kinase. We suggest that, similar to the situation with beef heart mitochondria, it is the mitochondria-like subunit-IV of the cyanobacterial aa3-type cytochrome-c oxidase that confers allosteric properties to the cyanobacterial enzyme, the H+/e- ratios of cytochrome c oxidation being significantly lowered by ATP (intravesicular or intraliposomal) but enhanced by ADP. Therefore, the antagonistic action of ATP and ADP was in a way that the redox reaction proper, was always significantly less affected than the coupled proton translocation. Evolutionary and ecological implications of the unusual allosteric regulation of a prokaryotic cytochrome-c oxidase is discussed.

Overexpression of chicken interferon regulatory factor-1 (Ch-IRF-1) induces constitutive expression of MHC class I antigens but does not confer virus resistance to a permanent chicken fibroblast cell line.

The chicken fibroblast cell line C32 has been transfected with the chicken homolog (Ch-IRF-1) of the mammalian transcription factor IRF-1. Stable transfectants were generated, constitutively overexpressing Ch-IRF-1 mRNA and protein. Cells overexpressing Ch-IRF-1 showed enhanced constitutive expression of MHC class I (B-F, beta-microglobulin) antigens. With increasing number of passages cells with normal B-F IV surface antigen expression accumulated. In the revertants, the amount of Ch-IRF-1 mRNA was reduced. Overexpression of Ch-IRF-1 had no effect on the constitutive expression and the induction by chicken interferon type-I and type-II (Ch-IFN) of guanylate-binding protein (GBP). Susceptibility to vesicular stomatitis virus, sindbis virus, Newcastle disease virus and vaccinia virus was not altered by overexpression of Ch-IRF-1. An antiviral state could be induced against all viruses tested by similar amounts of Ch-IFN type I in clone 20-18 expressing Ch-IRF-1 and cells transfected with empty vector.

The genomic structure of the chicken ICSBP gene and its transcriptional regulation by chicken interferon.

The chicken interferon consensus sequence binding protein (ChICSBP) gene spans over 9 kb of DNA and consists, as its murine homolog, of nine exons. The first untranslated exon was identified by 5'-RACE technology. The second exon contains the translation initiation codon. Canonical consensus splice sites are found on every exon/intron junction. The introns are generally smaller than their mammalian counterparts. The ChICSBP and ChIRF-1 genes have been mapped by fluorescence in situ hybridization to different microchromosomes. The transcription start site has been mapped by primer extension. Inspection of the DNA sequence of a genomic clone containing the first exon and the region 1700-bp upstream revealed several potential cisregulatory elements of transcription. The ChICSBP mRNA is induced by recombinant ChIFN type I and ChIFN-gamma. A palindromic IFN regulatory element (pIRE) with high sequence homology to gamma activation site (GAS) sequences was functionally required in transient transfection assays for the induction of transcription by ChIFN-gamma.

Cloning of chicken interferon regulatory factor-2 (IRF-2) cDNA: expression and mapping of the IRF-2 gene.

A cDNA clone encoding a member of the avian interferon regulatory factor (IRF) family homologous to mammalian IRF-2 was isolated from cDNA library from poly[rI:rC]-induced chicken embryo fibroblasts (CEF). The deduced amino acid sequence shows a characteristic DNA binding domain of 124 amino acids at the amino-terminal end with 96.8% identity to human and 96% to mouse IRF-2. Identities in the C-terminal part are 77.5% and 77%, respectively. Identity to all other known members of the chicken IRF (Ch-IRF) family is distinctly lower. In C32 cells, an IRF-2 mRNA of 2.4 kb is constitutively expressed in very low amounts but is inducible by Ch-IFN in the absence or presence of cycloheximide. The Ch-IRF-2 gene is a single copy gene and was mapped by fluorescence in situ hybridization to the long arm of chromosome 4.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Interferon sensitivity of expression of histone H5/H1(0)-vaccinia thymidine kinase fusion genes expressed by recombinant vaccinia viruses is enhanced by shortening the histone sequence.

To elucidate the structural basis responsible for the reduced IFN sensitivity of expression of the histone H1(0) and H5 gene, integrated into the vaccinia virus genome, vaccinia virus thymidine kinase (VV-TK)-histone H1(0)/H5 fusion genes were constructed and translocated into the TK locus of the VV genome. The chimeric genes, consisting of parts of either of the two histone genes and the 5' or 3' half of the TK gene, respectively, were expressed as histone-TK fusion proteins under the control of either the VV-TK promoter or the early sequences of the VV 7.5K promoter. IFN sensitivity of the expression of histone-TK fusion genes was shown to be influenced by the relative length of the histone sequence. Expression of fusion genes containing more than 45% cellular sequence either from the 5' or the 3' part of one of the two histone genes showed clearly reduced IFN sensitivity compared to the expression of VV-TK. On the other hand, by further reducing the relative amount of histone H5 or H1(0) sequence to 32%, the IFN sensitivity of expression of the corresponding fusion gene was drastically enhanced to levels indistinguishable from those of VV-TK.

Interferon induction of chicken MHC class I gene expression: phylogenetic conservation of the interferon-responsive element.

The 5' upstream region of a chicken MHC class I gene BF-IV contains sequence motifs similar to the interferon consensus sequences (ICS) contained in promoters of many mammalian interferon-regulated genes. To study a possible functional role of this putative chicken ICS, an oligonucleotide spanning the upstream sequences of the BF-IV gene (-174/-194) was cloned singly or in multiple copies before the herpes TK promoter controlling the chloramphenicol acetyl transferase (CAT) gene (pBLCAT2). Transient expression studies performed with primary chicken fibroblasts (CEF) showed that the chicken ICS represses constitutive promoter activity. The chicken ICS, however, enhanced CAT activity up to 20-fold following treatment with chicken interferon (IFN). Deletion analysis of the BF-IV promoter also confirms that the upstream DNA sequences (-174/-194) contain a functional ICS recognized by chicken interferon. The murine ICS of the H2-Ld gene was also activated by chicken interferon when introduced into CEF. IFN activation of chicken ICS containing reporters was also observed in transformed chicken fibroblast lines. We show that the chicken ICS binds two specific nuclear factors present in chicken fibroblasts which are induced by interferon. These factors were also capable of recognizing the mouse ICS, suggesting the conservation of a relevant DNA-binding protein. Taken together, these data indicate that the chicken ICS motif contained in a sequence from -174 to -194 of the BF-IV gene acts as a strong interferon-response element, which has been functionally conserved during about 270 million years of separate evolution of mammals and birds.

Expression of authentic vaccinia virus-specific and inserted viral and cellular genes under control of an early vaccinia virus promoter is regulated post-transcriptionally in interferon-treated chick embryo fibroblasts.

The interferon sensitivity of the expression of an influenza-virus hemagglutinin (HA) gene cloned into the thymidine kinase (TK) gene of vaccinia virus was studied in chick embryo fibroblasts (CEF) and Madin-Darby bovine kidney (MDBK) cells. In CEF, the expression of the HA gene is inhibited by pretreatment of cells with homologous interferon. In MDBK cells, on the other hand, expression of the HA is not impaired by pretreatment with human interferon-alpha, and the synthesis of early vaccinia virus enzymes was also unaffected. These results indicate that the interferon sensitivity of HA gene expression is at least in part controlled by flanking regions of vaccinia virus DNA. In this report, we also address the question whether the expression of an influenza virus HA gene and the human histone H1 zero gene under control of a vaccinia virus immediate early promoter is affected in interferon-treated CEF by a post-transcriptional mechanism in the same way as the expression of the viral TK gene. In interferon-treated cells mRNA synthesis specific for all these genes was enhanced. Steady state mRNA levels 6 hr p.i. were, however, lower than the amounts expected from the rate of mRNA synthesis during the first 6 hr p.i., suggesting that part of the viral RNA was degraded. Degradation resistant mRNA accumulated in the interferon-treated cells in an amount comparable to that found in infected CEF. This RNA could be translated into viral protein in a cell-free system. Therefore the degradation of viral mRNA cannot solely be responsible for the inhibition of viral protein synthesis in interferon-treated cells.

Interferon-gamma-induced assembly block in the replication cycle of adenovirus 2: augmentation by tumour necrosis factor-alpha.

Replication of adenovirus 2 (Ad-2) is inhibited in A 549 cells pretreated with interferon-gamma (IFN-gamma). The antiviral effect is synergistically enhanced by the simultaneous presence of tumor necrosis factor-alpha (TNF-alpha) before infection. Under conditions of strong inhibition of virus progeny formation, viral DNA synthesis and [35S]methionine incorporation into most late viral proteins are only marginally impaired. Pulse chase experiments indicate a partial inhibition of processing of viral proteins. Viral proteins are not degraded and capsomeres accumulate in the inhibitor-treated cells. Capsid formation, on the other hand, is strongly inhibited in the cytokine-treated cells. The inhibition of Ad-2 replication in A 549 cells by IFN-gamma and TNF-alpha is caused, therefore, by a block in the maturation of Ad-2.

Regulation of histone H5 and H1 zero gene expression under the control of vaccinia virus-specific sequences in interferon-treated chick embryo fibroblasts.

The duck histone H5 and human H1 zero were inserted into the thymidine kinase (TK) gene of vaccinia virus and the interferon sensitivity of their expression under the control of the viral TK and P7.5 promoters in chick embryo fibroblasts (CEF) was compared to the interferon sensitivity of vaccinia virus WR specific TK induction. Expression and transport of these histones to the nucleus in CEF infected with the appropriate vaccinia virus recombinants could be detected with antisera raised against chick histone H5. In CEF cultivated for 3 days, interferon treatment that completely inhibited TK synthesis had no or only a marginal inhibitory effect on the expression of the histone genes. Inhibition of the expression of the histones could be detected under conditions of increased interferon sensitivity in aged CEF. The magnitude of inhibition was, however, less pronounced than the inhibition of viral TK synthesis. These data indicate that flanking vaccinia virus DNA regions confer interferon sensitivity to the expression of these histone genes, but that they contain structural information that partially exempts their expression from the inhibitory activity of the interferon-induced regulatory system.

Two interferon sensitive steps in the replication cycle of Rous sarcoma virus.

De novo infection of Rous sarcoma virus (RSV) strains of receptor subgroups A, B, C, and D is inhibited by low doses of chick interferon. Adsorption and penetration into the cell are not marginally impaired by interferon treatment. Since the level of proviral DNA synthesis is strongly reduced in the interferon-treated cell it is concluded that uncoating or reverse transcription of the viral genome is inhibited. This inhibition of proviral DNA synthesis is not caused by an arrest of CEF in the stationary phase. Chronic infection of SR-RSV-A, -B, -C, but not SR-RSV-D is also sensitive to interferon. Chick interferon treatment (50 u/ml) also had no inhibitory effect on the amount of transcripts of the RSV-specific oncogene src or the cellular oncogenes src and myc.

Increased turnover of vaccinia virus-specific immediate early RNAs in interferon-treated chick embryo fibroblasts.

The synthesis and steady state level of immediate early vaccinia virus-specific RNAs in interferon-treated chick embryo fibroblasts were determined by blot hybridization analysis using the cloned restriction endonuclease fragment pEJ 18 containing the gene of vaccinia virus WR-specific DNA polymerase as a probe. Even though early vaccinia virus WR RNA was still synthesized, accumulation of immediate early viral RNAs was strongly inhibited. Accumulation of beta-actin RNA was not affected. This indicated an enhanced degradation of vaccinia virus WR-specific early RNAs in interferon-treated chick embryo fibroblasts. This notion was supported by Northern blot analysis which revealed degradation of residual RNA of vaccinia virus WR-specific DNA polymerase. In contrast to interferon-treated mouse L 929 cells, ribosomal RNA is not degraded in interferon-treated vaccinia WR-infected chick embryo fibroblasts.

Reduced steady-state levels of vaccinia virus-specific early mRNAs in interferon-treated chick embryo fibroblasts.

The molecular mechanism of interferon action on vaccinia virus-specific immediate early protein synthesis was studied in interferon-treated chick cells. In line with previous observations, the synthesis of total vaccinia WR virus-specific mRNA, thymidine kinase (TK) mRNA, and several other early mRNAs was detectable by short [3H]uridine pulses. Under conditions of over 90% inhibition of poxvirus-specific TK induction, accumulation of TK mRNA was strongly inhibited. Northern blot analysis revealed strong degradation of residual TK mRNA prepared from interferon-treated chick embryo fibroblasts (CEF). Blot hybridization analysis using total vaccinia DNA and restriction fragment N as probes demonstrated a generally reduced steady-state amount of vaccinia virus-specific early mRNAs in interferon-treated CEF. When CEF were infected with a recombinant vaccinia virus strain into the TK gene of which the chloramphenicol acetyltransferase gene had been inserted, CAT activity was far lower in interferon-treated than in untreated CEF. We conclude that signals that specify rapid breakdown of viral TK mRNA in interferon-treated CEF are located in the regions flanking the coding sequences of the viral TK gene.

Sensitivity of ortho- and paramyxovirus replication to human interferon alpha.

Replication of the influenza virus strains Influenza Ao/WSN (H0N1), fowl plague (Hav1N1) and B-Lee/40 (ATCC) and the paramyxovirus, New Castle disease virus (Victoria) are highly sensitive to human interferon type alpha in Madin Darby bovine kidney cells. Pretreatment of cells with human interferon type alpha resulted in protection of the cells against viral cytopathic effect. The inhibition of the orthomyxovirus strains used in this study and New Castle disease virus replication is mediated by an inhibition of viral protein synthesis. Residual WSN virus particles released from interferon treated cells showed the same structural protein pattern as virus particles isolated from control cells. Glycosylation of the viral structural components appeared to be unaffected by interferon.

Vinculin and 36 kDa protein are not tyrosine-phosphorylated in Rous sarcoma virus infected cells which have been treated with interferon.

The expression of membrane-associated transformation-specific parameters was analyzed in de novo Rous sarcoma virus (strain SR-RSV-D) infected chicken embryo fibroblasts pretreated with homologous interferon. Cellular morphology, hexose transport, microfilament organization, and tyrosine-phosphate content of two primary substrates of the transformation-generating viral kinase, pp60src, were found indistinguishable from non-infected controls. These observations support the hypothesis that vinculin and possibly 36 kDa protein are involved in microfilament organization and that tyrosine-phosphorylation of these structural proteins is a prerequisite for the rearrangement of microfilaments during transformation. In de novo infection, interferon pretreatment reduces viral protein synthesis and pp60src activity as compared to non-treated, SR-RSV-D infected cells. However, the phosphotyrosine content of total cellular proteins as measured under steady state conditions is as high in interferon-pretreated as in nontreated transformed cells.

Purification of chick interferon by zinc chelate affinity chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

An improved purification method for chick interferon from the allantoic fluid of embryonated chick eggs is described. Interferon prepurified by perchloric acid treatment, zinc acetate precipitation, and chromatography on SP-Sephadex C-25 was further enriched by column chromatography on zinc chelate. Analysis on sodium dodecylsulfate polyacrylamide gel electrophoresis of the interferon preparation with a specific activity of 8 X 10(5) units/mg protein shows that the major antiviral activity migrated in a broad band in the range of 20-29 kD molecular weight. Several protein bands were stainable with Coomassie blue and silver nitrate in this molecular weight range. Between 80 and 95% of the total protein charged to the gel could be removed from the interferon containing fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis.