Systematic analysis of the intracellular localization of human herpesvirus 8-encoded proteins

Human herpesvirus-8 (HHV-8) is the etiologic agent of Kaposi’s sarcoma (KS). The HHV-8 genome encodes for about 86 proteins. As yet, many of these proteins have never been studied. Therefore, we investigated the subcellular localization of all HHV-8-encoded proteins to gain a more comprehensive understanding of the functions of each protein. To this goal we generated a mammalian expression library consisting of all HHV-8-encoded genes. All viral open reading frames (ORFs) were cloned with a Myc tag in expression plasmids. The identity of the cloned genes was confirmed by full-length sequencing and their expression was controlled in HEK293T and HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Our study showed that 51% of all HHV-8-encoded proteins were localized in the cytoplasm, 22% were found in the nucleus and 27% were detected in both compartments, the cytoplasm and the nucleus. Surprisingly, we detected the HHV-8-encoded viral Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (vFLIP/K13) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This indicated that vFLIP/K13 may exert additional or alternative functions as compared to cellular FLIPs. In addition, it has been shown recently in a yeast-two-hybrid analysis that HHV-8 K10 interacts with 15 different HHV-8-encoded proteins. From our localization data we noticed that K10 but only five of its putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly in co-expression experiments K10 co-localized with 87% (13 of 15) of its putative binding partners. Co-localization was induced by translocation of K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework the intracellular localization map may provide a valuable reference to further elucidate the function of HHV-8-encoded genes in human diseases.Das humane Herpesvirus 8 (HHV-8) ist verantwortlich für die Entstehung des Kaposi- Sarkoms (KS). Die molekularen Mechanismen wie HHV-8 die Entstehung des KS induziert sind bislang unklar. Das HHV-8 Genom besteht aus 86 Genen. Während bisher nur die Funktionen einiger dieser Gene ausführlich untersucht wurden, sind die Funktionen vieler Gene noch völlig unklar. Um eine Grundlage für vertiefende Funktionsanalysen zu schaffen wurde in dieser Arbeit die Lokalisation aller HHV-8-kodierten Proteine in der Zelle untersucht. Zu diesem Zweck wurde eine Expressionsbibliothek aller HHV-8-kodierten Gene erstellt. Alle viralen Leserahmen wurden dazu C-terminal mit einem Myc Tag versehen und in Expressionsplasmide kloniert. Die Richtigkeit aller Plasmide wurde mit Hilfe von Sequenzierungen überprüft und ihre Expression in HEK293T Zellen mittels Western blot Analysen kontrolliert. Die Lokalisation der einzelnen Proteine wurde mit Hilfe von Immunofluoreszenzmikroskopie bestimmt. Es zeigte sich, dass 51% aller HHV-8-kodierten Proteine im Zytoplasma lokalisieren, 22% wurden im Kern detektiert und 27% aller Proteine lokalisierten sowohl im Kern als auch im Zytoplasma. Überraschender Weise wurde das HHV-8-kodierte virale „Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein“ (vFLIP/K13) im Kern detektiert, wohingegen zelluläre FLIPs ausschließlich im Zytoplasma zu finden sind. Dies deutete darauf hin, dass das HHV-8-kodierte vFLIP/K13 zusätzliche oder unterschiedliche Funktionen als die zellulären FLIPs ausüben könnte. Des Weiteren wurde von anderen in einer „Yeast-two-Hybrid“ Analyse gezeigt, dass HHV-8 K10 mit 15 verschiedenen HHV-8-kodierten Proteinen interagiert. Unsere Lokalizationsdaten zeigten, dass K10, wenn es alleine exprimiert wird im Kern lokalisiert, aber überraschender Weise nur fünf seiner Interaktionspartner bei isolierter Expression allein ebenfalls im Kern zu finden sind. Im Falle einer Koexpression zeigte sich jedoch, dass K10 mit 87% seiner möglichen Interaktionspartner Kolokalisiert. Hierbei wurde die Kolokalisation induziert entweder durch eine Translokation von K10 alleine oder durch eine Translokation beider Proteine. Diese Ergebnisse deuten auf aktive intrazelluläre Translokationsprozesse in Virus-infizierten Zellen hin. In diesem Zusammenhang bietet die Lokalisationskarte eine nützliche Basis, um zukünftig die Funktion HHV-8-kodierter Gene im Zusammenhang mit humanen Erkrankungen näher zu untersuchen

Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays ▿

Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses

Viral Inhibitor of Apoptosis vFLIP/K13 Protects Endothelial Cells against Superoxide-Induced Cell Death▿

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes an antiapoptotic viral Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (vFLIP/K13). The antiapoptotic activity of vFLIP/K13 has been attributed to an inhibition of caspase 8 activation and more recently to its capability to induce the expression of antiapoptotic proteins via activation of NF-κB. Our study provides the first proteome-wide analysis of the effect of vFLIP/K13 on cellular-protein expression. Using comparative proteome analysis, we identified manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant and an important antiapoptotic enzyme, as the protein most strongly upregulated by vFLIP/K13 in endothelial cells. MnSOD expression was also upregulated in endothelial cells upon infection with HHV-8. Microarray analysis confirmed that MnSOD is also upregulated at the RNA level, though the differential expression at the RNA level was much lower (5.6-fold) than at the protein level (25.1-fold). The induction of MnSOD expression was dependent on vFLIP/K13-mediated activation of NF-κB, occurred in a cell-intrinsic manner, and was correlated with decreased intracellular superoxide accumulation and increased resistance of endothelial cells to superoxide-induced death. The upregulation of MnSOD expression by vFLIP/K13 may support the survival of HHV-8-infected cells in the inflammatory microenvironment in KS

A Systems Biology Approach To Identify the Combination Effects of Human Herpesvirus 8 Genes on NF-κB Activation▿

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma and primary effusion lymphoma. Activation of the cellular transcription factor nuclear factor-kappa B (NF-κB) is essential for latent persistence of HHV-8, survival of HHV-8-infected cells, and disease progression. We used reverse-transfected cell microarrays (RTCM) as an unbiased systems biology approach to systematically analyze the effects of HHV-8 genes on the NF-κB signaling pathway. All HHV-8 genes individually (n = 86) and, additionally, all K and latent genes in pairwise combinations (n = 231) were investigated. Statistical analyses of more than 14,000 transfections identified ORF75 as a novel and confirmed K13 as a known HHV-8 activator of NF-κB. K13 and ORF75 showed cooperative NF-κB activation. Small interfering RNA-mediated knockdown of ORF75 expression demonstrated that this gene contributes significantly to NF-κB activation in HHV-8-infected cells. Furthermore, our approach confirmed K10.5 as an NF-κB inhibitor and newly identified K1 as an inhibitor of both K13- and ORF75-mediated NF-κB activation. All results obtained with RTCM were confirmed with classical transfection experiments. Our work describes the first successful application of RTCM for the systematic analysis of pathofunctions of genes of an infectious agent. With this approach, ORF75 and K1 were identified as novel HHV-8 regulatory molecules on the NF-κB signal transduction pathway. The genes identified may be involved in fine-tuning of the balance between latency and lytic replication, since this depends critically on the state of NF-κB activity

Genome Wide Methylome Alterations in Lung Cancer.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents

The genome compartment represented on the HELP Nimblegen microarray and statistically significant DM loci.

<p>(A) Approximately 91% of the 1.2 million loci represented on the HELP microarray are located in gene body (GB) and intergenic (IG) regions, with a small minority (9%) of the loci located within promoters (PR). (B) Statistical significance (Y-axis) vs. delta (X-axis) (magnitude) of DM. Delta (X-axis) indicates the difference in methylation between tumor (T) vs non-tumor (NT) at a given locus. Loci hypermethylated in T relative to NT have delta < 0. P-value (Y-axis) is calculated based on Benjamini Hochberg adjusted FDR. At FDR p < 0.05, 433,505 loci across all genomic compartments are found to be differentially methylated in T vs NT. Red dots indicate statistically significant DM loci.</p

Methylation vs Expression in Promoter regions.

<p>Analysis of DM loci within promoter regions and their overlap with differential gene expression. (Left panel A) All 21 pairs (all NSCLC histologies), overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 3.32E-4. <i>(Right panel B)</i> Within the set of adenocarcinomas overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 1.10E-7. Majority of DM promoter loci are associated with hypermethylation when the DM loci are within CG islands. This effect is more pronounced among adenocarcinomas, where the DM loci in CG islands are mostly associated with downregulation of the gene. KEY: “M” = methylation, “E” = expression. Upward arrow indicates increase and downward arrow indicates decrease.</p

Heat Map of the Top 50 DM Loci within Adenocarcinomas.

<p>(A) Promoter regions; and (B) Gene body regions. Several genes show differential methylation (DM) at more than one locus and appear multiple times in the heatmap. Blue = Non Tumor, Red = Tumor.</p