Coactivators p300 and PCAF physically and functionally interact with the foamy viral trans-activator

BACKGROUND: Foamy virus Bel1/Tas trans-activators act as key regulators of gene expression and directly bind to Bel1 response elements (BRE) in both the internal and the 5'LTR promoters leading to strong transcriptional trans-activation. Cellular coactivators interacting with Bel1/Tas are unknown to date. RESULTS: Transient expression assays, co-immunoprecipitation experiments, pull-down assays, and Western blot analysis were used to demonstrate that the coactivator p300 and histone acetyltransferase PCAF specifically interact with the retroviral trans-activator Bel1/Tas in vivo. Here we show that the Bel1/Tas-mediated trans-activation was enhanced by the coactivator p300, histone acetyltransferases PCAF and SRC-1 based on the crucial internal promoter BRE. The Bel1/Tas-interacting region was mapped to the C/H1 domain of p300 by co-immunoprecipitation and pull-down assays. In contrast, coactivator SRC-1 previously reported to bind to the C-terminal domain of p300 did not directly interact with the Bel1 protein but nevertheless enhanced Bel1/Tas-mediated trans-activation. Cotransfection of Bel1/Tas and p300C with an expression plasmid containing the C/H1domain partially inhibited the p300C-driven trans-activation. CONCLUSIONS: Our data identify p300 and PCAF as functional partner molecules that directly interact with Bel1/Tas. Since the acetylation activities of the three coactivators reside in or bind to the C-terminal regions of p300, a C/H1 expression plasmid was used as inhibitor. This is the first report of a C/H1 domain-interacting retroviral trans-activator capable of partially blocking the strong Bel1/Tas-mediated activation of the C-terminal region of coactivator p300. The potential mechanisms and functional roles of the three histone and factor acetyltransferases p300, PCAF, and SRC-1 in Bel1/Tas-mediated trans-activation are discussed

The Epstein-Barr Virus Protein Kinase BGLF4 and the Exonuclease BGLF5 Have Opposite Effects on the Regulation of Viral Protein Production▿

The Epstein-Barr virus BGLF4 and BGLF5 genes encode a protein kinase and an alkaline exonuclease, respectively. Both proteins were previously found to regulate multiple steps of virus replication, including lytic DNA replication and primary egress. However, while inactivation of BGLF4 led to the downregulation of several viral proteins, the absence of BGLF5 had the opposite effect. Using recombinant viruses that lack both viral enzymes, we confirm and extend these initial observations, e.g., by showing that both BGLF4 and BGLF5 are required for proper phosphorylation of the DNA polymerase processivity factor BMRF1. We further found that neither BGLF4 nor BGLF5 is required for baseline viral protein production. Complementation with BGLF5 downregulated mRNA levels and translation of numerous viral genes, though to various degrees, whereas BGLF4 had the opposite effect. BGLF4 and BGLF5 influences on viral expression were most pronounced for BFRF1 and BFLF2, two proteins essential for nuclear egress. For most viral genes studied, cotransfection of BGLF4 and BGLF5 had only a marginal influence on their expression patterns, showing that BGLF4 antagonizes BGLF5-mediated viral gene shutoff. To be able to exert its functions on viral gene expression, BGLF4 must be able to escape BGLF5's shutoff activities. Indeed, we found that BGLF5 stimulated the BGLF4 gene's transcription through an as yet uncharacterized molecular mechanism. The BGLF4/BGLF5 enzyme pair builds a regulatory loop that allows fine-tuning of virus protein production, which is required for efficient viral replication

A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus.

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas

B cells infected by viruses that lack the BART miRNAs express higher levels of caspase 3 and LMP1 and are more resistant to drugs that induce mitochondria-mediated apoptosis.

<p>(A) LCLs transformed by M81 or M81/ΔAll from 4 independent donors were subjected to immunoblotting with antibodies specific to viral latent proteins (EBNA3A, 3B, 3C, EBNA2, LMP1 and LMP2A), viral lytic proteins (BZLF1), caspase 3, and actin. The levels of expression of these proteins are also represented in a bar chart. (B and C) Apoptosis was induced in five pairs of LCLs transformed by M81 or M81/ΔAll. Scatter plots represent the percentage of apoptotic cells as determined by TUNEL assays (B) or by immunostaining with antibodies directed against cleaved caspase 3 (C). DMSO or ethanol-treated samples were used as controls. P values lower than 0.05 obtained after paired t-student tests are indicated. Please also refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005344#ppat.1005344.s007" target="_blank">S7</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005344#ppat.1005344.s008" target="_blank">S8</a> Figs.</p

Deletion of the BART miRNAs enhances spontaneous lytic replication and tumor progression in the humanized mice model.

<p>Viral titers in peripheral blood of infected mice were determined by quantitative PCR at (A) 5 weeks post-infection. (B) The pictures show tumors that developed in the spleen. Continuous tissue sections were stained with hematoxylin and eosin (H&E), immunostained with antibodies specific for BZLF1, gp350, LMP1, EBNA2, or subjected to an <i>in situ</i> hybridization with an EBER-specific probe. Among five M81-Wt-infected mice, 3 mice (referred to as group I) had very few whilst the other 2 mice (referred to as group II) exhibited a higher percentage of BZLF1-positive cells. (C) The number of EBER positive cells per 0.04μm² (surface of the field at high magnification) is given in this boxplot. (D-G) The boxplots display the ratio between (D) BZLF1-, (E) gp350-, (F) LMP1-, or (G) EBNA2-positive cells versus EBER-positive cells. The data collected from the mice euthanized at week 5 are shown as open squares. (H) This graph shows the tumor incidence for humanized mice investigated in this study. We used a one-tailed Chi-square analysis in figure H and two-tailed unpaired student t test for all other results.</p

Replicating cells produce lower levels of EBV viral miRNAs than non-replicating counterparts.

<p>(A) CD2-positive cells were isolated from LCLs generated with a M81 mutant that expresses a truncated form of rat CD2 behind an EA-D-responsive promoter. CD2-positive or CD2-negative cell populations were submitted to RT-qPCR to assess BZLF1 mRNA expression (top graph) and to a western blot analysis with a BZLF1-specific antibody (Bottom picture). (B) The scatter plot shows expression of 6 viral microRNAs extracted from CD2-positive or CD2-negative cell populations obtained from 6 different LCLs generated with the CD2-expressing virus. (C) The experiment described in (B) was repeated with 3 cellular miRNAs expressed in EBV-transformed B cells. P values lower than 0.05, 0.01, 0.001, and 0.0001 obtained after paired t-student tests are indicated as *, **, ***, and **** in the figure.</p

The deletion of BART miRNAs enhances virus production in infected B cells.

<p>(A) This figure shows a western blot analysis performed a different time points on B cells from the same donor transformed with M81, M81/ΔAll or ΔZR with antibodies specific for gp350 and actin. The upper picture shows expression of gp350 and of its alternative spliced form gp220 in these LCLs. The relative intensity of the signals was quantified using the ImageJ software and is depicted in a graph of bars. (B) We generated LCLs by exposing B cells from 6 different donors to M81 or M81/ΔAll. These cells were immunostained with antibodies specific for gp350 as exemplified in the top pictures. The adjacent scatter plot shows the percentage of gp350-positive cells, including cells producing gp350 and B cells covered by virions, in these LCLs at different days post infection. The figure also shows the p values obtained from paired t tests performed with the two types of LCLs. (C) We quantified the EBV DNA load in supernatants from three couple of LCLs obtained by infection with M81 or M81/ΔAll by qPCR and show the results in this scatterplot. The p values of paired t tests performed with the different types of supernatants are indicated. (D) This graph gives the result of B-cell transformation assays that were performed by exposing primary B cells to supernatants from three different LCLs obtained with M81 or M81/ΔAll virions.</p

The miRNA subcluster1 is mainly but not exclusively responsible for the control of BZLF1 expression.

<p>The figure shows Western blot analyses of LCLs generated with M81, M81/ΔAll, M81/ΔC1, M81/ΔC2, M81/ΔC1C2, M81/Δb2, M81/ΔZR with a BZLF1-specific antibody. The LCLs were stained at 42 (A) and 101 (B) days post-infection. The relative intensity of the signals were quantified using the ImageJ software and are also displayed as a graph of bars. One more sample is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005344#ppat.1005344.s005" target="_blank">S5 Fig</a>. (C) and (D) We determined BALF5 expression level in multiple LCLs transformed with M81 and M81/ΔAll (C) and M81/Δb2 by western blot (D), and depict the results as a graph of bars after Image J quantification. All LCLs shown in (C) and (D) were investigated between 40–43 days post infection.</p