Specialized DNA structures act as genomic beacons for integration by evolutionarily diverse retroviruses

Retroviral integration site targeting is not random and plays a critical role in expression and long-term survival of the integrated provirus. To better understand the genomic environment surrounding retroviral integration sites, we performed an extensive comparative analysis of new and previously published integration site data from evolutionarily diverse retroviruses from seven genera, including different HIV-1 subtypes. We showed that evolutionarily divergent retroviruses exhibited distinct integration site profiles with strong preferences for non-canonical B-form DNA (non-B DNA). Whereas all lentiviruses and most retroviruses integrate within or near genes and non-B DNA, MMTV and ERV integration sites were highly enriched in heterochromatin and transcription-silencing non-B DNA features (e.g. G4, triplex and Z-DNA). Compared to -derived HIV-1 integration sites, -derived sites are significantly more enriched in transcriptionally silent regions of the genome and transcription-silencing non-B DNA features. Integration sites from individuals infected with HIV-1 subtype A, C or D viruses exhibited different preferences for non-B DNA and were more enriched in transcriptionally active regions of the genome compared to subtype B virus. In addition, we identified several integration site hotspots shared between different HIV-1 subtypes with specific non-B DNA sequence motifs present at these hotspots. Together, these data highlight important similarities and differences in retroviral integration site targeting and provides new insight into how retroviruses integrate into genomes for long-term survival.