The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein

BACKGROUND: The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required. METHODS: Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities. RESULTS: Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM. DISCUSSION: Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients

p53 loss promotes acute myeloid leukemia by enabling aberrant self-renewal

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here, we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML), an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models, Cre-lox technology, and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML, while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells, such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently, myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells, resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation, and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity

Dynamics and Scaling of 2D Polymers in a Dilute Solution

The breakdown of dynamical scaling for a dilute polymer solution in 2D has been suggested by Shannon and Choy [Phys. Rev. Lett. {\bf 79}, 1455 (1997)]. However, we show here both numerically and analytically that dynamical scaling holds when the finite-size dependence of the relevant dynamical quantities is properly taken into account. We carry out large-scale simulations in 2D for a polymer chain in a good solvent with full hydrodynamic interactions to verify dynamical scaling. This is achieved by novel mesoscopic simulation techniques

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle

Counteracting effects of cellular Notch and Epstein-Barr virus EBNA2 : implications for stromal effects on virus-host interactions

A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of infection with Epstein-Barr virus (EBV), which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen 2 (EBNA2) both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-Jκ. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function both in newly infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand 1 inhibits EBNA2-mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 expression from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into the lytic cycle in a B cell non-Hodgkin's lymphoma line by upregulating the cellular transcription factor Zeb2, which represses the transcription of BZLF1. These results support the concept that in vivo, cumulative signals from the microenvironment downregulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood. IMPORTANCE Experimental infection of resting B cells by Epstein-Barr virus leads to the growth transformation program of virus gene expression and the outgrowth of lymphoblastoid cell lines. Previous studies at the single-cell level revealed complex cellular and viral signaling networks regulating transcription of the viral genome. This study demonstrates that viral gene expression can also be radically altered by molecules expressed on stromal cells in the microenvironment of lymphoid tissue, specifically, Delta-like ligand 1 on stromal cells ligating Notch-2 on infected B cells. Activation of Notch interferes with the transactivation function of EBNA2, downregulates the expression of LMP1 and LMP2a, and inhibits the activation of lytic virus replication in a B cell non-Hodgkin's lymphoma line by preventing expression of BZLF1. The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression, enabling establishment of true latency in memory B cells

mspecLINE: bridging knowledge of human disease with the proteome

<p>Abstract</p> <p>Background</p> <p>Public proteomics databases such as PeptideAtlas contain peptides and proteins identified in mass spectrometry experiments. However, these databases lack information about human disease for researchers studying disease-related proteins. We have developed mspecLINE, a tool that combines knowledge about human disease in MEDLINE with empirical data about the detectable human proteome in PeptideAtlas. mspecLINE associates diseases with proteins by calculating the semantic distance between annotated terms from a controlled biomedical vocabulary. We used an established semantic distance measure that is based on the co-occurrence of disease and protein terms in the MEDLINE bibliographic database.</p> <p>Results</p> <p>The mspecLINE web application allows researchers to explore relationships between human diseases and parts of the proteome that are detectable using a mass spectrometer. Given a disease, the tool will display proteins and peptides from PeptideAtlas that may be associated with the disease. It will also display relevant literature from MEDLINE. Furthermore, mspecLINE allows researchers to select proteotypic peptides for specific protein targets in a mass spectrometry assay.</p> <p>Conclusions</p> <p>Although mspecLINE applies an information retrieval technique to the MEDLINE database, it is distinct from previous MEDLINE query tools in that it combines the knowledge expressed in scientific literature with empirical proteomics data. The tool provides valuable information about candidate protein targets to researchers studying human disease and is freely available on a public web server.</p

The Murid Herpesvirus-4 gH/gL Binds to Glycosaminoglycans

The first contact a virus makes with cells is an important determinant of its tropism. Murid Herpesvirus-4 (MuHV-4) is highly dependent on glycosaminoglycans (GAGs) for cell binding. Its first contact is therefore likely to involve a GAG-binding virion glycoprotein. We have previously identified two such proteins, gp70 and gp150. Gp70 binds strongly to GAGs. However, deleting it makes little difference to MuHV-4 cell binding or GAG-dependence. Deleting gp150, by contrast, frees MuHV-4 from GAG dependence. This implies that GAGs normally displace gp150 to allow GAG-independent cell binding. But the gp150 GAG interaction is weak, and so would seem unlikely to make an effective first contact. Since neither gp70 nor gp150 matches the expected profile of a first contact glycoprotein, our understanding of MuHV-4 GAG interactions must be incomplete. Here we relate the seemingly disconnected gp70 and gp150 GAG interactions by showing that the MuHV-4 gH/gL also binds to GAGs. gH/gL-blocking and gp70-blocking antibodies individually had little effect on cell binding, but together were strongly inhibitory. Thus, there was redundancy in GAG binding between gp70 and gH/gL. Gp150-deficient MuHV-4 largely resisted blocks to gp70 and gH/gL binding, consistent with its GAG independence. The failure of wild-type MuHV-4 to do the same argues that gp150 is normally engaged only down-stream of gp70 or gH/gL. MuHV-4 GAG dependence is consequently two-fold: gp70 or gH/gL binding provides virions with a vital first foothold, and gp150 is then engaged to reveal GAG-independent binding

Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32

Response gene to complement-32 (RGC-32) activates cyclin-dependent kinase 1, regulates the cell cycle and is deregulated in many human tumours. We previously showed that RGC-32 expression is upregulated by the cancer-associated Epstein-Barr virus (EBV) in latently infected B cells through the relief of translational repression. We now show that EBV infection of naïve primary B cells also induces RGC-32 protein translation. In EBV-immortalised cell lines, we found that RGC-32 depletion resulted in cell death, indicating a key role in B cell survival. Studying RGC-32 translational control in EBV-infected cells, we found that the RGC-32 3′untranslated region (3′UTR) mediates translational repression. Repression was dependent on a single Pumilio binding element (PBE) adjacent to the polyadenylation signal. Mutation of this PBE did not affect mRNA cleavage, but resulted in increased polyA tail length. Consistent with Pumilio-dependent recruitment of deadenylases, we found that depletion of Pumilio in EBV-infected cells increased RGC-32 protein expression and polyA tail length. The extent of Pumilio binding to the endogenous RGC-32 mRNA in EBV-infected cell lines also correlated with RGC-32 protein expression. Our data demonstrate the importance of RGC-32 for the survival of EBV-immortalised B cells and identify Pumilio as a key regulator of RGC-32 translation