Epstein-Barr Virus Nuclear Antigen 3C Facilitates G1-S Transition by Stabilizing and Enhancing the Function of Cyclin D1

EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines

Probing the Antiprotease Activity of λCIII, an Inhibitor of the Escherichia coli Metalloprotease HflB (FtsH)▿

The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor λCII, thereby helping the phage to lysogenize the host bacterium. λCIII, a small (54-residue) protein of unknown structure, also protects σ32, another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CIIIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CIIIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII. On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII

Lytic burst of EBV virus during early infection of GFP-EBV.

<p>PBMC cells were infected with GFP-EBV and at specific times postinfection the supernanat was collected and used infect fresh PBMC cells. (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV infected PBMC cells are shown after specific times postinfection (24 hrs, 72 hrs, 120 hrs and 168 hours) (left panel). Phase-contrast (left) and fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection (from 24hrs, 72hrs, 120hrs and 168h) (right panel). B. Flow cytometry analysis of GFP expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) (left panel), and after infection of fresh PBMC from supernatant of infected cells (right panel).</p

Transfection of Full length BAC GFP-EBV into 293T cells.

<p>(A) BAC GFP-EBV DNA was transfected to 293T and stable cell line generated as screened by puromycin. Different genes encoding by EBV (BamHI T, BamHI K, BamHI E, BamHI H and BamHI C) as well as puromycin and GFP were analyzed by PCR amplification taking 5 different transfected clones (4, 10, 11, 12 and 15). BJAB and LCL were taken as negative and positive control, respectively. (B) Phase contrast (left panel) and Flourescence images (right panel) showed two indifferent (number 4 and 12) containing BAC GFP-EBV transfected into 293T stable cell line screened by puromycin.</p

Latent gene expression.

<p>Latent proteins expressions were analyzed in cells infected with in BAC GFP-EBV stably transfected 293T and LCLs made from BAC GFP-EBV infection. Expression of EBV EBNA-1, EBNA-3C and LMP-1 were determined by Western blotting.</p

Glycoprotein expression during early stage of infection in presence of acyclovir.

<p>Endogenous expression of (A) gp110 and (B) gp350 were detected using mouse monoclonal antibody (1∶200 dilution), and rabbit respectively (1∶250 dilution). Primary antibodies were diluted in blocking buffer and incubated with fixed cells for 1 h at RT. Slides were washed three times (5 min each) with PBS and incubated with appropriate secondary antibody (1∶2000) for 1 h at RT followed by three times washes (5 min each) with PBS. The last wash contained 4′, 6′-diamidino-2-phenylindole (DAPI; Promega Inc., Madison, WI) for nuclear staining. Goat anti-mouse antibody Alexa Fluor 594 and goat anti-rabbit antibody Alexa Fluor 594 were purchased from Molecular Probes Inc. (Carlsbad, CA). Slides were then washed in PBS and mounted using Prolong anti-fade (Molecular Probes Inc, Carlsbad, CA). Fluorescence was viewed by confocal microscopy and analyzed with Fluoview 300 software from Olympus Inc. (Melville, NY). The images were sequentially captured using an Olympus confocal microscope. All panels are representative pictures from similar repeat experiments.</p