Uncovering viral gene functions requires the modulation of gene expression through overexpression or loss-of-function. CRISPR interference (CRISPRi), a modification of the CRISPR-Cas9 gene editing technology, allows specific and efficient transcriptional silencing without genetic ablation. CRISPRi has been used to silence eukaryotic and prokaryotic genes at the single-gene and genome-wide levels. Here, we report the use of CRISPRi to silence latent and lytic viral genes, with an efficiency of ~80–90%, in epithelial and B-cells carrying multiple copies of the Kaposi’s sarcoma-associated herpesvirus (KSHV) genome. Our results validate CRISPRi for the analysis of KSHV viral elements, providing a functional genomics tool for studying virus–host interactions.
The macula and fovea comprise a highly sensitive visual detection tissue that is susceptible to common disease processes like age-related macular degeneration (AMD). Our understanding of the molecular determinants of high acuity vision remains unclear, as few model organisms possess a human-like fovea. We explore transcription factor networks and receptor-ligand interactions to elucidate tissue interactions in the macula and peripheral retina and concomitant changes in the underlying retinal pigment epithelium (RPE)/choroid. Poly-A selected, 100 bp paired-end RNA-sequencing (RNA-seq) was performed across the macular/foveal, perimacular, and temporal peripheral regions of the neural retina and RPE/choroid tissues of four adult Rhesus macaque eyes to characterize region- and tissue-specific gene expression. RNA-seq reads were mapped to both the macaque and human genomes for maximum alignment and analyzed for differential expression and Gene Ontology (GO) enrichment. Comparison of the neural retina and RPE/choroid tissues indicated distinct, contiguously changing gene expression profiles from fovea through perimacula to periphery. Top GO enrichment of differentially expressed genes in the RPE/choroid included cell junction organization and epithelial cell development. Expression of transcriptional regulators and various disease-associated genes show distinct location-specific preference and retina-RPE/choroid tissue-tissue interactions. Regional gene expression changes in the macaque retina and RPE/choroid is greater than that found in previously published transcriptome analysis of the human retina and RPE/choroid. Further, conservation of human macula-specific transcription factor profiles and gene expression in macaque tissues suggest a conservation of programs required for retina and RPE/choroid function and disease susceptibility.
Kaposi sarcoma (KS), caused by Kaposi sarcoma herpesvirus (KSHV), is a multicentric tumor characterized by abnormal vasculature and proliferation of KSHV-infected spindle cells. KS commonly involves the skin but in severe cases KS can also involve the gastrointestinal tract (GI). Here, we sought to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions. Skin and GI KS were compared to normal matched samples using bulk RNA sequencing.Twenty-two paired samples of KS and normal tissue were obtained (skin (10 pairs) and GI (12 pairs)) from 19 patients with KS of whom 17 had concurrent HIV infection. Seven paired samples were from patients who had received prior KS therapy. Three patients provided both skin and GI samples at the same timepoint. These analyses identified 370 differentially expressed genes unique to cutaneous KS and 58 DEGs unique to GI KS compared to normal skin or GI tissues. Twenty-six differentially expressed genes overlapped between skin and GI KS, which included FLT4, which encodes for a VEGF-C and VEGF-D receptor, and STC1. KSHV infection of primary lymphatic endothelial cells (LECs) resulted in increased angiogenesis, and repression of STC1 or FLT4 inhibited angiogenesis. The analyses of KSHV expression from KS lesions identified certain lytic genes, specifically ORF75, that were consistently expressed, and these expression patterns differed from laboratory infection of LECs with KSHV and KSHV gene expression in PEL cell lines. This study demonstrates that complex patterns of gene expression are found in KS tissue that differ from the canonical latent/lytic programs seen in KSHV cell lines and also demonstrates differences in viral gene and clinically relevant host gene expression in skin and GI KS that may offer insights into the pathogenesis of these forms of KS.
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