Ingemar Ernberg

HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.

The HIV‐1 trans‐activator protein, tat, is an RNA binding protein with a high affinity for a U‐rich bulge near the tip of the stem in the RNA stem‐loop structure encoded by the trans‐activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one‐to‐one complex with HIV‐1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6‐ to 8‐fold lower affinity than wild‐type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem‐loop structure reduced tat binding 10‐fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson‐Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.

Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma

The BamHI fragment containing the Epstein-Barr virus (EBV) LMP1 gene was cloned from a genomic library of the nude mouse-propagated Chinese nasopharyngeal carcinoma CAO. The sequence of the LMP1 gene and its promoter and enhancer was determined. The nucleotide sequence of the CAO isolate differed from those of the B95-8 and Raji isolates in the promoter/enhancer region; the amino acid sequence of the protein also differed. Structural differences in the protein were located mainly in the 20 N-terminal residues and the array of repeated amino acids in the C-terminal part of the protein, in which the CAO isolate displays a cluster of seven perfect repeats of 11 amino acids (aa). Three of these repeats have no counterpart in the other virus strains. This, together with two deletions of five and 10 aa in the C-terminal part, yields a protein of 404 aa, compared to 386 aa for B95-8 and Raji. The larger LMP1 protein was detected on immunoblots of tissue samples from the CAO nude mouse tumour, and was also present in EBV-negative B cell lines and immortalized keratinocytes transfected with the cloned gene. A XhoI restriction site in exon 1 of the B95-8 BNLF-1 gene was absent from the CAO EBV isolate, as well as from 36 of 37 Chinese NPC biopsies tested. In contrast, 17 of 19 NPC biopsies of African origin retained this XhoI site.

EBV gene expression in an NPC-related tumour.

A nasopharyngeal carcinoma tumour (designated C15) propagated in nude mice has been used to generate a large cDNA library that we have analysed for Epstein‐Barr virus (EBV) gene expression. No gross alterations exist in viral DNA from C15 relative to other human isolates and the large deletion present in the B95‐8 ‘prototype’ viral strain established in marmoset cells is not found; C15 contains no linear virion DNA. In the cDNA library, of the six EBV nuclear antigens (EBNAs) expressed in latently infected B‐lymphocytes, only clones for EBNA‐1 are found. These data are confirmed by immunoblotting. Sequence analysis shows the EBNA‐1 mRNA splicing pattern in the carcinoma to differ from that observed in B‐lymphocytes. Further, contrary to observations with B‐cell lines, most viral transcription in the tumour is localized onto the ‘rightmost’ region of the conventional EBV physical map. Transcripts identified corresponding to known genes include those for the latent membrane protein (LMP), the alkaline DNA exonuclease and probably the terminal protein; major transcripts are also derived from the BamHI D fragment and the region deleted in B95‐8 EBV DNA. Novel transcripts have also been identified that proceed in an anti‐sense direction to genes encoding functions associated with replication, such as the viral DNA polymerase. They contain a large, hitherto unidentified, open reading frame in the viral genome that is complementary to the putative function known as BALF3 and a smaller open reading frame complementary to BALF5 (the DNA polymerase gene). From the present studies we can conclude that: (i) EBV transcription patterns in the epithelial cells vary markedly from those identified previously in B‐cells, reflecting differential use of promoters or splicing patterns. (ii) Transcription is tightly regulated and restricted in the C15 tumour with many latent genes, notably EBNAs 2‐6, being ‘switched off.’ (iii) A family of cytoplasmic RNAs are transcribed in an antisense direction to a number of existing open reading frames in the EBV genome. (iv) There are a number of mutations in C15 transcripts relative to the B95‐8 genome, some of which could result in amino acid alterations in proteins.

WW Domains Provide a Platform for the Assembly of Multiprotein Networks

ABSTRACT WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs and phosphorylated serine/threonine-proline sites. To pursue the functional properties of WW domains, we employed mass spectrometry to identify 148 proteins that associate with 10 human WW domains. Many of these proteins represent novel WW domain-binding partners and are components of multiprotein complexes involved in molecular processes, such as transcription, RNA processing, and cytoskeletal regulation. We validated one complex in detail, showing that WW domains of the AIP4 E3 protein-ubiquitin ligase bind directly to a PPXY motif in the p68 subunit of pre-mRNA cleavage and polyadenylation factor Im in a manner that promotes p68 ubiquitylation. The tested WW domains fall into three broad groups on the basis of hierarchical clustering with respect to their associated proteins; each such cluster of bound proteins displayed a distinct set of WW domain-binding motifs. We also found that separate WW domains from the same protein or closely related proteins can have different specificities for protein ligands and also demonstrated that a single polypeptide can bind multiple classes of WW domains through separate proline-rich motifs. These data suggest that WW domains provide a versatile platform to link individual proteins into physiologically important networks.

Latent Membrane Protein 2A of Epstein-Barr Virus Binds WW Domain E3 Protein-Ubiquitin Ligases That Ubiquitinate B-Cell Tyrosine Kinases

ABSTRACT 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.

Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin's Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

In approximately 50% of patients with Hodgkins lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Comparison of plasma Epstein–Barr virus (EBV) DNA levels and serum EBV immunoglobulin A/virus capsid antigen antibody titers in patients with nasopharyngeal carcinoma

Serologic measurement of antibodies to Epstein–Barr virus (EBV) immunoglobulin A/viral capsid antigen (IgA/VCA) and early antigen (IgA/EA) has been used widely to screen for nasopharyngeal carcinoma (NPC) in China. Recently, it was found that plasma EBV DNA concentration is an indicator for the staging and prognosis of patients with NPC. To determine whether there is a correlation between plasma EBV DNA levels and serum levels of IgA/VCA, the authors measured both in patients with NPC and in a control group.

Epstein-Barr virus: adaptation to a life within the immune system

Epstein-Barr virus has developed multiple strategies to ensure its long-term persistence in the infected B cells of immunocompetent hosts. These include the establishment of cell-phenotype-specific programs of viral gene expression and the transduction of cellular genes that modulate immune responses. Cytotoxic T cells may specifically influence the evolution of this genetically stable virus.

Appearance of Epstein-Barr virus-associated antigens in infected Raji cells

Abstract The appearance of EBV-associated early and membrane antigens (EA and MA) was studied in EBV-infected Raji cells. Newly synthetized MA appeared on approximately 50% of the cells 20–24 hours after infection. Around the same time intranuclear EA appeared in 2–5% of the cells, spreading later into the cytoplasm. The appearance of MA and EA was not inhibited by Ara C and iododeoxyuridine, but was completely prevented by puromycin. Viral capsid antigen (VCA) synthesis was not observed during an observation period of 4 days. Unless DNA synthesis was inhibited, the frequency of MA- and EA-positive cells decreased continuously after 1 or 2 days; the infectious cycle was thus abortive. Exposure of EBV to sera with high anti-MA titers neutralized the virus, as judged by its inability to induce EA. The virusneutralizing titers could be reduced by absorbing the sera with an EBV carrying cell line containing a high frequency of MA-positive cells, but not with a line with few MA-positives. The frequency of VCA + EA + cells was approximately equal in both lines. This supports the hypothesis that MA may represent viral envelope components inserted into the cell membrane, in analogy with the HSV system.

Genomic sequence analysis of Epstein-Barr virus strain GD1 from a nasopharyngeal carcinoma patient

ABSTRACT To date, the only entire Epstein-Barr virus (EBV) genomic sequence available in the database is the prototype B95.8, which was derived from an individual with infectious mononucleosis. A causative link between EBV and nasopharyngeal carcinoma (NPC), a disease with a distinctly high incidence in southern China, has been widely investigated. However, no full-length analysis of any substrain of EBV from this area has been reported. In this study, we analyzed the entire genomic sequence of an EBV strain from a patient with NPC in Guangdong, China. This EBV strain was termed GD1 (Guangdong strain 1), and the full-length sequence of GD1 was submitted to the GenBank database. The assigned accession number is AY961628 . The entire GD1 sequence is 171,656 bp in length, with 59.5% G+C content and 40.5% A+T content. We detected many sequence variations in GD1 compared to prototypical strain B95.8, including 43 deletion sites, 44 insertion sites, and 1,413 point mutations. Furthermore, we evaluated the frequency of some of these GD1 mutations in Cantonese NPC patients and found them to be highly prevalent. These findings suggest that GD1 is highly representative of the EBV strains isolated from NPC patients in Guangdong, China, an area with the highest incidence of NPC in the world. Furthermore, these findings provide the second full-length sequence analysis of any EBV strain as well as the first full-length sequence analysis of an NPC-derived EBV strain.