HIV-1 virions selectively package circESYT2 to sculpt an actin scaffold that constraints egress

Retroviruses such as HIV-1 package both viral and host RNAs, yet whether circular RNAs (circRNAs) enter virions has remained unknown. Here, we capture the HIV-1 RNA packageome and show the evidence that circRNAs represent a previously unrecognized class of selectively encapsidated host RNAs. Using nanopore sequencing of purified virions, we identify fourteen host-encoded circRNAs that are packaged and focus on the abundant species circESYT2. Expression of HIV-1 Gag alone is sufficient to drive circESYT2 incorporation into virus-like particles, indicating that circRNA incorporation is an intrinsic property of the assembly machinery. Proximity-labeling, mass spectrometry and network analysis of the proteins reveal that circESYT2 is embedded in a filamentous cytoskeleton and interacts predominantly with actin, and functional perturbation in a T-cell line shows that circESYT2 depletion destabilizes actin assembly, reduces actin incorporation into virions and enhances viral egress. Extending this analysis to other retroviruses demonstrates that circESYT2 is more efficiently packaged by murine leukaemia virus than by HIV-1, whereas foamy virus excludes it, indicating virus-specific selectivity in circRNAs capture. These findings suggest circRNAs as bona fide components of retroviral particles and uncover a host RNA-cytoskeleton axis in late replication as an unanticipated layer to host-virus crosstalk.