The biological activity of interferon alpha is influenced by two distinct regions in the protein

With the aim to assign differences in activity between murine interferon-alpha 1 and -alpha 4 to specific amino acids, we have constructed hybrid genes and analysed the antiviral properties of the corresponding hybrid proteins. The hybrid genes were constructed by means of homologous recombination between the alpha 1 and alpha 4 genes in Escherichia coli. Hybrids in which the N-terminal part is derived from alpha 1 show that two regions have a major effect on the activity: amino acid 10-20 and 55-67. When comparing hybrids with N-terminal alpha 4 sequences, transitions in activity are found in the same regions. Interestingly, the curves for the two sets of hybrids are exactly each others mirror image.     

Synthesis of fusion and mature murine alpha interferons in Escherichia coli

Four murine interferon-alpha (MuIFN-alpha) genes (alpha 1, alpha 4, alpha 5, alpha 6) were previously identified and characterized. The coding regions of these IFN-alpha genes were inserted into bacterial expression vectors behind the lpp promoter under the control of the lac promoter-operator region, resulting in fusion peptides containing additional N-terminal amino acids (aa). Plasmids coding for the expression of mature IFN-alpha 1 and alpha 5 were also constructed using the same vector system, by inserting a 30-bp synthetic oligodeoxynucleotide, which contains a stop codon for the lpp gene, a ribosome-binding sequence and an ATG start codon for the IFN peptides. The amounts of IFN polypeptides synthesized in Escherichia coli were estimated in the maxi-cell system and their biological activities were measured on mouse and other mammalian cells. The yields of mature IFN produced in this vector were 2 to 4 X 10(6) units/liter; the antiviral activity of the majority of the MuIFNs on human and bovine cells was 100- to 1000-fold lower than on mouse cells. IFN-alpha 4, which contains an internal deletion of 5 aa, showed a lower antiviral activity than other MuIFNs on mouse cells.     

Characterization of a mouse interferon gene locus II. Differential expression of alpha-interferon genes.

A cluster of four MuIFN-alpha genes was recently isolated and characterized (1); one of the genes in this cluster had, in the coding region, an internal deletion of 5 amino acids. Bacterial expression plasmids were constructed to examine the effect of this deletion on the antiviral activity of the MuIFN-alpha 4 peptide and it was found that the alpha 4 interferon peptide had a 100-fold lower antiviral activity than full length alpha-interferon proteins when expressed in E. coli. Three of the four MuIFN-alpha genes identified were expressed coordinately in L-cells infected with NDV. The relative levels of alpha 4 mRNA were substantially higher than the levels of the other alpha mRNAs. Comparison of the 5' end flanking sequences of these four alpha interferon genes revealed that the promoter sequences of alpha 1, alpha 5 and alpha 6 are more homologous to each other than to the alpha 4 promoter which also contains a G rich cluster not seen in the other three promoters.     

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.     

Organization, structure and expression of murine interferon alpha genes.

Using a human interferon-alpha probe we have isolated recombinant phages containing murine interferon-alpha (Mu IFN-alpha) genes from a genomic library. One of these phages contained two complete Mu IFN-alpha genes and part of a third gene. The insert of a second phage held two IFN genes. This indicates that the Mu IFN-alpha genes are clustered in the genome as is the case for the analogous human genes. The nucleotide sequences of these 5 genes were determined. They show that the genes are all different, albeit highly homologous. The deduced amino acid sequences show that four of the five genes contain a putative glycosylation site. Three genes were transiently expressed in COS cells and they gave rise to protein products showing antiviral properties. The expression of the five Mu IFN-alpha genes and the Mu IFN-beta gene was studied in virus-induced mouse L cells. The individual mRNAs were visualized in a nuclease S1 experiment, using a specific probe for each gene. In RNA preparations from induced cells mRNAs for each of the five alpha genes and the beta gene were present. However, substantial differences in the amounts of the individual mRNAs were observed.     

Two transactivation domains of hypoxia-inducible factor-1alpha regulated by the MEK-1/p42/p44 MAPK pathway

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transactivator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric transactivator that is composed of alpha and beta subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the alpha subunit, whereas the HIF-1beta subunit is expressed constitutively. Our previous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1alpha. In this paper, we dissected the transactivation domain of HIF-1alpha in more detail, and tested the correlation between specific domains of HIF-1alpha and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1alpha that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven reporter system, we tested the transactivation activities of the Gal4/HIF-1alpha fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1alpha. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1alpha. Treatment of PD98059, a MEK-1 inhibitor, blocked the hypoxia-induced transactivation abilities of both the 522-649 and 650-822 amino acids of the C-terminal half of HIF-1alpha. This implies that the MEK-1/p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.     

The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.     

C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1

Vaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L(-)C7L(-)). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L(-)C7L(-) in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L(-)C7L(-) but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L(-)C7L(-) resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells.     

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1 alpha 1 was deduced from a 2.2-kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1 beta was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1 alpha 1 is at 96A2-5 on chromosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1 beta is localized at 9C1-2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1 alpha 1 shows 96% amino acid sequence identity to PP1 alpha 2 (302 amino acids), an isoform whose gene is located in the 87B6-12 region of chromosome 3 [Dombrádi, V., Axton, J. M., Glover, D.M. Cohen, P.T.W. (1989) Eur. J. Biochem. 183, 603-610]. PP1 beta shows 85% identity to PP1 alpha 1 and PP1 alpha 2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1 alpha 1 and PP1 beta show 89% amino acid sequence identity to rabbit PP1 alpha (330 amino acids) [Cohen, P.T.W. (1988) FEBS Lett. 232, 17-23] and PP1 beta (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1 alpha and PP1 beta, which have been preserved from insects to mammals, implies that the alpha and beta isoforms may have distinct biological functions.     

TAFA: a novel secreted family with conserved cysteine residues and restricted expression in the brain

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus using a novel database searching strategy. The family is composed of five highly homologous genes referred to as TAFA-1 to -5. The TAFA genes encode proteins of approximately 100 amino acids that contain conserved cysteine residues at fixed positions. TAFA-1 to -4 are more closely related to each other than to TAFA-5, in which a conserved motif including CC in TAFA-1 to -4 is not present. In H. sapiens, TAFA-3 has two isoforms formed by alternative splicing. Sequence homology analyses reveal that TAFA proteins appear distantly related to MIP-1alpha, a member of the CC-chemokine family. TAFA mRNAs are highly expressed in specific brain regions, with little expression seen in other tissues.     

Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors.     

Antiviral activity of interferon-alpha against hepatitis B virus can be studied in non-hepatic cells and Is independent of MxA.

It is well established that interferon-alpha can induce non-cytotoxic intracellular suppression of hepatitis B virus replication, but the mechanisms involved are unclear. Cell culture studies to characterize these mechanisms are restricted, in part because hepatitis B virus replicates almost exclusively in liver-derived cells. To overcome this limitation we used a cytomegalovirus promoter-controlled hepatitis B virus expression system, which leads to intracellular viral replication even in non-hepatic cell lines. In this experimental system interferon-alpha treatment specifically suppressed viral replication demonstrating that antiviral activities against hepatitis B virus are not restricted to hepatic cells. Furthermore, the interferon-inducible MxA protein was recently reported to play a key role in the antiviral action of interferon-alpha against hepatitis B virus. Our data demonstrate that interferon-alpha also suppresses hepatitis B virus replication in MxA-deficient HEp2 cells, indicating that MxA is not essential for these activities. Taken together, our data imply that the experimental approach presented can also be adapted to established cell lines which are deficient in parts of the signal transduction pathway or other elements located further downstream, providing important insights into mechanisms specifically suppressing hepatitis B virus.     

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.     

The primary structure of the alpha subunit of human elongation factor 1. Structural aspects of guanine-nucleotide-binding sites

The primary structure of the alpha subunit of elongation factor 1 (EF-1 alpha) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF-1 alpha. An inventory of amino acid sequences around the guanine-nucleotide-binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved sequence elements. Region I has the sequence apolar-Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Yaa-Xaa-Gly-LYs-Thr(Ser)- -Xaa-Xaa-Xaa-Xaa-X-apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar-apolar-Xaa-Xaa-Asn-Lys-Xaa-Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.     

Folding of homologous proteins: conservation of the folding mechanism of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids

The equilibrium and kinetic properties for the urea-induced unfolding of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids were compared to determine the role of protein folding in evolution. The parent proteins differ at 40 positions in the sequence of 268 amino acids, and the hybrids differ by up to 15 amino acids from the Escherichia coli alpha subunit. The results show that all the proteins follow the same folding mechanism and are consistent with a previously proposed hypothesis [Hollecker, M., & Creighton, T. E. (1983) J. Mol. Biol. 168, 409; Krebs, H., Schmid, F. X., & Jaenicke, R. (1983) J. Mol. Biol. 169, 619] that the folding mechanisms are conserved in homologous proteins. Analysis of the kinetic data suggests that the 15 positions at which the parent proteins differ in the amino folding unit, residues 1-188, do not play a role in a rate-limiting step in folding that has been previously identified as the association of the amino and carboxyl folding units [Beasty, A. M., Hurle, M. R., Manz, J. T., Stackhouse, T. S., Onuffer, J. J., & Matthews, C. R. (1986) Biochemistry 25, 2965]. One or more of the 25 positions at which the parent proteins differ in the carboxyl folding unit, residues 189-268, do appear to play a role in this same rate-limiting step.     

Two distinct families of human and bovine interferon-alpha genes are coordinately expressed and encode functional polypeptides.

The classical human interferon-alpha (HuIFN-alpha) gene family is estimated to consist of 15 or more nonallelic members which encode proteins sharing greater than 77% amino acid sequence homology. Low-stringency hybridization with a HuIFN-alpha cDNA probe permitted the isolation of two distinct classes of bovine IFN-alpha genes. The first subfamily (class I) is more closely related to the known HuIFN-alpha genes than to the second subfamily (class II) of bovine IFN-alpha genes. Extensive analysis of the human genome has revealed a HuIFN-alpha gene subfamily corresponding to the class II bovine IFN-alpha genes. The class I human and bovine IFN-alpha genes encode mature IFN polypeptides of 165 to 166 amino acids, whereas the class II IFN-alpha genes encode 172 amino acid proteins. Expression in Escherichia coli of members of both gene subfamilies results in polypeptides having potent antiviral activity. In contrast to previous studies which found no evidence of class II IFN-alpha protein or mRNA expression, we demonstrate that the class I and class II IFN-alpha genes are coordinately induced in response to viral infection.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.