The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein-Barr virus LMP2A membrane protein.

The Epstein-Barr virus latency-associated membrane protein LMP2A has been shown to activate the survival kinase Akt in epithelial and B cells in a phosphoinositide 3-kinase-dependent fashion. In this study, we demonstrate that the signalling scaffold Shb associates through SH2 and PTB domain interactions with phosphorylated tyrosine motifs in the LMP2A N-terminal tail. Additionally, we show that mutation of tyrosines in these motifs as well as shRNA-mediated downregulation of Shb leads to a loss of constitutive Akt-activation in LMP2A-expressing cells. Furthermore, utilization by Shb of the LMP2A ITAM motif regulates stability of the Syk tyrosine kinase in LMP2A-expressing cells. Our data set the precedent for viral utilization of the Shb signalling scaffold and implicate Shb as a regulator of LMP2A-dependent Akt activation.

C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal.

The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipid-enriched membrane microdomains.

Capturing whole-genome characteristics in short sequences using a naïve Bayesian classifier.

Bacterial genomes have diverged during evolution, resulting in clearcut differences in their nucleotide composition, such as their GC content. The analysis of complete sequences of bacterial genomes also reveals the presence of nonrandom sequence variation, manifest in the frequency profile of specific short oligonucleotides. These frequency profiles constitute highly specific genomic signatures. Based on these differences in oligonucleotide frequency between bacterial genomes, we investigated the possibility of predicting the genome of origin for a specific genomic sequence. To this end, we developed a naïve Bayesian classifier and systematically analyzed 28 eubacterial and archaeal genomes. We found that sequences as short as 400 bases could be correctly classified with an accuracy of 85%. We then applied the classifier to the identification of horizontal gene transfer events in whole-genome sequences and demonstrated the validity of our approach by correctly predicting the transfer of both the superoxide dismutase (sodC) and the bioC gene from Haemophilus influenzae to Neisseria meningitis, correctly identifying both the donor and recipient species. We believe that this classification methodology could be a valuable tool in biodiversity studies.

Definition of a divergent epitope that allows differential detection of early protein p41 from human herpesvirus 6 variants A and B.

The human herpesvirus 6 (HHV-6) early protein, p41, encoded by the U27 gene has been detected in oligodendrocytes of multiple sclerosis (MS) patients by using a monoclonal antibody (MAb to p41/38). We here report the antigenic epitope of HHV-6 p41 recognized by this MAb. First, we established that the MAb to p41/38 recognizes a nuclear antigen in HHV-6A strain GS-infected cells but not in HHV-6B strain Z29-infected cells. Secondly, we compared the reactivity of the MAb to p41/38 to that of another p41-specific MAb (MAb to p41) on immunoblots with purified p41-glutathione S-transferase fusion protein from strains GS and Z29 and GS- and Z29-infected-cell lysates. The two MAbs were tested in an enzyme-linked immunosorbent assay against a panel of synthetic peptides covering the amino acid substitutions between the GS- and Z29-derived p41 proteins, as determined by DNA sequencing of our cloned isolates of the U27 gene. The MAb to p41/38 reacted specifically with a peptide comprising p41 residues 321 to 340 from strain GS. The critical residue in this peptide was serine 328, as the substitution S328N in the Z29 strain rendered the corresponding peptide nonreactive. The p41 S328 marker was present in three of three HHV-6A strains, while four of four sequenced p41 genes from HHV-6B strains had N328. Our findings are of value for the interpretation of previous findings of p41 expression in brains of MS patients and may allow a more detailed analysis of the role of HHV-6 variants in other disorders.

Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases.

The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.

Cell phenotype-dependent splicing reflecting differential promoter usage for EBNA transcripts in EBV-carrying cells.

Three types of virus-host cell interactions have been described in cells latently infected with EBV: EBNA 1 expression in type I Burkitt's lymphoma cell lines (BL), EBNA 1, LMP1 and 2 expression in most nasopharyngeal carcinomas (NPC) and EBNA 1-6 with LMP 1 and 2 expression in group III BL-lines as well as lymphoblastoid cell lines (LCL). Two group I BL lines that express only EBNA 1 were found to initiate their EBNA 1 mRNA transcription from a promoter in the Bam HI Q-fragment. They use a sequence at +210 bp relative to the Fp transcription initiation site in group I BL cell lines. The Fp promoter-region seems to be activated in the lytic cycle. LCLs initiate their transcription from one of several upstream sites, usually the Cp promoter or, less frequently, one of several Wp-promoters. Using RNA-reverse transcription polymerase chain reaction (RT-PCR), we have now shown that EBV carrying cells that do not express EBNA 2-6 always splice their EBNA mRNA at the Q-exon, while EBNA 2-6 positive cells use either the Cp or one of the Wp promotors. When EBNA 2-6 are downregulated by somatic cell hybridisation between EBNA 1-6 positive B-cell lines and non B-cells of hematopoetic, epithelial or fibroblastic origin that express the phenotype of the non-B cell parent, the parental usage of Cp/Wp is switched off, and the Q-exon is activated. NPC cells show the same pattern of promoter usage as the hybrids with non-B phenotype. Group III BL cells use both promoter regions. Thus, the virus can use two alternative programs, depending on the cell phenotype. The "EBNA-1 only" program is activated from the Q-promoter. In cells with an immunoblastic (LCL or BL group III) phenotype, the upstream Cp/Wp promoters generate a 100 kb. long pre-mRNA, from which all the EBNAs are spliced. As a rule, only one of the two programs is used for each phenotype, except for the BL group III cells that began as group I but subsequently developed into a more LCL-like cell. Such cells used both promoter regions, with or without activation of the lytic cycle.

Degradation of the epstein-barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue.

The latent membrane protein 1 (LMP1) of the Epstein-Barr virus is a constitutively active receptor essential for B lymphocyte transformation by the Epstein-Barr virus. It is a short-lived protein, but the proteolytic pathway involved in its degradation is not known. The ubiquitin pathway is a major system for specific protein degradation in eukaryotes. Most plasma membrane substrates of the pathway are internalized upon ubiquitination and delivered for degradation in the lysosome/vacuole. Here we show that LMP1 is a substrate of the ubiquitin pathway and is ubiquitinated both in vitro and in vivo. However, in contrast to other plasma membrane substrates of the ubiquitin system, it is degraded mostly by the proteasome and not by lysosomes. Degradation is independent of the single Lys residue of the protein; a lysine-less mutant LMP1 is degraded in a ubiquitin- and proteasome-dependent manner similar to the wild type protein. Degradation of both wild type and lysine-less protein is sensitive to fusion of a Myc tag to the N terminus of LMP1. In addition, deletion of as few as 12 N-terminal amino acid residues stabilizes the protein. These findings suggest that the first event in LMP1 degradation is attachment of ubiquitin to the N-terminal residue of the protein. We present evidence suggesting that phosphorylation is also required for degradation of LMP1.

NotI clones in the analysis of the human genome.

Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.

Effective construction of DNA vaccines against variable influenza genes by homologous recombination.

We demonstrate the potential of cloning by homologous recombination as a rapid method to construct DNA molecules encoding newly developing hemagglutinins (HA) of influenza A virus. The variable parts of the HA genes were cloned into a basic construct containing the HA gene from an H3N2 strain. The recombinant DNAs thus created encode different variable domains with neutralising epitopes from four recently circulating influenza A H3 strains. The technology allows rapid production of DNA constructs for vaccines that can induce antibody and, particularly, cellular immune responses. These new constructs were also capable of conferring protection to challenge in mice. The technology may hence be a valuable tool for rapid adaptation of influenza vaccines to changes in the circulating influenza strains.

Augmentation of leukocyte infiltration in murine tumors expressing B-cell derived but not nasopharyngeal carcinoma derived EBV membrane protein LMP1.

The Epstein-Barr virus (EBV) encoded latent membrane protein of B cell origin, B-LMP1 (B95-8 prototype) and nasopharyngeal carcinoma (NPC) derived C-LMP1 (CAO prototype) were transfected individually in S6C adenocarcinoma cells of ACA (H-2f) origin. We have shown previously that inoculation of B-LMP1 expressing S6C cells led to tumor rejection in pre-immunized, immunocompetent syngeneic ACA mice, whereas the C-LMP1 transfectants were not immunogenic. Furthermore, B-LMP1 but not C-LMP1 expressing S6C cells grew with necrosis and extensive skin damage in non-immunized mice. A study was carried out to determine whether the in vivo growth pattern of S6C cells expressing two different LMP1 isolates could be correlated to any immunomodulatory mechanism. An increased infiltration of CD45+ leukocytes was found in B-LMP1 expressing S6C tumors originating in non-immunized, syngeneic ACA mice. The C-LMP1 expressors, vector transfectants and untransfected parental tumors had significantly lower number of infiltrating leukocytes. The immunoaccessory molecules ICAM-1, B7-1 and MHC Class I and II expression was unaltered in both B- and C-LMP1 transfectants. The data suggest that B-LMP1 but not C-LMP1 induce anti-tumor immune response.

DNA-encoding enzymatically active HIV-1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV-1 challenge.

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.

Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

Identification of new tumor suppressor genes based on in vivo functional inactivation of a candidate gene.

As a step towards developing a new functional test for the identification of tumor suppressor genes, human wild type and mutant RB genes were expressed in the mouse A9 fibrosarcoma cell line under the transcriptional regulation of the tetracycline repressor using two new vectors: pLNCtTA and pETI. Following passage of the transfectants in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumors tested. In contrast, a non-functional mutant RB gene was maintained in all 10 tumors studied. These results suggest that tests for the identification of tumor suppressor genes may be based on their functional inactivation in vivo, rather than on growth suppression.

A new picornavirus isolated from bank voles (Clethrionomys glareolus).

A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype.

Differential immunogenicity of Epstein-Barr virus (EBV) encoded growth transformation-associated antigens in a murine model system.

The strong immunosurveillance of humans against EBV transformed immunoblasts, mediated by CD8+ cytotoxic T cells, is based on the recognition of peptides derived from eight of the nine growth transformation-associated proteins, the nuclear antigens EBNA2-6 and the membrane proteins LMP1, -2A and -2B. The ninth protein, EBNA1, required for maintenance of the viral episomes, and expressed in a cell phenotype independent manner, has not been found to generate a cytotoxic lymphocyte (CTL) response in humans. We tested whether EBNA1 has a similar immunologically privileged status in a species that has not encountered the virus in nature, the mouse. Non-immunogenic murine mammary carcinoma cells were transfected with the appropriate viral gene. Rejection responses were assayed in syngeneic mice following repeated immunisation with irradiated cells. Previously, we found that LMP1 expression in S6C, a murine mammary carcinoma of ACA (H-2f) origin, induces high rejectability, whereas corresponding EBNA1 transfectants remained non-immunogenic. In order to test whether this finding could be reproduced on another MHC class 1 background, we expressed LMP1 and EBNA1 in another non-immunogenic mammary carcinoma, SBfnHd of CBA (H-2k) origin. LMP1 but not EBNA1 transfectants were immunogenic in this system. In order to investigate whether other growth transformation-associated EBV proteins were immunogenic in the mouse, we also transfected the S6C cells with EBNA4, EBNA5, LMP2A and -2B. All four proteins induced strong rejection reactions. These findings are fully consistent with corresponding findings in the human system. They also show that the immunologically privileged status of EBNA1 is not due to some peculiarity of the long-standing co-existence between EBV and the human species, nor to any specific features of the human MHC class I system. They are consistent with the suggestion that EBNA1 may not be properly processed and/or transported, due to specific features of the protein itself.

Re-expression of interleukin 1 in human papillomavirus 18 immortalized keratinocytes inhibits their tumorigenicity in nude mice.

The ability to form tumors in nude mice developed spontaneously in the human papillomavirus (HPV)-18 immortalized keratinocyte cell line, 18-11, and is shown here to be accompanied by a loss of interleukin 1 (IL-1) alpha and beta expression at both the RNA and protein level. In addition, a separate tumorigenic 18-11 derivative and two cervical carcinoma-derived cell lines, HeLa and Caski, were found to have significantly decreased or lost IL-1 alpha and IL-1 beta expression. Using retroviral expression vectors, we re-established IL-1 expression in tumorigenic 18-11 cells (18-11S3) in an effort to evaluate whether loss of IL-1 expression represented an important phenotypic change in the development of tumorigenicity in these cells. IL-1-expressing 18-11S3 cells showed a range of tumorigenic potential, depending on the type and combination of IL-1 alpha and IL-1 beta expressed. Although 18-11S3 expressing the precursor forms of both IL-1 alpha and IL-1 beta normally found in keratinocytes showed moderate inhibition of tumorigenicity, other IL-1-expressing lines showed complete inhibition of tumor formation. Co-injection of nontumorigenic, IL-1-expressing 18-11S3 with parental 18-11S3 also inhibited tumor formation. These results suggest that maintenance of IL-1 expression may play an important role in preventing progression to tumorigenicity in cervical carcinoma and other epithelial cancers.

Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen.

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.

NotI jumping and linking clones as a tool for genome mapping and analysis of chromosome rearrangements in different tumors.

Long-range restriction site maps are of central importance for mapping the human genome. The use of clones from linking and jumping libraries for genome mapping offers a promising alternative to the laborious procedures used up until now. In the present review, this research field is analyzed with particular emphasis on the implementation of a shot-gun sequencing strategy for genome mapping and the use of NotI linking clones for analysis of rearrangements in tumors and tumor cell lines.

Antibodies to LMP2A/2B in EBV-carrying malignancies.

Antibodies to the Epstein-Barr virus (EBV)-encoded membrane proteins, LMP2A and LMP2B, were assayed in 540 individuals, including 154 patients with nasopharyngeal carcinoma, 16 with African Burkitt's lymphoma, 113 with Hodgkin's disease, 14 with EBV-carrying gastric carcinoma, 14 with oral hairy leucoplakia (HIV+ patients), 37 with non-Hodgkin's lymphoma, 49 with tumours of the head/neck, 19 with infectious mononucleosis, 62 with chronic illnesses with EBV titres consistent with re-activations, and 62 healthy controls. A novel assay, mouse monoclonal enhanced indirect immunofluorescence assay (MIFA) was designed and used to test the sera for antibodies to the LMP2A and 2B proteins, expressed in human keratinocytes. Antibody to both LMP2A and LMP2B was strikingly specific to NPC. Virtually all (99 of 101) of the LMP2 antibody positive individuals were NPC patients, 95% of whom had antibodies that reacted both with the LMP2A- and LMP2B-transfected indicator cells, while the remaining 5% reacted only with the LMP2B expressing cells.