Multilayered network analysis reveals the centrality of newly discovered Nucleocytoviricota in wastewater treatment plant communities.

Viruses of the phylum Nucleocytoviricota are remarkable entities due to their exceptionally large genomes and complex gene repertoires, which blur the lines between viral and cellular life. Previous research has leveraged computational approaches to map their extensive diversity. Experimental work has, in parallel, begun to elucidate their intricate interactions with hosts, bacterial symbionts, co-infecting virophages, and other mobile genetic elements.Although unlikely to replicate all simultaneously within the same host cell, the convergence of these entities within a single environment creates a dense hub of potential interactions that calls for a holistic, topology-based analysis. Here, we recovered high-quality genomes of 61 Nucleocytoviruses, 15 virophages and 14 polinton-like viruses from deeply sequenced metagenomes of 23 Danish wastewater treatment plants, and integrated them into a multilayered interaction network together with microbial contigs. This network recovered putative interactions across multiple levels, including Nucleocytoviruses co-occurring with hyperparasitic elements, associations with abundant activated sludge bacteria, and read-level signals of integration events. We show that the giants among viruses act as centrality-inducing entities, elevating others into highly central positions within the community and therefore are key players shaping the microbial community structure, ecology and evolution in wastewater treatment plants.