Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells

The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus.     

Full resistance of herpes simplex virus type 1-infected primary human cells to alpha interferon requires both the Us11 and gamma(1)34.5 gene products

The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells.

The interferon induced double-stranded RNA-activated kinase, PKR, has been suggested to act as a tumor suppressor since expression of a dominant negative mutant of PKR causes malignant transformation. However, the mechanism of transformation has not been elucidated. PKR phosphorylates translation initiation factor eIF-2 alpha on Ser51, resulting in inhibition of protein synthesis and cell growth arrest. Consequently, it is possible that cell transformation by dominant negative PKR mutants is caused by inhibition of eIF-2 alpha phosphorylation. Here, we demonstrate that in NIH 3T3 cells transformed by the dominant negative PKR mutant (PKR delta 6), eIF-2 alpha phosphorylation is dramatically reduced. Furthermore, expression of a mutant form of eIF-2 alpha, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH 3T3 cells. These results are consistent with a critical role of phosphorylation of eIF-2 alpha in control of cell proliferation, and indicate that dominant negative PKR mutants transform cells by inhibition of eIF-2 alpha phosphorylation.     

Sequence and expression of a novel murine interferon alpha gene--homology with enhancer elements in the regulatory region of the gene

A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library. The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7. The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter. A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene. The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes.     

Genetic transfer of a functional human interferon alpha receptor into mouse cells: cloning and expression of its cDNA

A cDNA coding for the human interferon alpha receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon alpha. The transfected cells expressed the human interferon alpha receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA defects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of the cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon alpha, demonstrating that the cloned cDNA encodes a functional human interferon alpha receptor.     

Dephosphorylation of eIF-2alpha mediated by the gamma(1)34.5 protein of herpes simplex virus type 1 is required for viral response to interferon but is not sufficient for efficient viral replication

The gamma(1)34.5 protein of herpes simplex virus type 1 (HSV-1) functions to block the shutoff of protein synthesis involving double-stranded RNA-dependent protein kinase (PKR). In this process, the gamma(1)34.5 protein recruits cellular protein phosphatase 1 (PP1) to form a high-molecular-weight complex that dephosphorylates eIF-2alpha. Here we show that the gamma(1)34.5 protein is capable of mediating eIF-2alpha dephosphorylation without any other viral proteins. While deletion of amino acids 1 to 52 from the gamma(1)34.5 protein has no effect on eIF-2alpha dephosphorylation, further truncations up to amino acid 146 dramatically reduce the activity of the gamma(1)34.5 protein. An additional truncation up to amino acid 188 is deleterious, indicating that the carboxyl-terminal domain alone is not functional. Like wild-type HSV-1, the gamma(1)34.5 mutant with a truncation of amino acids 1 to 52 is resistant to interferon, and resistance to interferon is coupled to eIF-2alpha dephosphorylation. Intriguingly, this mutant exhibits a similar growth defect seen for the gamma(1)34.5 null mutant in infected cells. Restoration of the wild-type gamma(1)34.5 gene in the recombinant completely reverses the phenotype. These results indicate that eIF-2alpha dephosphorylation mediated by the gamma(1)34.5 protein is required for HSV response to interferon but is not sufficient for viral replication. Additional functions or activities of the gamma(1)34.5 protein contribute to efficient viral infection.     

Expression in Escherichia coli cells of mutant human interferon alpha2

cDNA library was obtained from mRNA isolated from human leukocytes induced by Newcastle disease virus. Clones containing cDNA for alpha 2-interferons were identified by colony hybridization with two synthetic hexadecanucleotides. One of the positive clones contained a NH2-terminal part of cDNA of human interferon identical to cDNA for IFN-alpha 2. The only difference between these two clones was the Ser-8 leads to Asn-8 substitution in deduced sequenced of mature interferons. This mutant interferon, named alpha 2, was expressed in E. coli and its properties were compared with those of interferon alpha 2.     

Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.     

A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon.

2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.     

West Nile virus noncoding subgenomic RNA contributes to viral evasion of the type I interferon-mediated antiviral response

We previously showed that a noncoding subgenomic flavivirus RNA (sfRNA) is required for viral pathogenicity, as a mutant West Nile virus (WNV) deficient in sfRNA production replicated poorly in wild-type mice. To investigate the possible immunomodulatory or immune evasive functions of sfRNA, we utilized mice and cells deficient in elements of the type I interferon (IFN) response. Replication of the sfRNA mutant WNV was rescued in mice and cells lacking interferon regulatory factor 3 (IRF-3) and IRF-7 and in mice lacking the type I alpha/beta interferon receptor (IFNAR), suggesting a contribution for sfRNA in overcoming the antiviral response mediated by type I IFN. This was confirmed by demonstrating rescue of mutant virus replication in the presence of IFNAR neutralizing antibodies, greater sensitivity of mutant virus replication to IFN-α pretreatment, partial rescue of its infectivity in cells deficient in RNase L, and direct effects of transfected sfRNA on rescuing replication of unrelated Semliki Forest virus in cells pretreated with IFN-α. The results define a novel function of sfRNA in flavivirus pathogenesis via its contribution to viral evasion of the type I interferon response.     

The interferon alpha induced protein ISG12 is localized to the nuclear membrane.

Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon alpha inducible genes of unknown function. We have determined the 5' end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon alpha inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon alpha by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope.     

Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.     

Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.