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

Retroviruses are responsible for significant pathology 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 (Ψ) 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 location of the highest concentration of USvRNA molecules in the cell. In support of this model, previous studies using fixed cells infected with RSV revealed that Gag co-localizes with large USvRNA nuclear foci representing viral transcriptional burst sites. To test this idea, we used single molecule labeling and imaging techniques to visualize fluorescently-tagged, actively transcribing viral genomes, and Gag proteins in living cells. Gag condensates were observed in the nucleus, transiently co-localized with USvRNA at transcriptional burst sites, forming co- localized viral ribonucleoprotein complexes (vRNPs). These results support a novel paradigm for retroviral assembly in which Gag traffics to transcriptional burst sites and interacts through a dynamic kissing interaction to capture nascent gRNA for incorporation into virions.