Dynamic interactions of retroviral Gag condensates with nascent viral RNA at transcriptional burst sites: implications for genomic RNA packaging

Retroviruses cause significant diseases in humans and animals, including the acquired immunodeficiency syndrome and a wide range of malignancies. A crucial yet poorly understood step in the replication cycle is the recognition and selection of unspliced viral RNA (USvRNA) by the retroviral Gag protein, which binds to the psi ({Psi}) packaging sequence in the 5 leader, to package it as genomic RNA (gRNA) into nascent virions. It was previously thought that Gag initially bound gRNA in the cytoplasm. However, previous studies demonstrated that the Rous sarcoma virus (RSV) Gag protein traffics transiently through the nucleus, which is necessary for efficient gRNA packaging. These data formed a strong premise for the hypothesis that Gag selects nascent gRNA at transcription sites in the nucleus, the highest concentration of USvRNA molecules in the cell. To test this hypothesis, we used single molecule labeling and imaging techniques to visualize fluorescently-tagged, actively transcribing viral genomes, and Gag proteins in living cells. Gag foci were observed in the nucleus, transiently co-localizing with USvRNA at transcriptional burst sites, and forming viral ribonucleoprotein complexes (vRNPs). Furthermore, we found that RSV Gag interacts with Med26 and CTCF in the nucleus, suggesting a possible role for these factors in trafficking Gag to transcription sites. These results support a novel paradigm for retroviral assembly in which Gag traffics to transcriptional burst sites and interacts with nascent USvRNA through a dynamic kissing interaction to capture gRNA for incorporation into virions. ImportanceRetroviruses depend upon host cell transcription machinery to synthesize USvRNA, which serves as the genome selected by Gag for packaging. We previously reported that RSV Gag undergoes transient nucleocytoplasmic trafficking, which is needed for optimal genome packaging and co-localizes with USvRNA in the nucleus. Here, using live cell imaging, we found that the association of Gag with USvRNA at the transcriptional burst site is transient and dynamic. Both Gag and the RSV transcriptional burst are located near the periphery of the nucleus, which may facilitate viral RNA export. Our data also suggest that host transcription-associated factors may play a role in trafficking Gag to transcription sites.