The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies. microparticles | microvesicles | oncosomes

Cryptochromes are blue-light photoreceptors that regulate a variety of responses such as growth and circadian rhythms in organisms ranging from bacteria to humans. Cryptochromes share a high level of sequence identity with the light-activated DNA repair enzyme photolyase. Photolyase uses energy from blue light to repair UV-induced photoproducts in DNA through cyclic electron transfer between the catalytic flavin adenine dinucleotide cofactor and the damaged DNA. Cryptochromes lack DNA repair activity, and their mechanism of signal transduction is not known. It is hypothesized that a light-dependent signaling state in cryptochromes is created as a result of an intramolecular redox reaction, resulting in conformational rearrangement and effector binding. Plant and animal cryptochromes possess 30-250 amino acid carboxy-terminal extensions beyond the photolyase-homology region that have been shown to mediate phototransduction. We analyzed the structures of C-terminal domains from an animal and a plant cryptochrome by computational, biophysical, and biochemical methods and found these domains to be intrinsically unstructured. We show that the photolyase-homology region interacts with the C-terminal domain, inducing stable tertiary structure in the C-terminal domain. Importantly, we demonstrate a light-dependent conformational change in the C-terminal domain of Arabidopsis Cry1. Collectively, these findings provide the first biochemical evidence for the proposed conformational rearrangement of cryptochromes upon light exposure.

MicroRNAs (miRNAs) are stable, small non-coding RNAs that modulate many downstream target genes. Recently, circulating miRNAs have been detected in various body fluids and within exosomes, prompting their evaluation as candidate biomarkers of diseases, especially cancer. Kaposi's sarcoma (KS) is the most common AIDS-associated cancer and remains prevalent despite Highly Active Anti-Retroviral Therapy (HAART). KS is caused by KS-associated herpesvirus (KSHV), a gamma herpesvirus also associated with Primary Effusion Lymphoma (PEL). We sought to determine the host and viral circulating miRNAs in plasma, pleural fluid or serum from patients with the KSHV-associated malignancies KS and PEL and from two mouse models of KS. Both KSHV-encoded miRNAs and host miRNAs, including members of the miR-17–92 cluster, were detectable within patient exosomes and circulating miRNA profiles from KSHV mouse models. Further characterization revealed a subset of miRNAs that seemed to be preferentially incorporated into exosomes. Gene ontology analysis of signature exosomal miRNA targets revealed several signaling pathways that are known to be important in KSHV pathogenesis. Functional analysis of endothelial cells exposed to patient-derived exosomes demonstrated enhanced cell migration and IL-6 secretion. This suggests that exosomes derived from KSHV-associated malignancies are functional and contain a distinct subset of miRNAs. These could represent candidate biomarkers of disease and may contribute to the paracrine phenotypes that are a characteristic of KS.

The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Ö zgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and fourway junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2 ⌬B ) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junctionspecific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop.Telomeres are nucleoprotein structures that protect the ends of chromosomes and are essential for regulating the replicative lifespan of somatic cells. The DNA component of the mammalian telomere consists of long double-stranded (ds) 2 tracts of the hexameric repeat unit TTAGGG (2) that ends with a G-rich 3Ј single-stranded (ss) overhang (3). Telomeric DNA is thought to be organized into a t-loop "end-capping" structure by the telomere-binding proteins TRF1, TRF2, and POT1 and the proteins that bind to them, TIN2, TPP1, and Rap1 (4, 5). This higher order structure may enable cells to distinguish chromosome ends from random double-strand breaks. Large blocks of telomere repeat sequences can be lost when these end-capping proteins are disrupted, or problems are encountered during DNA replication or repair (for review, see Ref. 6). This typically results in p53-and Rb-mediated senescence or cellular crisis, as evidenced by end-to-end fusions of chromosomes, ATM-dependent activation of p53, and apoptosis (for review, see Ref. 7).Much has been learned about the properties of TRF1 and TRF2 including their binding to DNA and the effects of their ablation or overexpression in the cell. We observed that TRF1 forms filamentous structures on long tracts of telomeric DNA in vitro (8), whereas TRF2 binds preferentially to the telomeric DNA at the junction between the duplex repeats and the ss overhang (9). Both TRF1 and TRF2 contain a similar Myb domain at their COOH terminus that mediates their binding to ds telomeric DNA (10). TRF1 and TRF2 differ in their NH 2 termini, however, which are rich in either acidic residues in TRF1 or basic residues in TRF2. The function of the basic domain of TRF2 is poorly understood. Deletion of this domain (TRF2 ⌬B ) does not impede the DNA binding activity of TRF2 or its localization to telomeres in vivo, but expression of TRF2 ⌬B resulted in stochastic deletions of telomeric DNA, generation of t-loop-sized telomeric circles, cell cycle arrest, and induction of senescence in human cells (11,12). In addition, recent evidence suggested that the...

Among several different types of repetitive sequences found in the human genome, this study has examined the telomeric repeat, necessary for the protection of chromosome termini, and the disease-associated triplet repeat (CTG)·(CAG)n. Evidence suggests that replication of both types of repeats is problematic and that a contributing factor is the repetitive nature of the DNA itself. Here we have used electron microscopy to investigate DNA structures formed at replication forks on large model DNAs containing these repeat sequences, in an attempt to elucidate the contributory effect that these repetitive DNAs may have on their replication. Visualization of the DNA revealed that there is a high propensity for a paused replication fork to spontaneously regress when moving through repetitive DNAs, and that this results in a four-way chickenfoot intermediate that could present a significant block to replication in vivo, possibly leading to unwanted recombination events, amplifications or deletions.