Kaddis Maldonado, R. J. | bioRxiv Science

bioRxiv2025

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.

microbiology↗

bioRxiv2024

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome

Retroviruses exploit a variety of host proteins to assemble and release virions from infected cells. To date, most studies that examined possible interacting partners of retroviral Gag proteins focused on host proteins that localize primarily to the cytoplasm or plasma membrane. Given the recent findings that several full-length Gag proteins localize to the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings that reveal previously unknown host processes. In this study, we systematically compared nuclear factors identified in published HIV-1 proteomic studies which had used a variety of experimental approaches. In addition, to contribute to this body of knowledge, we report results from a mass spectrometry approach using affinity-tagged (His6) HIV-1 and RSV Gag proteins mixed with nuclear extracts. Taken together, the previous studies--as well as our own--identified potential binding partners of HIV-1 and RSV Gag involved in several nuclear processes, including transcription, splicing, RNA modification, and chromatin remodeling. Although a subset of host proteins interacted with both Gag proteins, there were also unique host proteins belonging to each interactome dataset. To validate one of the novel findings, we demonstrated the interaction of RSV Gag with a member of the Mediator complex, Med26, which is required for RNA polymerase II-mediated transcription. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.

microbiology↗