TY - JOUR
T1 - Visualizing association of the retroviral gag protein with unspliced viral RNA in the nucleus
AU - Maldonado, Rebecca J.Kaddis
AU - Rice, Breanna
AU - Chen, Eunice C.
AU - Tuffy, Kevin M.
AU - Chiari, Estelle F.
AU - Fahrbach, Kelly M.
AU - Hope, Thomas J.
AU - Parent, Leslie J.
N1 - Funding Information:
We acknowledge those who kindly aided in this work. Robert Singer, Megerditch Kiledjian, Jan Karlseder, and Yaron Shav-Tal generously supplied plasmids. Luke Lavis (Janelia Research Campus) provided the SNAP JF646 ligand. Stephen Lockett generously provided the MatLab colocalization scripts. Ping Du (Penn State College of Medicine) provided guidance for statistical analysis. Alan Cochrane (University of Toronto) provided technical advice. Confocal microscopy images were generated using Leica SP8 (1S10OD010756-01A1), Leica SP2, and DeltaVision Elite microscopes, and microscopy image analysis and processing were conducted using the Imaris imaging processing system (Bitplane) located in the Penn State College of Medicine Microscopy Imaging Core Facility. We appreciate the technical assistance provided by Malgorzata Sudol in the Department of Medicine at the Penn State College of Medicine. This work was supported by grants from the National Institutes of Health, R01 CA76534 (to L.J.P.), P50 GM103297 CRNA (to L.J.P.), F31 CA171862 (to R.J.K.M.), F31 CA196292 (to B.R.), F30 CA214010 (to E.C.C.), and 2P50GM08254511 (to T.J.H.), grant 1S10OD01077701A1 (to T.J.H.) supporting the purchase of the DeltaVision OMX, and under a grant with the Pennsylvania Department of Health using Tobacco Settlement CURE Funds (to L.J.P.)
Funding Information:
This work was supported by grants from the National Institutes of Health, R01 CA76534 (to L.J.P.), P50 GM103297 CRNA (to L.J.P.), F31 CA171862 (to R.J.K.M.), F31 CA196292 (to B.R.), F30 CA214010 (to E.C.C.), and 2P50GM08254511 (to T.J.H.), grant 1S10OD01077701A1 (to T.J.H.) supporting the purchase of the DeltaVision OMX, and under a grant with the Pennsylvania Department of Health using Tobacco Settlement CURE Funds (to L.J.P.).
Publisher Copyright:
© 2020 Maldonado et al.
PY - 2020/3/1
Y1 - 2020/3/1
N2 - 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.
AB - 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.
KW - Gag proteins
KW - Genomic RNA packaging
KW - Live cell imaging
KW - Nucleocytoplasmic trafficking
KW - RNA trafficking
KW - Retrovirus assembly
KW - Rous sarcoma virus
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U2 - 10.1128/mBio.00524-20
DO - 10.1128/mBio.00524-20
M3 - Article
C2 - 32265329
AN - SCOPUS:85083071490
SN - 2161-2129
VL - 11
JO - mBio
JF - mBio
IS - 2
M1 - e00524-20
ER -