Biology subjects

Tibbetts, S.

Publications and source records attributed to Tibbetts, S..

4 recordsLinked to original sources

bioRxiv2026

KSHV miR-K12-9 Induces Transformation of Immortalized and Primary Endothelial Cells

Like most herpesviruses, KSHV encodes multiple microRNAs (miRNAs). Collectively, they comprise an important mechanism through which the virus maintains latency and persists in cells. At the same time, individual miRNAs can also play distinct, nonredundant roles. Past experiments with single miRNA knockout viruses showed that miR-K12-9, in particular, filled a unique niche. Endothelial cells latently infected with the miR-K12-9 knockout grew to be many times larger than WT-infected cells and proliferated at a significantly slower rate. Their ability to migrate was slowed as well. RNA-seq identified nearly 8,500 differentially expressed genes between miR-K12-9 knockout- and WT-infected cells. To further study miR-K12-9, we generated Telomerase-Immortalized Microvascular Endothelial (TIME) cells expressing either miR-K12-9 or a control miRNA from a lentivirus. Unexpectedly, after approximately one month in culture, unmistakable morphological changes began to occur in two of the three miR-K12-9-expressing cell lines. These smaller, more rounded cells proliferated rapidly and swiftly took over the two cultures. Given this result, we proceeded to characterize all the lentivirus-transduced cell lines in various assays focused on oncogenesis. When looking at colony formation in soft agar, only those two miR-K12-9-expressing cell lines produced colonies, indicating a loss of contact inhibition. NOD/SCID mice injected with the two cell miR-K12-9-expressing cell lines developed tumors while those receiving other cell lines did not. To confirm reproducibility of these results, we transduced both TIME and primary endothelial cells (HUVECs) with the miR-K12-9 and control lentiviruses. Once again, approximately half of the cell lines expressing miR-K12-9 showed hallmark phenotypes of transformation. We are currently characterizing the miR-K12-9 targetome in the transduced cell lines and mouse tumors using bulk and single-cell RNA-seq. This should yield insights into the underlying mechanism and required cofactors of miR-K12-9-induced transformation. To our knowledge, this is the first description of transformation of endothelial cells by a viral miRNA.

cancer biology↗

bioRxiv2026

scDEcrypter: Uncertainty-aware differential expression analysis for viral infection in scRNA-seq

Single-cell RNA-seq studies of viral infection are limited by sparse viral reads, under-labeled infected cells, and bystander responses that confound differential expression (DE) analysis. We introduce scDEcrypter, a penalized two-way mixture model that leverages partial labels for infection status and additional variables such as cell type. Our approach employs data-splitting to avoid double-dipping and enables fast, likelihood-based inference for DE analysis. Through simulations and applications on two different viral infection datasets, scDE-crypter demonstrated improved recovery of infected cell states and identified more biologically coherent infection-associated genes and enriched pathways.

genomics↗

bioRxiv2023

DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM NATIVE VIRAL tRNA PROMOTER

Gammaherpesviruses are associated with multiple types of tumour development and understanding the pathogenesis of these viruses has been the subject of many different studies. Throughout the lytic and latent life cycle, these viruses utilize numerous virally encoded miRNAs to regulate the key mechanisms of the infected cell in their favour. Therefore it is important to understand the miRNA and their mRNA target interactions for developing better therapeutics. In this study, the strategy and design of a short hairpin encoding RNA element targeting Blimp1 transcript in the context of viral infection is evaluated. This proof of principle experiment provides a mean to study important miRNA mRNA interactions in vivo. The very short promoter size of the Murine gammaherpesvirus 68 (MHV68) vtRNA4 and its ability to generate two shRNAs from a ~180 nucleotide sequence is useful if there is a size limit for the shRNA construct.

microbiology↗

bioRxiv2020

EBV miRNAs are potent effectors of tumor cell transcriptome remodeling in promoting immune escape.

Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitts Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and a BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposis sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in TCGA GC dataset. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing the host immune response. AUTHOR SUMMARYBurkitts Lymphoma and gastric cancer are both associated with EBV, a prolific DNA tumor virus that latently resides in nearly all human beings. Despite mostly restricting viral gene expression to noncoding RNAs, EBV has important influences on the fitness of infected tumor cells. Here, we show that the miRNA class of viral noncoding RNAs are a major viral contributor to remodeling the tumor cell regulatory machinery in patient tumor samples. First, an assessment of miRNA expression in clinical tumor samples showed that EBV miRNAs are expressed at unexpectedly high levels relative to cell miRNAs. Using a highly specific miRNA target identification approach, CLASH, we comprehensively identified both viral and cellular microRNA targets and the relative abundance of each microRNA-mRNA interaction. We also show that viral microRNAs bind to and alter the expression of their mRNA targets more effectively than their cellular microRNA counterparts. Pathway analysis of the most effectively targeted mRNAs revealed enrichment of immune signaling pathways and we show a corresponding inverse correlation between EBV miRNA expression and infiltrating immune cells in EBV positive primary tumors. Altogether, this study shows that EBV miRNAs are key regulators of the tumor cell phenotype and the immune cell microenvironment.

cancer biology↗