Biomedical subjects

E Kieff

Publications and source records attributed to E Kieff.

At least 163 records · Page 9Linked to original sources

PubMed1986

Epstein-Barr virus (EBV) antigen-specific leukocyte migration inhibition (LMI) in infectious mononucleosis (IM). I. Kinetics and response to a membrane protein on EBV-transformed cells.

Cell-mediated immune response of mononucleosis (IM) patients to Epstein-Barr virus (EBV)-determined antigens was measured by the leukocyte migration inhibition (LMI) assay. Patients in the acute phase of the disease failed to respond to partially purified nuclear antigen, EBNA, or to cell extracts that contained EBNA as the predominant EBV antigen. They showed a strong specific response to cell extracts enriched in early antigen (EA) and virus capsid antigen (VCA). The LMI response to EBNA appeared in convalescence in parallel with EBNA-specific antibodies, slightly later in children than in adults. Membrane fractions of EBV-carrying, virus nonproducer Raji cells induced an EBV-specific LMI at approximately the same time. A bacterial fusion protein containing the hydrophilic part of the virus-coded membrane antigen of latently EBV-infected cells also induced an EBV-specific response that parallelled the LMI reaction elicited by the Raji membrane fraction. This is in line with our previous finding (D. Sulitzeanu et al., J. Virol. 58, 230, 1986) that this fusion protein shares an epitope with Raji cell membranes.

Adolescent↗

PubMed1986

Nucleotide sequences of mRNAs encoding Epstein-Barr virus nuclear proteins: a probable transcriptional initiation site.

Three cDNA clones of the second Epstein-Barr virus nuclear antigen (EBNA2) mRNA and two of the EBNA1 mRNA were analyzed. Two EBNA2 clones begin 42 bases 3' to a promoter in the Epstein-Barr virus long internal repeat, which is likely to be the EBNA2 promoter. Surprisingly, the first splice creates an AUG at the beginning of the first of two nonoverlapping open reading frames. The second open reading frame encodes EBNA2. Two incomplete EBNA1 mRNA cDNA clones begin with parts of two of the EBNA2 exons and contain two other exons that map 19 and 59 kilobases 3' to the EBNA2 coding domain. The 3' exon of this mRNA encodes EBNA1. A model for regulation of transcription of these RNAs is presented.

Antigens, Viral↗

PubMed1986

Definitive identification of a member of the Epstein-Barr virus nuclear protein 3 family.

Some Epstein-Barr virus (EBV) immune human antisera are known to react with a 142-kDa protein, EBV-encoded nuclear antigen 3 (EBNA3), which, like EBNA1 and EBNA2, is likely to be involved in the establishment of latent infection or growth transformation. We have now constructed gene fusions between Escherichia coli lacZ and an EBV DNA open reading frame (BERF1; BamHI E fragment rightward open reading frame 1), which is transcribed into an mRNA in latently infected cells. Purified hybrid protein from one of these constructs, chosen because of its reactivity with EBNA3-positive human antisera, was used to affinity purify the specific antibody from human antiserum. This specific antibody was used to prove that EBNA3 is encoded, at least in part, by BERF1, and that EBNA3 is in the nucleus of each latently infected cell. In rodent cells, BERF1 encodes a 120- to 130-kDa protein, which translocates to the nucleus and is recognized by EBNA3-positive human antisera. Two other proteins similar in size to EBNA3 are detected in latently infected cells by EBV immune human antisera. Two EBV open reading frames related to BERF1 may encode these proteins.

Animals↗

PubMed1986

Leukocyte migration inhibition demonstrates a human T-cell response to a membrane protein expressed in latent Epstein-Barr virus infection.

Leukocyte migration inhibition tests show that lymphocytes of Epstein-Barr virus-seropositive individuals recognize a Raji cell membrane antigen and a membrane protein encoded by Epstein-Barr virus in latently infected cells. Antiserum against the latter blocks the leukocyte migration inhibition triggered by both preparations, suggesting that the two antigens are associated with the same protein complex.

Antigens, Surface↗

PubMed1986

Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus.

Epstein-Barr virus is known to encode three nuclear proteins and one membrane protein (LMP) in latently infected growth-transformed cells. Studies of the plasma membrane localization and orientation of LMP by protease digestion of live cells and by immunofluorescence indicated the following. (i) At least 30% of LMP is in the plasma membrane, as opposed to other cytoplasmic membranes. (ii) A small LMP domain which corresponds to a previously proposed outer reverse turn between the first two transmembrane domains is exposed on the outer cell surface (and two other proposed outer-reverse-turn domains may be exposed), whereas all or almost all of the rest of the protein is not exposed on the outer cell surface. (iii) LMP is present in patches in the cell plasma membrane.

Antigens, Viral↗

PubMed1986

Expression of the Epstein-Barr virus nuclear protein 2 in rodent cells.

A 3.0-kilobase-pair Epstein-Barr virus (EBV) DNA segment necessary for lymphocyte immortalization encodes at least part of a nuclear protein (EBNA2) which is characteristically expressed in latently infected, immortalized cells. A 1.5-kilobase open reading frame within this DNA segment has now been inserted into a murine leukemia virus (MuLV)-derived expression vector (pZIP-NEO-SV(X)1) which provides for transcription of heterologous DNA but not for translational start. Transfection of the recombinant DNA into NIH 3T3 cells resulted in expression of a full-sized EBNA2 which localized to the cell nucleus. Significant new evidence is thereby provided that this 1.5 kilobase open reading frame includes a translational start site and encodes the entire EBNA2 protein. Transfection of the recombinant DNA into a helper cell line (psi am22b) providing amphotropic MuLV-packaging functions resulted in the release of a recombinant MuLV carrying the EBNA2 gene. This recombinant virus can infect rodent cells and convert them to stable EBNA2 expression. Rat-1 cells infected with the MuLV EBNA2 recombinant expressed EBNA2 and grew more rapidly in medium supplemented with 1 or 0.5% fetal calf serum than did Rat-1 cells infected with MuLV vector lacking EBNA2. The Rat-1 cells expressing EBNA2 remained contact inhibited, anchorage dependent, and nontumorigenic in nude mice. Different EBV isolates have one of at least two EBNA2 alleles. Despite divergence between the two alleles, a human serum recognized the prototype EBNA2 allele (EBNA2A) as well as the variant EBNA2B allele characteristic of some Burkitt tumor EBV isolates. The EBNA2B allele was also expressed from the MuLV-derived vector. The reproducible expression of EBNA2A or EBNA2B from these recombinant vectors will facilitate analysis of the EBNA2A and EBNA2B phenotypes.

Animals↗

PubMed1985

A second nuclear protein is encoded by Epstein-Barr virus in latent infection.

A region of the Epstein-Barr virus (EBV) genome that is important in inducing cell proliferation includes a single long open reading frame. Part of this open reading frame has been fused to the lacZ gene and expressed in Escherichia coli. Antisera to the fusion protein identify a protein in the nuclei of latently infected growth-transformed lymphocytes and in Burkitt tumor cells grown in vitro. This nuclear protein is encoded by a different virus-gene than that which encodes the previously described EBV nuclear antigen, EBNA.

Animals↗

PubMed1985

An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells.

Epstein-Barr virus expresses a cytoplasmic and plasma membrane protein (LMP) in latently infected growth transformed lymphocytes. The gene specifying LMP has now been expressed in NIH3T3 and Rat-1 cells. Expression of the gene in these cells resulted in altered cell morphology and some resistance to the growth inhibiting effect of medium containing low serum. In Rat-1 cells, LMP expression often led to loss of contact inhibition and anchorage-independent growth in soft agar. Rat-1 cells expressing LMP were uniformly tumorigenic in nude mice. Thus, LMP is a transforming gene which is likely to account for many aspects of EBV induced cell transformations. This is the first demonstration of a transforming gene in Epstein-Barr virus, a ubiquitous human pathogen associated with neoplasia.

Animals↗

PubMed1985

A third viral nuclear protein in lymphoblasts immortalized by Epstein-Barr virus.

Most sera from patients with rheumatoid arthritis as well as some sera from normal Epstein-Barr virus (EBV)-infected people detect a 140-kDa protein on immunoblots of EBV-infected lymphoblasts. The 140-kDa protein is a nuclear protein characteristic of latent EBV infection. Sera reactive with this protein identify a distinctive globular nuclear antigen. Although the 140-kDa protein is encoded by EBV, it is not encoded by genes that encode the two previously described EBV nuclear antigens (EBNA) or the latent-infection membrane protein. The 140-kDa protein is therefore designated EBNA3. The EBV genes, including the gene encoding EBNA3, that are characteristically expressed in latent infection are likely to play a role in the maintenance of persistent latent viral infection or in the cell proliferation caused by virus infection.

Antigens, Viral↗

PubMed1985

Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the same gene.

Two major outer envelope glycoproteins of Epstein-Barr virus, gp350 and gp220, are known to be encoded by 3.2- and 2.5-kilobase RNAs which map to the same DNA fragment (M. Hummel, D. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417). These RNAs have the same 5' and 3' ends. The larger RNA is encoded by a 2,777-base DNA segment which is preceded by TATTAAA, has AATAAA near its 3' end, and contains a 2,721-base open reading frame. The smaller RNA has one internal splice which maintains the same open reading frame. Translation of the 3.2- and 2.5-kilobase RNAs yielded proteins of 135 and 100 kilodaltons (Hummel et al., J. Virol. 49:413-417). The discrepancy between the 907 codons of the open reading frame and the 135-kilodalton size of the gp350 precursor is due to anomalous behavior of the protein in gel electrophoresis, since a protein translated from most of the Epstein-Barr virus open reading frame in Escherichia coli had similar properties. Antisera raised in rabbits to the protein expressed in E. coli specifically immunoprecipitated gp350 and gp220, confirming the mapping and sequencing results and the translational reading frame. The rabbit antisera also reacted with the plasma membranes of cells that were replicating virus and neutralized virus, particularly after the addition of complement. This is the first demonstration that the primary amino acid sequence of gp350 and gp220 has epitopes which can induce neutralizing antibody. We propose a model for the gp350 protein based on the theoretical analysis of its primary sequence.

Amino Acid Sequence↗

PubMed1985

Repeat arrays in cellular DNA related to the Epstein-Barr virus IR3 repeat.

We isolated clones and determined the sequence of portions of mouse and human cellular DNA which cross-hybridize strongly with the IR3 repetitive region of Epstein-Barr virus. The sequences were found to be tandem arrays of a simple sequence based on the triplet GGA, very similar to the IR3 repeat. The cellular repeats have distinct differences from the viral repeat region, however, and their sequences do not appear capable of being translated into a purely glycine-plus-alanine protein domain like the portion of the Epstein-Barr nuclear antigen coded by IR3. Although the relationship between IR3 and the cellular repeats is left unclear, the cellular repeats have many interesting features. The tandem arrays are about 1 to several kilobases long, much shorter than satellite tandem repeats and larger than other interspersed, tandem repeats. Each of the repeats is a distinct variation, perhaps diverged from a common sequence, (GGA)n. This family is present in the genomes of all species tested and appears to be a ubiquitous feature of all higher eucaryotic genomes.

Animals↗

PubMed1985

Biochemistry of latent Epstein-Barr virus infection and associated cell growth transformation.

There is sufficient knowledge of the biochemistry of Epstein-Barr virus (EBV) persistence and gene expression in latent growth-transforming infection and of the persistence and expression of other oncogenic viruses to permit interesting and possibly useful comparisons. Most smaller oncogenic viral genomes usually persist solely as integrated DNAs despite their ability to circularize. Papilloma and hepatitis viruses may persist as episomes, and parts of their genomes may integrate. Usually, only the oncogenic fragment of adenovirus DNA is integrated into cell DNA. In contrast, the entire EBV genome persists in cells as an episome or as integrated DNA. Thus, EBV may have novel mechanisms to maintain its complete genome as an episome or as a complete integrated virus DNA. Three viral genes are expressed in latently EBV-infected growth-transformed cells, each of which encodes one RNA and one protein. Two of the proteins are probably nuclear DNA-binding proteins; the third is probably a membrane protein. Thus, the repertoire of genes expressed is similar in complexity and intracellular distribution to that expressed by papova and adenoviruses in cellular transformation. The papova and adenovirus-transforming genes are partially analogous to retrovirus oncogenes. This similarity cannot as yet be extended to EBV. There is no homology at the DNA-sequence or protein-sequence level between EBV and other viral or cell oncogenes. Thus, it remains important to pursue analysis of the EBV-transforming genes. Identification of these genes is a first step in discerning their function in latent growth-transforming cell infection. Parts of each of these genes are being made in bacteria. The bacterial products enable us to make antisera that are specific for each of the viral proteins. These antisera can also be used to identify the viral proteins within latently infected growth-transformed cells or within cells stably expressing transfected virus genes. The antisera can also be used to study the association of Epstein-Barr nuclear antigen (EBNA) 1 and 2 with DNA and of the lymphocyte-determined membrane antigen (LYDMA) with the cell membrane. The three genes must be introduced into nontransformed cells to determine whether, alone or in combination, they are sufficient to accomplish cell growth transformation.

Amino Acid Sequence↗

PubMed1984

Persistence of the entire Epstein-Barr virus genome integrated into human lymphocyte DNA.

The entire Epstein-Barr virus genome is integrated into Burkitt tumor cell DNA at the terminal direct repeat sequence of the virus. There is no homology between the GC-rich (G, guanine; C, cytosine) terminal repeat and the AT-rich (A, adenine; T, thymine) cell sequences with which it has recombined. More than 15 kilobases of cell DNA have been deleted and 236 base pairs are duplicated at one virus-cell junction site.

Base Sequence↗

PubMed1984

Expression of Epstein-Barr virus (EBV) DNA and cloned DNA fragments in human lymphocytes following Sendai virus envelope-mediated gene transfer.

Purified EBV DNA and cloned DNA fragments were trapped in Sendai virus (SV) envelopes during envelope reconstitution. The DNA-loaded reconstituted envelopes (RSVE/DNA) served as gene-transfer vehicles using the capability of RSVE to fuse with normal and tumor cells. The efficiency of RSVE-mediated EBV DNA transfer into lymphoid tumor cells and fresh human lymphocytes was 5-10% of the enveloped 3H-labeled EcoRI fragment B of EBV DNA. Purified intracellular EBV (B95-8 strain) DNA induced EBV nuclear antigen (EBNA) in 0.2-1% of human lymphocytes, transiently stimulated cellular DNA synthesis, but did not fully transform cells. Cloned Sal I F1 fragment [approximately equal to 9 kilobase pairs (kbp)] and a smaller BamHI K (5.2 kbp) fragment from the same region of B95-8 EBV DNA induced EBNA in 2-4% of human lymphocytes but did not stimulate DNA synthesis nor transform cells. Cloned BamHI D1 fragment (approximately equal to 9 kbp) from AG-876 virus DNA, or a combination of cloned BamHI X and H fragments (approximately equal to 2 and 7 kbp, respectively) from the similar region of B95-8 virus DNA, significantly stimulated lymphocyte DNA synthesis, but EBNA could not be detected and transformation was not achieved. Early antigen and viral capsid antigen were not observed with any of the fragments tested. Our results suggest that the induction of EBNA and stimulation of lymphocyte proliferation are not controlled by the same region of EBV DNA.

Base Sequence↗

PubMed1984

A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection.

The nucleotide sequence of an Epstein-Barr virus gene expressed in latently infected growth-transformed cells is known to include a long open reading frame containing a 33-base-pair repeat element. A bacterial fusion protein constructed from a portion of the reading frame and Escherichia coli beta-galactosidase was used to produce sera in rabbits against the previously unidentified gene product. The viral protein detected with these sera in latently infected cells varies in size with the number of copies of the DNA repeat element. Translation of the RNA in vitro yields a protein of similar size. As expected from its primary sequence, the protein is a membrane protein. Immunofluorescence studies with the rabbit antisera suggest that the protein is in the plasma membrane. Thus, this protein could be the lymphocyte-determined membrane antigen (LYDMA) responsible for the generation of T-cell immunity to latently infected cells.

Cell Membrane↗

PubMed1984

U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2.

Sequence analysis of the U2 regions of the B95-8 and AG876 Epstein-Barr virus (EBV) isolates reveals divergence within a long open reading frame previously identified as encoding 1.5 kilobases of the 3' end of a viral RNA expressed in latently infected, growth-transformed, B-lymphocyte cell lines. Differences among EBV isolates within the U2 open reading frame are shown to correlate with differences in an EBV nuclear antigen, EBNA2. B95-8, W91, Raji, Cherry, and Lamont EBV isolates have similar U2 domains and encode similar-size EBNA2 proteins, while AG876, Jijoye, and P3HR-1 have variant or absent U2 domains and variant or absent EBNA2 proteins. The AG876 U2 open reading frame and EBNA2 protein are both shorter than those of B95-8. These data indicate that the U2 open reading frame encodes EBNA2.

Animals↗

PubMed1984

An Epstein-Barr virus DNA fragment encodes messages for the two major envelope glycoproteins (gp350/300 and gp220/200).

The genes encoding the two major Epstein-Barr virus glycoproteins (gp350/300 and gp220/200) have been mapped to a 5-kilobase fragment of the viral genome (BamHI-L). This fragment encodes 3.4- and 2.8-kilobase RNAs which translate proteins of 135 and 100 kilodaltons, respectively. Both proteins react with antiserum specific for gp350/300 and gp220/200. The 135-kilodalton protein is identical in size to the nascent polypeptide precursor to gp350/300, and the 100-kilodalton protein is the expected size of the polypeptide precursor to gp220/200.

Animals↗

PubMed1984

Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein.

The most abundant Epstein-Barr virus mRNA in a latently infected cell line, IB4, established by in vitro growth transformation with virus, was a 2,8-kilobase RNA encoded by largely unique DNA near the right end of the genome. The RNA was transcribed from right to left, and two introns were spliced out. This region of the genome was sequenced, and the exons of the RNA were identified by S1 analysis of DNA-RNA hybrids and primer extension. The first start codon in the RNA was 40 nucleotides from its 5' end. Beginning with the start codon, there was a 1,158-nucleotide open reading frame which crossed both introns. The important characteristics of the translated protein were as follows. (i) The amino terminus was highly charged and not suggestive of a leader sequence. (ii) There were six markedly hydrophobic alpha-helical domains, each having 21 amino acids and connected by 5 to 7 amino acid segments predicted to be reverse turns. (iii) The carboxy-terminal 200 amino acids were markedly acidic, containing 6 glutamic and 37 aspartic acids. The hydrophobic region is predicted to form six membrane-spanning regions, leaving the short charged amino terminus and long acidic carboxy terminus on the inside of the membrane. This protein could be responsible for the new antigen detected in the plasma membrane of Epstein-Barr virus-transformed cells, lymphocyte-determined membrane antigen. There were two other open reading frames in the RNA.

Amino Acid Sequence↗