Epstein-Barr virus LMP2A enhances B-cell responses in vivo and in vitro

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.     

Epstein-Barr virus LMP2A-induced B-cell survival in two unique classes of EmuLMP2A transgenic mice

Latent membrane protein 2A (LMP2A) is one of only two viral proteins expressed during latent Epstein-Barr virus (EBV) infections in human peripheral B cells. LMP2A blocks B-cell receptor (BCR) signal transduction in vitro by modulation of the Syk and Lyn protein tyrosine kinases. Five genetically unique LMP2A transgenic mouse lines (EmuLMP2A) with B-cell lineage expression of LMP2A were generated in this study to analyze the importance of LMP2A expression in vivo. These animals can be grouped into EmuLMP2A(BCR+) (TgB, Tg6, and TgC) and EmuLMP2A(BCR-) (Tg7 and TgE) lines based on B-cell phenotype. LMP2A expression in bone marrow cells of EmuLMP2A(BCR-) lines was associated with a bypass of normal B-lymphocyte developmental checkpoints inasmuch as immunoglobulin light-chain gene rearrangement occurred in the absence of complete immunoglobulin heavy-chain gene rearrangement. The resulting BCR-negative B cells were able to exit the bone marrow and colonize peripheral lymphoid organs. LMP2A expression in EmuLMP2A(BCR+) lines was not associated with altered B-cell development in a genetically wild-type background. When crossed into a recombinase activating null (RAG(-/-)) genetic background, LMP2A expression in either RAG(-/-) EmuLMP2A(BCR+) or RAG(-/-) EmuLMP2A(BCR-) animals was able to provide a survival signal to BCR-negative splenic B cells. Additionally, bone marrow cells from all EmuLMP2A animals were able to proliferate in response to interleukin-7-dependent developmental signals in vitro. These studies illustrate that LMP2A can provide a survival signal to BCR-negative B cells in two different groups of EmuLMP2A transgenic mice.     

Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been shown to be indispensable for immortalization of latently infected B lymphocytes, and it has been shown that EBNA-2 exists in a high-molecular-weight complex in these cells. In order to study the components of this protein machinery, we have purified baculovirus-expressed EBNA-2 from insect cells to greater than 95% homogeneity. We have shown by both gel filtration and sucrose gradient analysis that the purified material corresponds to a multimer containing eight EBNA-2 subunits. This multimeric complex is stable in 1.0 M NaCl, suggesting that the self-association is quite strong in vitro. By expressing portions of the EBNA-2 open reading frame to generate fusion proteins in yeast cells, we have used the two-hybrid system to demonstrate that this self-association occurs in vivo and is mediated at least in part by a domain of EBNA-2 encompassing amino acids 122 to 344. Mutational analysis of the self-association function suggests that two subdomains that flank amino acid 232 may each play a role in EBNA-2 protein-protein interaction.     

PY motifs of Epstein-Barr virus LMP2A regulate protein stability and phosphorylation of LMP2A-associated proteins

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A amino-terminal domain. In this study, LMP2A PY motif mutant viruses were constructed to investigate the role of the LMP2A PY motifs. AIP4 was found to specifically associate with the LMP2A PY motifs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our original observation to EBV-infected cells. Mutation of both of the LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa protein. In addition, there was a modest increase in the constitutive phosphorylation of Syk and an unidentified 60-kDa protein. These results indicate that the PY motifs contained within LMP2A are important in regulating phosphorylation in EBV-infected LCLs, likely through the regulation of Lyn activity by specifically targeting the degradation of Lyn by ubiquination by Nedd4 family E3s. Despite differences between PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still exhibited a block in B-cell receptor (BCR) signal transduction as measured by the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation. EBV-transformed LCLs with mutations in the PY motifs were not different from wild-type LCLs in serum-dependent cell growth. Protein stability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.     

WW- and SH3-domain interactions with Epstein-Barr virus LMP2A

Epstein-Barr virus (EBV) is a human herpesvirus which establishes a lifelong latent infection in B lymphocytes. Latent membrane protein 2A (LMP2A) is expressed in both humans with EBV latent infection and EBV immortalized cell lines grown in culture. Previous studies have shown that the amino terminal domain of LMP2A, which contains eight tyrosines, associates with a variety of cellular proteins via SH2-phosphotyrosine interactions. Also contained within the LMP2A amino terminal domain are five proline-rich regions, three of which possess the PxxP core consensus sequence required for interacting with SH3 domains and two of which possess the PPxY core consensus sequence (PY motif) required for interacting with class I type WW domains. In the current study, the ability of LMP2A to interact with either modular SH3 or WW domains was investigated. The results of these studies indicate that the two LMP2A PY motifs interact strongly with representative class I WW domains, but not with representative class II WW domains. In contrast, no interactions were detected between LMP2A and any of the five different SH3 domains tested. These data demonstrate that a subset of the conserved proline-rich motifs within the amino terminus of LMP2A can potentially mediate interactions with cellular proteins and may play a role in EBV-mediated latency and/or transformation.     

Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt

The Epstein-Barr virus LMP2A protein was expressed in a human keratinocyte cell line, HaCaT, and effects on epithelial cell growth were detected in organotypic raft cultures and in vivo in nude mice. Raft cultures derived from LMP2A-expressing cells were hyperproliferative, and epithelial differentiation was inhibited. The LMP2A-expressing HaCaT cells were able to grow anchorage independently and formed colonies in soft agar. HaCaT cells expressing LMP2A were highly tumorigenic and formed aggressive tumors in nude mice. The LMP2A tumors were poorly differentiated and highly proliferative, in contrast to occasional tumors that arose from parental HaCaT cells and vector control cells, which grew slowly and remained highly differentiated. Animals injected with LMP2A-expressing cells developed frequent metastases, which predominantly involved lymphoid organs. Involucrin, a marker of epithelial differentiation, and E-cadherin, involved in the maintenance of intercellular contact, were downregulated in LMP2A tumors. Whereas activation of the mitogen-activated protein kinase pathway was not observed, phosphatidylinositol-3-kinase (PI3-kinase)-dependent activation of the serine-threonine kinase Akt was detected in LMP2A-expressing cells and LMP2A tumors. Inhibition of this pathway blocked growth in soft agar. These data indicate that LMP2A greatly affects cell growth and differentiation pathways in epithelial cells, in part through activation of the PI3-kinase-Akt pathway.     

The only domain which distinguishes Epstein-Barr virus latent membrane protein 2A (LMP2A) from LMP2B is dispensable for lymphocyte infection and growth transformation in vitro; LMP2A is therefore nonessential.

Using second-site homologous recombination, Epstein-Barr virus (EBV) recombinants were constructed which carry an LMP2A mutation terminating translation at codon 19. Despite the absence of LMP2A or LMP2A cross-reactive protein, the recombinants were able to initiate and maintain primary B-lymphocyte growth transformation in vitro. EBNA1, EBNA2, and LMP1 expression was unaffected by the LMP2A mutation. The LMP2A mutant recombinant EBV-infected lymphoblastoid cell lines (LCLs) were identical to wild-type recombinant EBV-infected control LCLs with respect to initial outgrowth, subsequent growth, sensitivity to limiting cell dilution, sensitivity to low serum, and growth in soft agarose. The permissivity of LCLs for lytic EBV infection and virus replication was also unaffected by the LMP2A mutation.     

A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1.

Recent cDNA cloning and sequencing of two Epstein-Barr virus (EBV)-specific mRNAs from latently infected cultures revealed that these RNAs are encoded across the fused terminal repeats of the viral genome and that they are likely to encode two nearly identical proteins with the same transmembrane domains. The smaller predicted protein (LMP2B) lacks 119 amino-terminal amino acids found in the larger one (LMP2A). To test whether these proteins are expressed in latently infected lymphocytes, antibodies to the LMP2 proteins were derived by immunizing rabbits with TrpE-LMP2A fusion proteins. Affinity-purified LMP2-specific antibodies recognized 54- and 40-kilodalton proteins, corresponding to LMP2A and LMP2B, in immunoblots of rodent fibroblasts stably transfected with eucaryotic expression plasmids containing either the LMP2A or LMP2B cDNA. Similar-size proteins were also identified in immunoblots of latently infected lymphocytes. LMP2A localized to membranes in cellular fractionation studies. In immunofluorescent studies, LMP2 localized in the plasma membrane of EBV-infected lymphocytes, with the majority of reactivity confined to the region of the LMP1 patch. This reactivity was detected in almost all lymphoblastoid cells latently infected with EBV.     

Extending the cytoplasmic tail of the influenza a virus M2 protein leads to reduced virus replication in vivo but not in vitro

A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.     

Host-range restriction of vaccinia virus E3L deletion mutant can be overcome in vitro, but not in vivo, by expression of the influenza virus NS1 protein

During the last decades, research focused on vaccinia virus (VACV) pathogenesis has been intensified prompted by its potential beneficial application as a vector for vaccine development and anti-cancer therapies, but also due to the fear of its potential use as a bio-terrorism threat. Recombinant viruses lacking a type I interferon (IFN) antagonist are attenuated and hence good vaccine candidates. However, vaccine virus growth requires production in IFN-deficient systems, and thus viral IFN antagonists that are active in vitro, yet not in vivo, are of great value. The VACV E3 and influenza virus NS1 proteins are distinct double-stranded RNA-binding proteins that play an important role in pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. Based on the functional similarities between E3 and NS1, we investigated the ability of NS1 to replace the biological functions of E3 of VACV in both in vitro and in vivo systems. For this, we generated a VACV recombinant virus lacking the E3L gene, yet expressing NS1 (VVΔE3L/NS1). Our study revealed that NS1 can functionally replace E3 in cultured cells, rescuing the protein synthesis blockade, and preventing apoptosis and RNA breakdown. In contrast, in vivo the VVΔE3L/NS1 virus was highly attenuated after intranasal inoculation, as it was unable to spread to the lungs and other organs. These results indicate that there are commonalities but also functional differences in the roles of NS1 and E3 as inhibitors of the innate antiviral response, which could potentially be utilized for vaccine production purposes in the future.     

Comparative analysis of signal transduction by CD40 and the Epstein-Barr virus oncoprotein LMP1 in vivo

There is much evidence, based primarily on in vitro studies, indicating that the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1) mimics an activated CD40 receptor. In order to investigate the extent of similarity between LMP1 and CD40 functions in vivo, we analyzed the cytoplasmic signaling properties of LMP1 and CD40 in B cells in a directly comparable manner. For this purpose, we generated transgenic mice expressing either LMP1 or a chimeric LMP1CD40 molecule, which constitutively activates the CD40 pathway, under the control of the CD19 promoter. LMP1 and LMP1CD40 were expressed at similar levels in a B-lymphocyte-specific manner. Similar to LMP1, LMP1CD40 suppressed germinal center (GC) formation and antibody production in response to thymus-dependent antigens, albeit to a greater extent than LMP1. Furthermore, the avidity of the antibodies produced against thymus-dependent antigens was lower for LMP1CD40 transgenic mice than for wild-type and LMP1 transgenic mice. GC suppression was linked to the ability of LMP1CD40 and LMP1 to downregulate mRNA and protein levels of BCL6 and to suppress the activity of the BCL6 promoter. In contrast to LMP1, LMP1CD40 caused an upregulation of CD69, CD80, and CD86 in B cells and a dramatic increase in serum immunoglobulin M. In addition, LMP1CD40 but not LMP1 transgenic mice had elevated numbers of marginal-zone B cells and increased populations of polymorphonuclear cells and/or neutrophils. Consistent with these findings, LMP1CD40 but not LMP1 transgenic mice showed signs of spontaneous inflammatory reactions and the potential for autoimmunity.     

TAK1 is a component of the Epstein-Barr virus LMP1 complex and is essential for activation of JNK but not of NF-kappaB

Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-kappaB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-kappaB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-kappaB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IkappaB kinase gamma is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-kappaB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-kappaB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-kappaB activation in LMP1 signaling.     

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.     

A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus.

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.     

In vivo assessment of the relative contributions of deletion, anergy, and editing to B cell self-tolerance

In normal B cell development, a large percentage of newly formed cells bear receptors with high levels of self-reactivity that must be tolerized before entry into the mature B cell pool. We followed the fate of self-reactive B cells expressing high affinity anti-hen egg lysozyme (HEL) Ag receptors exposed in vivo to membrane HEL in a setting in which the anti-HEL L chain was "knocked-in" at the endogenous L chain locus. These mice demonstrated extensive and efficient L chain receptor editing responses and had B cell numbers comparable to those found in animals lacking membrane Ag. BrdU labeling indicated that the time required for editing in response to membrane HEL was approximately 6 h. In mice transgenic for soluble HEL, anti-HEL B cells capable of editing showed evidence for both editing and anergy. These data identify receptor editing as a major physiologic mechanism by which highly self-reactive B cells are tolerized to membrane and soluble self-Ags.     

HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro

Background

We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo.

Methodology/Principal Findings

The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60–67%).

Conclusions/Significance

The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.