Abstract
Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944-3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790-6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome.IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.
Highlights
IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes
Given that retroviral RNA synthesis occurs in the nucleus and that the Gag proteins of many retroviruses, including Rous sarcoma virus (RSV) [1, 2, 10,11,12,13,14,15], feline immunodeficiency virus [16], foamy virus [17,18,19,20,21,22,23], human immunodeficiency virus type 1 (HIV-1) [13], Mason-Pfizer monkey virus [24,25,26], mouse mammary tumor virus [13, 27], and murine leukemia virus [28], are present in the nucleus [29], it is plausible to hypothesize that retroviral Gag proteins associate with genomic vRNA (gRNA) in the nucleus
Genetic and biochemical evidence suggests that RSV Gag nuclear trafficking is necessary for efficient packaging of gRNA [2, 11], leading to the hypothesis that Gag binds the vRNA genome in the nucleus
Summary
IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. In vitro assays revealed that RNA facilitates the binding of RSV Gag to the nuclear export complex Crm1-RanGTP, suggesting that the Gag-nucleic acid association induces a conformational change exposing the NES in p10 [11] These data form the basis of a model in which RSV Gag enters the nucleus, locates and binds unspliced vRNA, undergoes a structural change allowing binding of the p10 NES to the Crm1-RanGTP export complex, and transports vRNA into the cytoplasm and, subsequently, to the plasma membrane [11]. We directly examined whether RSV Gag associates with vRNA in the nucleus using live-cell confocal microscopy to image vRNP complexes These data demonstrate that Gag and vRNA colocalized in the nucleus and moved together as a single vRNP across the nuclear membrane toward the cytoplasm. These data provide evidence in support of a novel mechanism by which retroviruses select their unspliced RNA genomes in the nucleus
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