Luftig, M. A. | bioRxiv Science

bioRxiv2017

The Epstein-Barr Virus miR-BHRF1 microRNAs Regulate Viral Gene Expression in cis

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant ({triangleup}123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of primiR-BHRF1-2 and 1-3 in the {triangleup}123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the {triangleup}123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.\n\nHighlightsO_LIThe EBV miR-BHRF1 microRNAs do not up upregulate B cell growth in trans.\nC_LIO_LIEBNA-LP expression is downregulated by pri-miR-BHRF1-2 and 1-3 acting in cis.\nC_LIO_LILoss of miR-BHRF1-2 and 1-3 causes EBNA-LP overexpression and inhibits B cell growth.\nC_LIO_LINovel alternative splicing of EBV Cp/Wp transcripts was identified.\nC_LI

microbiology↗

bioRxiv2017

Intracellular BH3 profiling reveals shifts in anti-apoptotic dependency in B-cell maturation and activation

Apoptosis is critical to B-cell maturation, but studies of apoptotic regulation in primary human B cells is lacking. Previously, we found that infecting human B cells with Epstein-Barr virus induces two different survival strategies (Price et al., 2017). Here, we sought to better understand the mechanisms of apoptotic regulation in normal and activated B cells. Using intracellular BH3 profiling (iBH3), we defined the Bcl2-dependency of B-cell subsets from human peripheral blood and tonsillar lymphoid tissue as well as mitogen-activated B cells. We found that naive and memory B cells were BCL-2 dependent, while germinal center B cells were MCL-1 dependent and plasma cells were BCL-XL dependent. Proliferating B cells activated by CpG or CD40L/IL-4 became more dependent upon MCL-1 and BCL-XL. As B-cell lymphomas often rely on survival mechanisms derived from normal and activated B cells, these findings offer new insight into potential therapeutic strategies for lymphomas.

immunology↗