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Richard Longnecker

Publications and source records attributed to Richard Longnecker.

At least 37 records · Page 2Linked to original sources
PubMed2004

Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion.

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.

Amino Acid Sequence↗
PubMed2004

LMP2A does not require palmitoylation to localize to buoyant complexes or for function.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed constitutively in lipid rafts in latently infected B lymphocytes. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids selective for specific protein association. Lipid rafts have been shown to be necessary for B-cell receptor (BCR) signal transduction. LMP2A prevents BCR recruitment to lipid rafts, thereby abrogating BCR function. As LMP2A is palmitoylated, whether this fatty acid modification is necessary for LMP2A to localize to lipid rafts and for protein function was investigated. LMP2A palmitoylation was confirmed in latently infected B cells. LMP2A was found to be palmitoylated on multiple cysteines only by S acylation. An LMP2A mutant that was not palmitoylated was identified and functioned similar to wild-type LMP2A; unmodified LMP2A localized to lipid rafts, was tyrosine phosphorylated, was associated with LMP2A-associated proteins, was ubiquitinated, and was able to block calcium mobilization following BCR cross-linking. Therefore, palmitoylation of LMP2A is not required for LMP2A targeting to buoyant complexes or for function.

B-Lymphocytes↗
PubMed2004

Latent membrane protein 2A inhibits transforming growth factor-beta 1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway.

Latent membrane protein 2A (LMP2A) blocks B-cell receptor signal transduction in vitro by binding the Syk and Lyn protein tyrosine kinases. As well as blocking B-cell signal transduction, LMP2A has been shown to activate the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which acts as a survival signal in both B cells and epithelial cells. Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. The loss of the growth-inhibitory response to the TGF-beta 1 signal is found in many cancers and is widely thought to promote tumor development. In this study, we found that LMP2A induced the phosphorylation of Akt (serine 473) in Burkitt's lymphoma cell line Ramos and in gastric carcinoma cell line HSC-39 and partially enhanced cell viability following TGF-beta 1 treatment. In addition, LMP2A partially inhibited TGF-beta 1-induced DNA fragmentation and cleavage of poly(ADP-ribose) polymerase (PARP). In the presence of LY294002, an inhibitor of PI3-K, the LMP2A-mediated inhibitory effects on TGF-beta 1-induced DNA fragmentation and cleavage of PARP were alleviated. Furthermore, LMP2A did not alter the levels of expression of type I and type II TGF-beta 1 receptors. Taken together, these results suggest that LMP2A may inhibit TGF-beta 1-mediated apoptosis through activation of the PI3-K/Akt pathway.

Apoptosis↗
PubMed2004

Inability of simian virus 40 to establish productive infection of lymphoblastic cell lines.

Lymphoblastic cell lines were infected with simian virus 40 (SV40) and then monitored for evidence of a productive infection. No evidence of early gene expression was found 2 days following infection, as determined by assaying viral mRNAs and early antigens. Furthermore, only small amounts of virus could be detected by plaque assay 2 days after infection, and levels slowly declined until they were undetectable after a few weeks in culture. Thus, human lymphocytes are not readily infectible with SV40 and do not provide a simple model for studying interactions of SV40 with a human cell type.

Cell Line↗
PubMed2003

Interference with T cell receptor-HLA-DR interactions by Epstein-Barr virus gp42 results in reduced T helper cell recognition.

Epstein-Barr virus (EBV) persists lifelong in infected hosts despite the presence of antiviral immunity. Many viral antigens are expressed during lytic infection. Thus, for EBV to spread, it must have evolved effective ways to evade immune recognition. Here, we report that HLA class II-restricted antigen presentation to T helper cells is hampered in the presence of the lytic-phase protein gp42. This interference with T cell activation involves association of gp42 with class II peptide complexes. Using HLA-DR tetramers, we identify a block in T cell receptor (TCR)-class II interactions imposed by gp42 as the underlying mechanism. EBV gp42 sterically clashes with TCR Valpha-domains as visualized by superimposing the crystal structures for gp42-HLA-DR1 and TCR-MHC class II complexes. Blocking TCR recognition provides a previously undescribed strategy for viral immune evasion.

Flow Cytometry↗
PubMed2003

Epstein-Barr Virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed-Sternberg cells of Hodgkin lymphoma.

Epstein-Barr virus (EBV) is associated with the development of a variety of malignancies, including Hodgkin lymphoma. One of the few viral transcripts expressed in EBV-positive Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma is latent membrane protein 2A (LMP2A). This viral protein blocks B-cell receptor (BCR)-signaling in vitro. Furthermore, expression of LMP2A in developing B cells in vivo induces a global down-regulation of genes necessary for proper B-cell development. In this study we have analyzed gene transcription in primary B cells from LMP2A transgenic mice, LMP2A-expressing human B-cell lines, and LMP2A-positive and -negative EBV-infected lymphoblastoid cell lines (LCLs). We demonstrate that LMP2A increases the expression of genes associated with cell cycle induction and inhibition of apoptosis, alters the expression of genes involved in DNA and RNA metabolism, and decreases the expression of B-cell-specific factors and genes associated with immunity. Furthermore, many alterations in gene expression induced by LMP2A are similar to those recently described in HRS cells of Hodgkin lymphoma and activated, proliferating germinal center centroblasts/centrocytes. These correlations suggest that LMP2A expression in EBV-infected B cells may lead to the induction and maintenance of an activated, proliferative state that could ultimately result in the development of Hodgkin lymphoma.

Animals↗
PubMed2003

Cholesterol-dependent infection of Burkitt's lymphoma cell lines by Epstein-Barr virus.

Epstein-Barr virus (EBV) infection is a multi-step process, first requiring virus binding to the host cell, followed by fusion of the viral envelope with the host cell plasma membrane. Efficient EBV entry into B cells requires, at the minimum, the interaction of the EBV-encoded glycoproteins gp350 with cellular CD21 and gp42 with MHC class II proteins. In this study, use of the cholesterol-binding drugs methyl-beta-cyclodextrin and nystatin efficiently inhibited EBV infection of target Burkitt's lymphoma B-cell lines, indicating an important role for cholesterol and suggesting the involvement of lipid rafts in EBV infection.

Burkitt Lymphoma↗
PubMed2003

Epstein-Barr virus LMP2A interferes with global transcription factor regulation when expressed during B-lymphocyte development.

Epstein-Barr virus (EBV) is associated with the development of malignant lymphomas and lymphoproliferative disorders in immunocompromised individuals. The LMP2A protein of EBV is thought to play a central role in this process by allowing the virus to persist in latently infected B lymphocytes. We have demonstrated that LMP2A, when expressed in B cells of transgenic mice, allows normal B-cell developmental checkpoints to be bypassed. To identify cellular genes targeted by LMP2A that are involved in this process, we have utilized DNA microarrays to compare gene transcription in B cells from wild-type versus LMP2A transgenic mice. In B cells from LMP2A transgenic mice, we observed decreased expression of many genes associated with normal B-cell development as well as reduced levels of the transcription factors that regulate their expression. In particular, expression of the transcription factor E2A was down-regulated in bone marrow and splenic B cells. Furthermore, E2A activity was inhibited in these cells as determined by decreased DNA binding and reduced expression of its target genes, including the transcription factors early B-cell factor and Pax-5. Expression of two E2A inhibitors, Id2 and SCL, was up-regulated in splenic B cells expressing LMP2A, suggesting a possible mechanism for E2A inhibition. These results indicate that LMP2A deregulates transcription factor expression and activity in developing B cells, and this likely allows for a bypass of normal signaling events required for proper B-cell development. The ability of LMP2A to interfere with B-cell transcription factor regulation has important implications regarding its role in EBV latency.

Animals↗
PubMed2003

Mutational analysis of the HLA class II interaction with Epstein-Barr virus glycoprotein 42.

Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.

Amino Acid Sequence↗
PubMed2003

Itchy, a Nedd4 ubiquitin ligase, downregulates latent membrane protein 2A activity in B-cell signaling.

Nedd4 family ubiquitin protein ligases (E3s) specifically associate with latent membrane protein 2A (LMP2A) of Epstein-Barr virus. Our previous studies analyzing LMP2A function in vitro have suggested that Nedd4 family E3s regulate LMP2A function. To determine the role of Nedd4 family E3s in LMP2A B-cell signaling, LMP2A transgenic (LMP2A(+)) mice were crossed with mice with the Itch-deficient (Itch(-/-)) background. Itchy, a mouse homologue of human AIP4, is a Nedd4 family E3 and is also the most abundant Nedd4 family E3 found in LMP2A affinity precipitates from B cells. There were significantly fewer B-cell receptor-positive B cells in spleen and bone marrow B cells in LMP2A(+) Itch(-/-) mice than in LMP2A(+) mice. In addition, LMP2A(+) Itch(-/-) bone marrow B cells formed larger colonies in cultures treated with interleukin-7 (IL-7) than control bone marrow B cells did. Finally, there was a dramatic increase in tyrosine phosphorylation of LMP2A and Syk in IL-7-cultured LMP2A(+) Itch(-/-) B cells. These results indicate that Nedd4 family E3s, in particular Itchy, downmodulate LMP2A activity in B-cell signaling.

Animals↗
PubMed2002

Lysine-independent ubiquitination of Epstein-Barr virus LMP2A.

Latent membrane protein 2A (LMP2A) of latent Epstein-Barr virus (EBV) specifically associates with HECT domain-containing Nedd4-family ubiquitin-protein ligases (E3s). Here we demonstrate that LMP2A is specifically ubiquitinated by the HECT domains of AIP4 and WWP2. Deletion and site-specific mutation of LMP2A indicates that LMP2A is ubiquitinated at its amino-terminus and is not ubiquitinated on lysine residues. LMP2A and LMP1, also encoded by EBV, are two of only four proteins that have been identified that are ubiquitinated at the amino-terminus, indicating that EBV may specifically target and utilize this host cell protein modification.

Animals↗
PubMed2002

Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation.

Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2002

The LMP2A signalosome--a therapeutic target for Epstein-Barr virus latency and associated disease.

Most adults are infected with Epstein-Barr virus (EBV), a virus that establishes a lifelong latent infection in B lymphocytes and is associated with a variety of cancers. In normal individuals, latent infection with EBV typically poses no health risk, but upon immunosuppression, either following organ transplantation or HIV infection, malignancies and lymphoproliferative diseases can result. We have utilized both transgenic mice and EBV transformed lymphoblastoid cell lines (LCLs) as models of EBV latent infection to explore the function of latent membrane protein 2A (LMP2A) of EBV. This has allowed us to identify important functional domains of LMP2A, essential host proteins necessary for LMP2A function, and the effect of LMP2A on normal B cell function. These studies have provided a more complete understanding of the role of LMP2A in EBV latency and tumorigenesis and may allow for the identification of novel therapeutics for the treatment or eradication of EBV latent infections and associated proliferative disorders.

Animals↗
PubMed2002

Inhibition of host kinase activity altered by the LMP2A signalosome-a therapeutic target for Epstein-Barr virus latency and associated disease.

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection in the majority of the human population. The virus resides in a latent state in B lymphocytes and is associated with a variety of cancers in the human host. In normal individuals, latent infection with EBV typically poses no health risk, but upon immunosuppression, either following organ transplantation or HIV infection, malignancies and lymphoproliferative diseases can result. Latent membrane protein 2A (LMP2A) is a virally encoded membrane protein that is expressed in EBV latent infection and in most of the tumors associated with EBV infection. Previous studies have indicated that LMP2A expression alters the activity of the Src family protein tyrosine kinases, the Syk protein tyrosine kinase, the Btk protein tyrosine kinase, and phosphatidylinositol 3-kinase (PI3-kinase). In this study, inhibitors of each of these kinases were tested using an in vitro system dependent on LMP2A expression for B cell colony formation in IL-7 containing methylcellulose media. Of the inhibitors tested, only piceatannol, a Syk tyrosine kinase inhibitor, demonstrated a specific effect on LMP2A expressing cells and not control cells. These studies provide a basis for targeting LMP2A function in EBV latency and may allow for the identification of novel therapeutics for the treatment or eradication of EBV latent infections and associated proliferative disorders.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2002

Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II receptor HLA-DR1.

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

Amino Acid Sequence↗