Abstract. Bacteriophages are powerful ecosystem engineers. They drive bacterial mortality rates and genetic diversity, and affect microbially mediated biogeochemical processes on a global scale. This has been demonstrated in marine environments; however, phage communities have been less studied in freshwaters, despite representing a potentially more diverse environment. Lake Michigan is one of the largest bodies of freshwater on the planet, yet to date the diversity of its phages has yet to be examined. Here, we present a composite survey of viral ecology in the nearshore waters of Lake Michigan. Sequence analysis was performed using a web server previously used to analyse similar data. Our results revealed a diverse community of DNA phages, largely comprising the order Caudovirales. Within the scope of the current study, the Lake Michigan virome demonstrates a distinct community. Although several phages appeared to hold dominance, further examination highlighted the importance of interrogating metagenomic data at the genome level. We present our study as baseline information for further examination of the ecology of the lake. In the current study we discuss our results and highlight issues of data analysis which may be important for freshwater studies particularly, in light of the complexities associated with examining phage ecology generally.
Kaposi′s sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 and modulation of NF-κB signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus MC159L, which are all strong caspase 8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or CXCR4, contribute to TRAIL-R1 expression. In sum, our work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.
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