Uncovering novel virophages and giant viruses in high-altitude Lake Namtso: diversity and evolution of host-virus-virophage tripartite interaction systems.

Virophages are small double-stranded DNA viruses that parasitize giant viruses, modulating virus–host interactions and influencing microbial community dynamics. Despite their ecological significance, virophages and giant viruses remain poorly studied in extreme environments. Here, we present the first metagenomic survey of virophage and giant virus diversity in Lake Namtso, a high-altitude saline lake on the Tibetan Plateau. Metagenomic assembly and phylogenomic analyses uncovered 93 virophage major capsid protein sequences spanning seven established families, alongside numerous unclassified lineages. Two nearly complete virophage genomes were reconstructed: Namtso Virophage 1 (NMV1), which encodes both replication- and integration-associated genes and likely represents a novel family, and Namtso Virophage 2 (NMV2), affiliated with Omnilimnoviroviridae but distinguished by duplicated protease genes and dual DNA methyltransferases. Parallel analyses identified over 18,000 giant virus marker genes, with DNA PolB affiliated to Imitervirales, Pimascovirales, Asfuvirales, Algavirales, and Chitovirales, as well as divergent lineages representing potential novel Nucleocytoplasmic large DNA viruses (NCLDVs). Homologous protein analysis and tetranucleotide clustering suggest extensive host–virus–virophage interactions. These findings significantly expand the known diversity and genomic repertoire of virophages and giant viruses, highlight their ecological roles in sustaining microbial resilience, and provide new insights into viral evolution and adaptation in extreme high-altitude ecosystems.