Transcriptome Analysis of KSHV during de novo primary infection of human CD14+ cells
ABSTRACT: The objective of this study was to identify the viral transcripts packaged into the virion particles and the transcription profiles of the viral genome during early infection, till the virus establishes latent infection in B-cells as well as in endothelial cells Samples from each time points were analyzed and the experiments were done in duplicate.
Project description:The objective of this study was to identify the viral transcripts packaged into the virion particles and the transcription profiles of the viral genome during early infection, till the virus establishes latent infection in human PBMCs Samples from each time points were analyzed and the experiments were done in duplicate.
Project description:The objective of this study was to identify the viral transcripts packaged into the virion particles and the transcription profiles of the viral genome during early infection, till the virus establishes latent infection in endothelial (TIVE) cells Samples from each time points were analyzed and the experiments were done in duplicate.
Project description:The objective of this study was to identify the transcription profiles of wild type KSHV and ORF59 deleted KSHV (Kaposi's sarcoma-associated herpesvirus) genome during early infection, till the virus establishes latent infection in human PBMCs Samples from each time points were analyzed and the experiments were done in duplicate.
Project description:The virion proteins of Kaposi Sarcoma Associated Herpesvirus (KSHV) were initially characterized in 2005 in two separate studies that combined detected 24 viral proteins and a few cellular components via LC-MS/MS or MALDI-TOF. Despite considerable advances in the sensitivity and specificity of mass spectrometry instrumentation in recent years, leading to significantly higher yields in detections, the KSHV virion proteome has not been revisited. In this study, we have re-examined the protein composition of purified KSHV virions via Ultra-High Resolution Qq-Time-Of-Flight mass spectrometry (UHR-QqTOF). Our results confirm the detection of all previously reported virion proteins, in addition to 17 other viral proteins, some of which have been characterized as virion-associated using other methods, and 10 novel proteins identified as virion-associated for the first time in this study. These results add KSHV ORF9, ORF23, ORF35, ORF48, ORF58, ORF72/vCyclin, K3, K9/vIRF1, K10/vIRF4, and K10.5/vIRF3 to the list of KSHV proteins that can be incorporated into virions. The addition of these proteins to the KSHV virion proteome provides novel and important insight into early events in KSHV infection mediated by virion-associated proteins.
Project description:We performed Ago HITS-CLIP to identify targets of viral and human miRNAs in latently KSHV-infected PEL cells Ago HITS-CLIP was performed in two latently infected PEL cell lines, BCBL-1 and BC-3; Argonaute-immunoprecipitation of UV cross-linked Ago-miRNA-mRNA complexes, followed by RNA isolation, library construction, and high-throughput sequencing (Illumina GAxII); we performed 3 biological replicates for each cell line, two technical (sequencing) replicates of BCBL-1 biological replicate 1
Project description:Kaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognizable by its distinctive red skin lesions. The lesions are KSHV infected spindle cells, most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. The effects of KSHV infection of LECs were assayed using Affymetrix hgu133plus2 chips at 6 and 72 hours post infection. There were n=4 each of lymphatic endothelial cells (LEC) following 6 hours of culture, LEC following 6 hours post KSHV infection, LEC following 72 hours of culture, and LEC following 72 hours post KSHV infection.
Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus which establishes latent infection in endothelial and B cells, as well as in primary effusion lymphoma (PEL). During latency, the viral genome exists as a circular DNA minichromosome (episome) and is packaged into chromatin analogous to human chromosomes. Only a small subset of promoters, those which drive latent RNAs, are active in latent episomes. In general, nucleosome depletion (“open chromatin”) is a hallmark of eukaryotic regulatory elements such as promoters and transcriptional enhancers or insulators. We applied formaldehyde-assisted isolation of regulatory elements (FAIRE) followed by next-generation sequencing to identify regulatory elements in the KSHV genome and integrated these data with previously identified locations of histone modifications, RNA polymerase II occupancy, and CTCF binding sites. We found that (i) regions of open chromatin were not restricted to the transcriptionally defined latent loci; (ii) open chromatin was adjacent to regions harboring activating histone modifications, even at transcriptionally inactive loci; and (iii) CTCF binding sites fell within regions of open chromatin with few exceptions, including the constitutive LANA promoter and the vIL6 promoter. FAIRE-identified nucleosome depletion was similar among B and endothelial cell lineages, suggesting a common viral genome architecture in all forms of latency. Ten total samples analyzed by FAIRE-seq from latent KSHV-infected cell lines. Two replicates were performed for BC1, KSHV-BJAB, KSHV-HUVEC, and L1-TIVE cells using the Illumina HiSeq 2000 platform. For BCBL1 cells, 1 FAIRE-seq sample and 1 non-cross-linked control BCBL1 sample was analyzed using the Illumina GAIIx
Project description:Human SLK cells were infected with wildtype (wt) and LANA knockout (KO) Kaposi's sarcoma-associated herpesvirus (KSHV), separately for 3 days. Cellular gene expression changes were identified upon the wild type and LANA KO KSHV virus infection compared to the uninfected SLK cells using the human gene expression microarray U133plus2.0. 2 independent biological replicates from uninfected SLK cells, wild type KSHV infected SLK cells at 72hrs post-infection (hpi) , and LANA KO infected SLK cells at 72 hrs post-infection were collected and RNA was prepared for microarray analysis.
Project description:Infectious bronchitis is a highly contagious respiratory disease of poultry caused by the coronavius infectious bronchitis virus (IBV). Until recently is was thought that coronavirus virions were composed of the structural proteins nucleocapsid, envelope, spike and membrane proteins, but investigations of TGEV and SARS-CoV have shown the proteome of coronavirus virions also includes viral non-structural and group specific proteins as well as host cell proteins. To study the proteome of IBV virions, virus was grown in embryonated chicken eggs and purified by sucrose gradient ultracentrifugation. Purified virus was analysed using sensitive gel-free proteomic techniques to determine the proteome of IBV. Analysis of three preparations of purified IBV yielded a list of 39 proteins commonly associated with the IBV virion. Three of these proteins were the viral structural proteins spike, membrane and nucleocapsid, but none of the viral non-strucutral or groups specific proteins could be identified. The other 35 proteins commonly associated to the IBV virion were all found to be host cell proteins. These proteins were classified into 12 categories using pantherdb (pantherdb.org). These proteins were involved in a diverse range of functions such as cytoskeletal proteins, nucleic acid binding proteins and chaperone proteins. Some of these proteins were unique to this study, whilst others were found to be orthologous to proteins identified in the SARS-CoV protein, and indeed some were also identified in association with virions from a number of other RNA and DNA viruses.