Biomedical subjects
Publications and source records attributed to R Longnecker.
Like other herpesviruses, Epstein-Barr virus persists in its host through its ability to establish a latent infection that periodically reactivates. Latent membrane protein 2A (LMP2A) regulates reactivation from latency by interfering with normal B cell signal transduction processes, and may define a new class of regulators of herpesvirus latency.
The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.
Epstein-Barr virus (EBV) recombinants which carry three different deletion mutations in the LMP2A cytoplasmic amino-terminal domain were constructed. The presence of each mutation, LMP2A delta 21-36, LMP2A delta 21-64, and LMP2A delta 21-85, in EBV-infected transformed lymphoblastoid cell lines was confirmed by PCR analysis and Southern blot hybridization. Confirmation of mutant LMP2A protein expression was by immunofluorescence and immunoblotting with a newly identified rat monoclonal antibody that recognizes each of the LMP2A deletion mutations. Lymphoblastoid cell lines infected with recombinant EBV DNAs containing the mutations were analyzed for loss of LMP2A's dominant-negative effect on surface immunoglobulin signal transduction by monitoring induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication following surface immunoglobulin cross-linking. Domains of LMP2A important for induction of tyrosine phosphorylation, calcium mobilization, and activation of lytic replication were identified.
An Epstein-Barr virus-encoded protein, LMP2, blocks the effects of surface immunoglobulin (slg) cross-linking on calcium mobilization and on lytic reactivation of EBV in latently infected and growth-transformed primary human B lymphocytes. In wild-type EBV-transformed cells, LMP2 is constitutively tyrosine phosphorylated and is associated with Lyn and Syk protein-tyrosine kinases (PTKs). Baseline Lyn PTK activity is substantially reduced, and slg cross-linking fails to activate Lyn, Syk, Pl3-K, PLC gamma 2, Vav, Shc, and MAPK. Syk, Pl3-K, PLC gamma 2, and Vav are constitutively tyrosine phosphorylated, and their tyrosine phosphorylation does not change following slg cross-linking. In contrast, cross-linking slg on cells transformed by LMP2 null mutant EBV recombinants triggers the same protein tyrosine kinase cascade as in noninfected B lymphocytes. These data are consistent with a model in which LMP2 is a constitutive dominant negative modulator of slg receptor signaling through its effects on Lyn, Syk, or regulators of these kinases.
The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.
Epstein-Barr Virus (EBV) causes infectious mononucleosis in normal adolescents and malignant B lymphocyte proliferation in immune compromised patients, in marmosets, or upon transfer of infected human B lymphocytes into SCID mice. EBV is also etiologically associated with African Burkitt's lymphoma, Hodgkin's Disease, and nasopharyngeal cancer. EBV transformed, latently infected B lymphocytes contain EBV episomes and eight virus encoded proteins. Six are nuclear proteins (EBNAs) and two are the integral membrane proteins, LMP1 and LMP2. These eight proteins are presumed to mediate latent virus infection or B lymphocyte proliferation and are thus under intense scrutiny. Besides EBNA1, which is required for episome maintenance, LMP1 and LMP2, are the two transformation associated proteins that are most consistently detected in EBV related malignancies, and the LMP2 message is the only message detected in PCR analysis of B lymphocytes from individuals harboring EBV latent infections. LMP2 associates with src family tyrosine kinases, a 70 kda cell phosphoprotein, LMP1 and several other unidentified cell proteins. LMP1 is a key mediator of EBV's effects on inducing B lymphocyte activation and adhesion molecules and is a transforming oncogene in rodent fibroblasts. The association of these two EBV encoded membrane proteins could create a macromolecular complex mediating constitutive B lymphocyte activation through normal cell signal transduction pathways. LMP2 might may control activation of lytic replication or down regulate the activation state of EBV infected cells allowing persistence in the human host.
A Burkitt lymphoma cell line infected in vitro with a transformation-defective mutant recombinant Epstein-Barr virus (EBV) was used to attempt marker rescue of transformation competence by transfection with cloned wild-type DNA. EBV replication was induced in the transfected cells, and wild-type EBV DNA recombined via flanking homologous sequences adjacent to the deletion, resulting in a virus which transformed primary B lymphocytes in vitro. This strategy should be useful for molecular genetic analysis of the role of part or all of any gene in cell growth transformation.
Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)
Specifically mutated Epstein-Barr virus (EBV) recombinants which truncate latent membrane protein 2A (LMP2A) and LMP2B after 260 of 497 amino acids and after 141 of 378 amino acids, respectively, were constructed. Despite truncation before the last seven transmembrane domains and the carboxy terminus, the mutant recombinants were not altered in initiation of primary B-lymphocyte infection or growth transformation, in expression of nuclear protein 1 or 2 or LMP1, or in induction of lytic EBV replication. Cells transformed by mutant virus recombinants were not different from wild-type virus transformants in initial or long-term outgrowth, sensitivity to limiting cell dilution, serum requirement, or clonogenic growth in soft agar. Together with similar analyses of a mutation stopping translation of the LMP2A amino-terminal cytoplasmic domain, these results indicate that LMP2 is not required for primary B-lymphocyte infection in vitro.
LMP2A is expressed in latent Epstein-Barr virus (EBV) infection and interacts with LMP1 and members of the src tyrosine kinase family in the plasma membrane. Since tyrosine kinase mediate receptor-induced changes in intracellular free calcium, the effect of LMP2A on receptor-mediated intracellular calcium mobilization was evaluated by stably expressing LMP2A in an EBV-negative Burkitt tumor cell line (BJAB) or in LMP1-converted BJAB cells. LMP2A significantly blocked calcium mobilization following class II, CD19, or immunoglobulin M cross-linking. LMP2A effects were partially reversed in LMP1-converted cell lines. These results are compatible with LMP2A acting in latent B-lymphocyte infection to downmodulate LMP1 effects on cell growth or to inhibit induction of lytic EBV infection in specific human tissues following receptor ligation.
A recombinant Epstein-Barr virus (EBV) was constructed, with a positive-selection marker inserted at the site of a deletion of a DNA segment which encodes the first five transmembrane domains of LMP2A and LMP2B. Despite the mutation, the mutant recombinant EBV was able to initiate and maintain primary B-lymphocyte growth transformation in vitro. Cells transformed with the mutant recombinant were not different from wild-type virus transformants in initial or long-term outgrowth, sensitivity to limiting cell dilution, or serum requirement. Expression of EBNA1, EBNA2, EBNA3A, EBNA3C, and LMP1 and permissivity for lytic EBV infection were also unaffected by the LMP2 deletion mutation. These results complete the molecular genetic studies proving LMP2 is dispensable for primary B-lymphocyte growth transformation, latent infection, and lytic virus replication in vitro.
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.
Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.
In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.
Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitt's lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.
Epstein-Barr virus (EBV) encodes two integral membrane proteins in latently infected growth-transformed cells. One of these, LMP1, can transform rodent fibroblasts and induce markers of B-lymphocyte activation. The second, LMP2, colocalizes with LMP1 in a constitutive patch in the EBV-transformed B-lymphocyte plasma membrane. The experiments reported here demonstrate that LMP2 may biochemically interact with LMP1 and that LMP2 closely associates with and is an important substrate for a B-lymphocyte tyrosine kinase in EBV-transformed B lymphocytes or in B-lymphoma cells in which LMP2 is expressed by gene transfer. LMP2 is also serine and threonine phosphorylated. LMP2 localizes to a peripheral membrane (presumably plasma membrane) patch in transfected B-lymphoma cells and colocalizes with much of the cellular tyrosine-phosphorylated proteins. LMP2 undergoes tyrosine phosphorylation in anti-LMP2 or antiphosphotyrosine immunoprecipitates from transfected B-lymphoma cells or EBV-transformed B lymphocytes. The first 167 of the 497 amino acids of LMP2 retain full ability to associate with and act as a substrate for a tyrosine kinase. A 70-kDa phosphotyrosine cell protein associates with LMP2 in transfected cells or in EBV-transformed B lymphocytes and could be a mediator of the effects of LMP2.
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.
We report the results of studies on the biologic properties of seven deletion mutants of herpes simplex virus 1 (HSV-1). The genes deleted from six of these mutants map in the S component of HSV-1 DNA and include those specifying the alpha protein 47, the glycoproteins G and E, the viral protein kinase, and two proteins whose functions are not yet known (open reading frames US2 and US11). The seventh virus [HSV-1(F) delta 305] contained a 700-bp deletion in the thymidine kinase gene. The results of intracerebral inoculation of Balb/c mice indicated that all but one of the deletion mutants in the S component were significantly attenuated. The PFU/LD50 ratios for these mutants ranged from 10(4)- to 10(5)-fold higher than that of the wild-type, HSV-1(F). The PFU/LD50 for mutant R7032, from which the glycoprotein E gene had been deleted, was less than 100-fold higher than that of the parent virus. All of the mutants, with one exception, were able to establish latency in mice; the exception, HSV-1(F) delta 305, was able to establish latency in rabbits.