Impaired antiviral response and alpha/beta interferon induction in mice lacking beta interferon.

We have generated mice lacking the gene for beta interferon and report that they are highly susceptible to vaccinia virus infection. Furthermore, in cultured embryo fibroblasts, viral induction of alpha interferon and of 2-5A synthetase genes is impaired. We also show that beta interferon does not prime its own expression.

Reconstitution of virus-mediated expression of interferon alpha genes in human fibroblast cells by ectopic interferon regulatory factor-7.

Type I interferons constitute an important part of the innate immune response against viral infection. Unlike the expression of interferon (IFN) B gene, the expression of IFNA genes is restricted to the lymphoid cells. Both IFN regulatory factor 3 and 7 (IRF-3 and IRF-7) were suggested to play positive roles in these genes expression. However, their role in the differential expression of individual subtypes of human IFNA genes is unknown. Using various IFNA reporter constructs in transient transfection assay we found that overexpression of IRF-3 in virus infected 2FTGH cells selectively activated IFNA1 VRE, whereas IRF-7 was able to activate IFNA1, A2, and A4. The binding of recombinant IRF-7 and IRF-3 to these VREs correlated with their transcriptional activation. Nuclear proteins from infected and uninfected IRF-7 expressing 2FTGH cells formed multiple DNA-protein complexes with IFNA1 VRE, in which two unique DNA-protein complexes containing IRF-7 were detected. In 2FTGH cells, virus stimulated expression of IFNB gene but none of the IFNA genes. Reconstitution of IRF-7 synthesis in these cells resulted, upon virus infection, in the activation of seven endogenous IFNA genes in which IFNA1 predominated. These studies suggest that IRF-7 is a critical determinant for the induction of IFNA genes in infected cells.

Activation of the interferon-inducible (2'-5') oligoadenylate synthetase by the Epstein-Barr virus RNA, EBER-1.

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.

A regulatory domain within the virus-response element of the interferon alpha 1 gene acts as a transcriptional repressor sequence and determinant of cell-specific gene expression.

Type-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN-beta is induced in a variety of cells of different origin, while not all of these are able to secrete IFN-alpha. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells.

Relative transcriptional inducibility of the human interferon-alpha subtypes conferred by the virus-responsive enhancer sequence.

This paper addresses the role of transcriptional regulation in the determination of the levels of expression of different interferon-alpha subtypes secreted from Namalwa cells following infection with Sendai virus. Using RT-PCR to determine the relative abundance of mRNA species coding for the various subtypes, we found a general correlation with corresponding protein levels, indicative of a role for transcriptional control in the determination of levels of individual subtypes. We have used reporter gene constructs to compare the inducibility of the virus-response elements from the IFNA1, A2, A4, and A14 subtype genes cloned upstream of a secreted alkaline phosphatase gene. The inducibility of these reporter gene constructs broadly correlated with the relative mRNA abundances in both transiently and stably transfected Namalwa cells. During work with stable cell lines, we found that G418, the drug used for the selection of transfected cells, inhibited the induction of interferon by both Sendai virus and double-stranded RNA. This inhibition was reversible when G418 was removed from the medium 24 h before the addition of virus.

Regulation of the interferon-inducible protein kinase PKR and (2'-5')oligo(adenylate) synthetase by a catalytically inactive PKR mutant through competition for double-stranded RNA binding.

The interferon-inducible double-stranded RNA-dependent protein kinase PKR has been suggested to function as a tumour suppressor gene product. Catalytically inactive mutants of PKR give rise to a tumorigenic phenotype when overexpressed in NIH-3T3 fibroblasts and this has been attributed to a dominant negative effect on the activity of the wild-type enzyme. Here we show that the mutant with Lys296 replaced by Arg, [K296R]PKR, not only inhibits the protein kinase activity of wild-type PKR but is also inhibitory towards another double-stranded RNA-dependent enzyme, the 40-kDa form of (2'-5')oligo(adenylate) synthetase. Inhibition of both wild-type PKR and (2'-5')oligo(adenylate) synthetase is reversed by adding higher concentrations of double-stranded RNA. These results suggest competition between [K296R]PKR and wild-type PKR or (2'-5')oligo(adenylate) synthetase for limiting amounts of double-stranded RNA. Moreover, the data imply that the tumorigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double-stranded RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.

Detection of rare allelic variants of the interferon-alpha 2 gene in human genomic DNA.

We have analyzed human donor DNA for the presence of sequences corresponding to allelic variants of the IFN-alpha 2 locus. Using both restriction enzyme digestion of PCR-amplified fragments and sequence analysis of these fragments, we have identified the three reported allelic variants, IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c, in genomic DNA derived from donors of African or Afro-Caribbean origin. This is the first report of the IFN-alpha 2a and IFN-alpha 2c alleles occurring in human donor DNA and supports the view that these are variants of the predominant IFN-alpha 2b allele rather than arising from mutations occurring in cultured cells.

Interferon (IFN)-alpha 2 genotype analysis of Chinese chronic hepatitis B patients undergoing recombinant IFN-alpha 2a therapy.

Sixteen Chinese chronic hepatitis B virus (HBV)-infected patients were treated with recombinant interferon-alpha 2a (rIFN-alpha 2a). Of these, 8 made a response to IFN, with titers of neutralizing antibody of 141-4525 as determined by an antiviral neutralization bioassay. To determine whether the immunogenicity of the IFN was directly linked to the patients' genotype, their genomic DNA was analyzed for the presence of the human IFN-alpha 2a gene. None of the patients possessed the gene for IFN-alpha 2a, but only 50% developed neutralizing antibodies. The hypothesis, therefore, of a direct link between antibody formation and genotype cannot be sustained. Alternative explanations of the immunogenicity of IFN-alpha 2a must be sought.

Comparison of expression of a humanized monoclonal antibody in mouse NSO myeloma cells and Chinese hamster ovary cells.

We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification. As expected, an increase in steady state mRNA levels of heavy chain correlated with an increase in expression of antibody whilst an increase in the steady state levels of mRNA of the selectable marker correlated with increased resistance to the selective agent. In NSO and CHO cells producing equivalent amounts of antibody, the copy number of the antibody genes and selectable marker was significantly higher in the CHO cells than in the NSO cells. However, the steady state mRNA levels of the heavy chain of the antibody were virtually identical. Rates of protein secretion in the two cell lines were also compared and found to be very similar. When the antibody purified from both systems was compared in a number of functional assays they behaved identically.

Molecular cloning of two new interferon-induced, highly related nuclear phosphoproteins.

During the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect homology over 612 base pairs and divergent at both ends. In addition, cDNA 75 presents an insertion of 150 base pairs highly homologous to a region common to both sequences. The 75 and 41 peptidic sequences are very hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinases. Furthermore, they present two protamine- and histone-like nuclear targeting sequences as well as some homology with helix-loop-helix motifs of some DNA-binding proteins. The 75-encoded product, which resolved as a 52-kDa protein after in vitro expression in rabbit reticulocyte lysates, was found to migrate as a 65-67-kDa protein after in vivo expression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells and was recovered from the membrane/nuclear pellet. In contrast, the 41-encoded product (30-kDa protein in reticulocyte lysates) could not be expressed in vivo, as it provoked a rapid and severe shut-off of protein synthesis in insect cells. The function of the 75 and 41 proteins and their relation to PKR remains to be determined. However, the presence of nuclear targeting sequences, phosphorylation sites, and helix-loop-helix motif is consistent with a role of these proteins in the mechanism of transduction of the interferon action.

The interferon-stimulable response elements of two human genes detect overlapping sets of transcription factors.

We have previously reported three types of DNA-protein complexes, formed specifically with the interferon-stimulable response elements (ISRE) in the 5' flanking DNA of the interferon-inducible 6-16 and 9-27 genes, a type-I interferon-inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type-I and type-II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band-shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe. With a 9-27 ISRE probe the M, G and C1/C2 complexes all contain the gamma subunit of ISGF3, which is present constitutively but is induced in response to IFN-alpha (to yield M) or IFN-gamma (to yield G). In contrast, a 6-16 ISRE probe forms band-shift complexes with IFN-alpha-inducible and IFN-gamma-inducible IRF1 and IRF2. With a 6-16 ISRE probe, therefore, M and G each correspond to two complexes which co-migrate in band-shift assays, one corresponding to IRF1, the other to IRF2. With this probe, the constitutive complex C1/C2 corresponds predominantly to IRF2. Consistent with this, IRF1 and IRF2 have lower affinity for the 9-27 ISRE than the 6-16 ISRE, whereas the reverse is true for E (ISGF3) and its gamma subunit. Relatively small differences in affinity appear sufficient to determine whether or not a band-shift complex is detected. In the case of IRF1 and IRF2, the different affinities for the 6-16 and 9-27 probes are dominated by a dinucleotide sequence in the centre of the 14-nucleotide 'core' ISRE. In contrast, preferential binding of E (ISGF3) by the 39-nucleotide 9-27 ISRE-containing sequence, although ISRE dependent, appears to be mediated by sequences 3' of the 'core' ISRE. Accordingly, these complexes can be simultaneously assayed using a hybrid probe consisting of the 5' flanking region and 'core' ISRE sequences from the 6-16 gene and sequences immediately 3' of the 'core' 9-27 ISRE sequence. No evidence was obtained for a modulatory role in factor binding for a pseudo-ISRE sequence close to ISRE in the 9-27 gene. The precise roles of IRF1 and IRF2 in the induction of IFN-beta and the control of interferon-inducible gene expression remain to be established.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversal of the double-stranded-RNA-induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR.

The interferon-inducible double-stranded-RNA(dsRNA)-dependent protein kinase PKR has been implicated in both the antiviral and cell growth-regulatory effects of the interferons. Over-expression of the wild-type form of this protein inhibits cell proliferation, whereas over-expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild-type protein kinase. We have investigated this possibility using the rabbit reticulocyte cell-free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor eIF-2. Addition of a highly purified inactive PKR mutant, synthesised in a baculovirus-infected insect cell system, rescues protein synthesis from inhibition by low concentrations of dsRNA in a dose-dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA or in the presence of another inhibitory protein kinase, the haem-controlled repressor. Inhibition of translation can be re-established in the presence of the mutant PKR by adding a higher concentration of dsRNA. These results suggest that inactive mutant PKR does exert a dominant negative effect on wild-type PKR and that this may be due to competition for dsRNA binding.

Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination.

We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses. A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward. We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants. Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required. We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis. Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.

Expression of the mouse c-abl type IV proto-oncogene product in the insect cell baculovirus system.

The cellular gene c-abl is the normal homologue of the transforming gene (v-abl) within the genome of the Abelson leukaemia virus. The cDNA sequence coding for the cellular form of the murine abl gene (c-abl type IV) has been inserted into the baculovirus transfer vector, pAc36C, so that the c-abl gene is under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with the recombinant transfer vector in the presence of wild type AcNPV DNA yielded recombinant, polyhedrin negative virus that expressed moderate levels of the c-Abl protein (representing approx. 0.5-1% of the stained cellular proteins as determined by densitometric scanning). The insect derived c-Abl protein was compared to the P210-BCR/ABL protein from K562 cells, a cell line derived from a patient with chronic myelogenous leukaemia. Antibodies raised against synthetic peptides based on c-abl encoded peptides react with the insect derived c-Abl. In addition, the baculovirus derived c-Abl protein has a tyrosine kinase activity as demonstrated by phosphorylation of a synthetic polypeptide and also by autophosphorylation. Phosphoamino acid analysis of immunoprecipitated, autophosphorylated baculovirus derived c-Abl protein indicates that the majority of label incorporated is on the tyrosine residues. Immunofluorescence microscopy has been used to show that the majority of the c-Abl protein expressed in cells infected with recombinant virus is located in the nuclear and plasma membranes.

Differential response of the human 6-16 and 9-27 genes to alpha and gamma interferons.

9-27 mRNA is expressed to a high level in response to both alpha and gamma interferons. In contrast, 6-16 mRNA is expressed well in response to alpha but very poorly in response to gamma interferon in human cells. The factors governing these different levels of expression were investigated. For both genes the major effect of both interferons is on transcription. A transcriptional bias in the 6-16 promoter/enhancer accounts in large part for the differential response of 6-16 to the two interferons. No single DNA element appears responsible; the smaller the 5' region analysed the lower the absolute activity and the smaller the differential response to alpha and gamma interferons observed. Both the 6-16 and 9-27 mRNAs are very stable and no effect of the interferons on stability was detected. Nor was any direct evidence obtained for preferential processing of the 9-27 mRNA. Nevertheless, differentials between the transcription and accumulation of mature mRNAs, particularly for 6-16 mRNA in response to gamma interferon, suggest that post-transcriptional control(s) must additionally operate. The 9-27 5' promoter/enhancer is much less active than that from 6-16 when placed 5' of a marker gene, despite the similar response of the two genes to alpha interferon.

Constitutive expression of a 2',5'-oligoadenylate synthetase cDNA results in increased antiviral activity and growth suppression.

The interferon (IFN)-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase has been implicated in the development of antiviral activity in human and animal cells. However, its role in IFN-mediated growth inhibition remains unclear. To elucidate the function of 2-5A synthetase, we have stably introduced a human 2-5A synthetase cDNA into a human glioblastoma cell line (T98G). Constitutive expression of the cDNA in these cells is associated with increased levels of resistance to infection by encephalomyocarditis virus. One transfected subclone, which expresses elevated levels of 2-5A synthetase enzyme activity, also shows a reduced rate of cellular proliferation.

A single DNA response element can confer inducibility by both alpha- and gamma-interferons.

Genomic and cDNA clones corresponding to 9-27, a member of the human 1-8 gene family highly inducible by alpha- and gamma-interferons (IFNs), have been isolated and characterized. A 1.7-kilobase genomic clone contains a complete functional gene with two exons, encoding a 125-amino acid polypeptide of unknown function. The 5' flanking region of the gene contains a 13-base-pair IFN-stimulable response element (ISRE), homologous to the ISREs of the 6-16, ISG 15, and ISG 54 genes, which are predominantly inducible by IFN-alpha, beta. Analysis of constructs containing native and mutated ISREs suggests that this motif is essential for the response of 9-27 to IFN-gamma as well as IFN-alpha. Furthermore, the 9-27 (GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT) ISREs can each confer a response to both types of IFN when placed on the 5' side of a marker gene. Since the 6-16 gene does not normally respond to IFN-gamma, the context of the ISRE must determine the specificity of the response.

Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA.

Human alpha or beta interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down-regulate expression of the c-myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2'5' oligoadenylate synthetase or the interferon-sensitive mRNAs, 6-16 or 9-27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP-ribosyl transferase activity by 3-methoxybenzamide impairs interferon- or TPA-induced differentiation of Daudi cells. This agent induces a higher level of c-myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c-myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.