Integration of an anellovirus genome in the SKNO-1 acute myeloid leukemia cell line

Anelloviruses are highly diverse, ubiquitous single-stranded DNA viruses whose replication, cellular reservoirs, and mechanisms of persistence remain poorly understood. Here, we identify and characterize a Alphatorquevirus homin 29 genome stably integrated into an rDNA locus of human chromosome 21 in the acute myeloid leukemia cell line SKNO-1. Large-scale mining of NCBI Sequence Read Archive datasets revealed unusually high anellovirus k-mer abundance specifically in sequencing runs of the SKNO-1 cell line from multiple laboratories. De novo assembly of RNA-seq data recovered a 3.25 kb viral genome, and long-read PacBio sequencing confirmed its integration within the RNA45SN2 gene on human chromosome 21. Digital droplet PCR (ddPCR) quantified [~]0.5 viral genomes per cell, and RT-ddPCR detected transcripts from ORF1 and viral terminal repeats, consistent with transcription of the integrant. The ORF1 coding sequence encoded for a truncated but structurally conserved capsid protein retaining jelly roll and P-domain features but lacking the C-terminal domain. Analysis of public ChIP-seq data from the SKNO-1 cell line demonstrated broad occupancy of the BRD4 transcriptional coactivator across the viral genome along with focal enrichment of hematopoietic ETS family transcription factors within the integrants [~]300 bp untranslated region upstream of viral open reading frames. Together, these findings demonstrate stable integration, chromatin accommodation, and transcriptional maintenance of an Alphatorquevirus in a human leukemia cell line, providing a unique model to study host tolerance and transcriptional regulation of anellovirus DNA and informing our understanding of anellovirus hematopoietic cell tropism. IMPORTANCEAnelloviruses are a curious group of highly genetically diverse single-stranded DNA viruses that have been found ubiquitously among humans and are hypothesized to be a potential commensal human virus. Their omnipresence has been matched only by the dearth of our understanding of their basic biological processes - how they persist, how they replicate, and how they spread. Here we identify a naturally integrated Alphatorquevirus genome in the widely used AML cell line SKNO-1 and show that it is stably maintained, transcribed, and embedded within a rDNA locus on human chromosome 21. This discovery highlights the ability to anelloviruses to integrate into human DNA, generates hypotheses around transcription factors used in anellovirus gene transcription, and suggests anelloviruses can persist in myeloblasts.