Coupled transcriptome and proteome analysis of human lymphotropic tumor viruses: insights on the detection and discovery of viral genes

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are related human tumor viruses that cause primary effusion lymphomas (PEL) and Burkitt's lymphomas (BL), respectively. Viral genes expressed in naturally-infected cancer cells contribute to disease pathogenesis; knowing which viral genes are expressed is critical in understanding how these viruses cause cancer. To evaluate the expression of viral genes, we used high-resolution separation and mass spectrometry coupled with custom tiling arrays to align the viral proteomes and transcriptomes of three PEL and two BL cell lines under latent and lytic culture conditions.</p> <p>Results</p> <p>The majority of viral genes were efficiently detected at the transcript and/or protein level on manipulating the viral life cycle. Overall the correlation of expressed viral proteins and transcripts was highly complementary in both validating and providing orthogonal data with latent/lytic viral gene expression. Our approach also identified novel viral genes in both KSHV and EBV, and extends viral genome annotation. Several previously uncharacterized genes were validated at both transcript and protein levels.</p> <p>Conclusions</p> <p>This systems biology approach coupling proteome and transcriptome measurements provides a comprehensive view of viral gene expression that could not have been attained using each methodology independently. Detection of viral proteins in combination with viral transcripts is a potentially powerful method for establishing virus-disease relationships.</p

The influence of student characteristics on the use of adaptive e-learning material

Adaptive e-learning materials can help teachers to educate heterogeneous student groups. This study provides empirical data about the way academic students differ in their learning when using adaptive e-learning materials. Ninety-four students participated in the study. We determined characteristics in a heterogeneous student group by collecting demographic data and measuring motivation and prior knowledge. We also measured the learning paths students followed and learning strategies they used when working with adaptive e-learning material in a molecular biology course. We then combined these data to study if and how student characteristics relate to the learning paths and strategies they used. We observed that students did follow different learning paths. Gender did not have an effect, but (mainly Dutch) BSc students differed from (international) MSc students in the intrinsic motivation they had and the learning paths and strategies they followed when using the adaptive e-learning material. (C) 2011 Elsevier Ltd. All rights reserved

The nested open reading frame in the Epstein-Barr virus nuclear antigen-1 mRNA encodes a protein capable of inhibiting antigen presentation in cis.

Herpesviruses employ many mechanisms to evade the immune response, allowing them to persist life-long in their hosts. The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) and, more recently, the latency-associated nuclear antigen 1 (LANA-1) of the Kaposi Sarcoma Herpesvirus have been shown to function as in cis-acting inhibitors of antigen presentation. In both proteins, long simple repeat elements are responsible for the inhibition, but the sequences of these repeats are strongly dissimilar. Intriguingly, EBNA-1 mRNA contains a large nested open reading frame that codes for a 40.7kDa strongly acidic protein, in addition to the full-length EBNA-1. This protein, here called pGZr, has a 230 amino-acids long glycine, glutamine, and glutamic acid-rich repeat ('GZ' repeat), highly similar (65% amino-acid identity) to the acidic repeat of LANA-1. To evaluate if pGZr, like EBNA-1 and LANA-1, can inhibit antigen presentation in cis, we fused the nested ORF with the E. coli-derived LacZ gene encoding beta-galactosidase. Whereas cells producing the unmodified beta-galactosidase readily present the H-2L(d)-restricted CTL epitope TPHPARIGL, which resides in the C-terminal region of beta-galactosidase, cells producing the pGZr-beta-galactosidase fusion protein do not. Also shorter fragments of the repeat can inhibit peptide presentation. Even though the physiological function of pGZr remains to be elucidated, the GZ-repeat protein may be valuable as inhibitor of presentation of antigenic peptides derived from transgenes in gene therapy

Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1

peer-reviewedViruses establishing persistent latent infections have evolved various mechanisms to avoid immune surveillance. The Epstein-Barr virus-encoded nuclear antigen, EBNA1, expressed in all EBV-associated malignancies, modulates its own protein levels at quantities sufficient to maintain viral infection but low enough so as to minimize an immune response by the infected host cell. This evasion mechanism is regulated through an internal purine-rich mRNA repeat sequence encoding glycine and alanine residues. In this study we assess the impact of the repeat's nucleotide versus peptide sequence on inhibiting EBNA1 self-synthesis and antigen presentation. We demonstrate that altered peptide sequences resulting from frameshift mutations within the repeat do not alleviate the immune-evasive function of EBNA1, suggesting that the repetitive purine-rich mRNA sequence itself is responsible for inhibiting EBNA1 synthesis and subsequent poor immunogenicity. Our comparative analysis of the mRNA sequences of the corresponding repeat regions of different gammaherpesvirus maintenance homologues to EBNA1 highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. These studies demonstrate the importance of gammaherpesvirus purine-rich mRNA repeat sequences on antigenic epitope generation and evasion from T-cell mediated immune control, suggesting novel approaches to prevention and treatment of latent infection by this class of virus.National Health & Medical Research Council (NH&MRC) Canberra, Australia (#496684 APP1005091); NH&MRC Career Development Award Research Fellowship (#496712

Left ventricular shear strain in model and experiment

Mathematical modeling of cardiac mechanics could be a useful clinical tool, both in translating measured abnormalities in cardiac deformation into the underlying pathology, and in selecting a propertreatment. We investigated to what extent a previously published model of cardiac mechanics could predict deformation in the healthy left ventricle, as measured using MR tagging. The model adequately predicts circumferential strain, but fails to accurately predict shear strain. However, the time course of shear strain proves to be that sensitive tomyofiber orientation, that agreement between model predictions and experiment may be expected if fiber orientation is changed by only a few degrees

Molecular Determinants of Peptide Binding to Two Common Rhesus Macaque Major Histocompatibility Complex Class II Molecules

Major histocompatibility complex class II molecules encoded by two common rhesus macaque alleles Mamu-DRB1*0406 and Mamu-DRB*w201 have been purified, and quantitative binding assays have been established. The structural requirements for peptide binding to each molecule were characterized by testing panels of single-substitution analogs of the two previously defined epitopes HIV Env242 (Mamu-DRB1*0406 restricted) and HIV Env482 (Mamu-DRB*w201 restricted). Anchor positions of both macaque DR molecules were spaced following a position 1 (P1), P4, P6, P7, and P9 pattern. The specific binding motif associated with each molecule was distinct, but largely overlapping, and was based on crucial roles of aromatic and/or hydrophobic residues at P1, P6, and P9. Based on these results, a tentative Mamu class II DR supermotif was defined. This pattern is remarkably similar to a previously defined human HLA-DR supermotif. Similarities in binding motifs between human HLA and macaque Mamu-DR molecules were further illustrated by testing a panel of more than 60 different single-substitution analogs of the HLA-DR-restricted HA 307–319 epitope for binding to Mamu-DRB*w201 and HLA-DRB1*0101. The Mamu-DRB1*0406 and -DRB*w201 binding capacity of a set of 311 overlapping peptides spanning the entire simian immunodeficiency virus (SIV) genome was also evaluated. Ten peptides capable of binding both molecules were identified, together with 19 DRB1*0406 and 43 DRB*w201 selective binders. The Mamu-DR supermotif was found to be present in about 75% of the good binders and in 50% of peptides binding with intermediate affinity but only in approximately 25% of the peptides which did not bind either Mamu class II molecule. Finally, using flow cytometric detection of antigen-induced intracellular gamma interferon, we identify a new CD4(+) T-lymphocyte epitope encoded within the Rev protein of SIV