Unveiling the cryptic diversity and distribution of elements related to virophage mavirus through deep mining of pPolB proteins

Virophages are unique double-stranded DNA (dsDNA) viruses that parasitize viruses of Nucleocytoviricota (NCVs). While conventionally viewed as a viral group, growing evidence suggests that "virophage" is better understood as a parasitic lifestyle rather than a natural group. Despite this conceptual shift, their diversity and evolution remain largely obscure and contentious. Through deep mining of pPolBs in 7,041 eukaryotic genomes and 12,053 metagenomes sampled globally, we expand the diversity of protein-primed type B DNA polymerase (pPolB)-carrying mavirus virophage-related elements (pMVREs), which include virophages, transpovirons, and Polinton-like viruses (PLVs). Our phylogenomic and metagenomic mining reveals the widespread distribution of pMVREs in eukaryotic genomes (97/7,041, 1.38%) and global environments (2,450/12,053, 20.33%). pMVREs possess genome architectures of high plasticity and promiscuity. The presence of pMVREs and NCVs is statistically correlated in both eukaryotic genomes and global metagenomes, supporting a specific co-occurrence association between pMVREs and NCVs. Moreover, pMVRE diversity and composition exhibit strong heterogeneity across global ecosystems. Together, this study unveils a vast diversity of virophage-related elements and provides insights into the intricate relationship among virophages, transpovirons, PLVs, pMVREs, and NCVs.