Detailed Mapping of the Nuclear Export Signal in the Rous Sarcoma Virus Gag Protein

ABSTRACT The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA 99:3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.

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Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

mBio ◽

10.1128/mbio.00524-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 3

Author(s):

Rebecca J. Kaddis Maldonado ◽

Breanna Rice ◽

Eunice C. Chen ◽

Kevin M. Tuffy ◽

Estelle F. Chiari ◽

...

Keyword(s):

Plasma Membrane ◽

Nuclear Envelope ◽

Rous Sarcoma Virus ◽

Nuclear Export ◽

Live Cell ◽

Viral Rna ◽

Wild Type ◽

Gag Protein ◽

Rous Sarcoma ◽

Sarcoma Virus

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.

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Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.062652199 ◽

2002 ◽

Vol 99 (6) ◽

pp. 3944-3949 ◽

Cited By ~ 83

Author(s):

Lisa Z. Scheifele ◽

Rachel A. Garbitt ◽

Jonathan D. Rhoads ◽

Leslie J. Parent

Keyword(s):

Rous Sarcoma Virus ◽

Nuclear Export ◽

Nuclear Entry ◽

Gag Polyprotein ◽

Rous Sarcoma ◽

Sarcoma Virus

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Faculty Opinions recommendation of Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005053.59504 ◽

2002 ◽

Author(s):

Anita Hopper

Keyword(s):

Rous Sarcoma Virus ◽

Nuclear Export ◽

Nuclear Entry ◽

Gag Polyprotein ◽

Rous Sarcoma ◽

Sarcoma Virus

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Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

Journal of Virology ◽

10.1128/jvi.00101-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6790-6797 ◽

Cited By ~ 45

Author(s):

Rachel Garbitt-Hirst ◽

Scott P. Kenney ◽

Leslie J. Parent

Keyword(s):

Nuclear Localization ◽

Rous Sarcoma Virus ◽

Rna Binding ◽

Genetic Evidence ◽

Genomic Rna ◽

Wild Type ◽

Nuclear Trafficking ◽

Gag Protein ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and trans-acting elements within the Gag polyprotein. The packaging signal ψ, at the 5′ end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.

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TNPO3-mediated nuclear entry of the Rous sarcoma virus Gag protein is independent of the cargo-binding domain

10.1101/2020.03.12.989608 ◽

2020 ◽

Author(s):

Breanna L. Rice ◽

Matthew S. Stake ◽

Leslie J. Parent

Keyword(s):

Nuclear Import ◽

Rous Sarcoma Virus ◽

Binding Domain ◽

Nuclear Entry ◽

Gag Protein ◽

Nuclear Localization Signals ◽

Rous Sarcoma ◽

Importin Α ◽

Sarcoma Virus ◽

Cargo Binding

AbstractRetroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLS) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS, which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functionality of importin α/β and importin-11 has been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 mediates nuclear entry of Gag and directly binds to Gag. To our surprise, this interaction did not require the cargo-binding domain of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3 including SR-domain containing splicing factors and tRNAs that re-enter the nucleus. These results suggest that RSV hijacks the host nuclear import pathway using a unique mechanism, potentially allowing other cargo to bind TNPO3 simultaneously.ImportanceRSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the cargo binding domain of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

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TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain

Journal of Virology ◽

10.1128/jvi.00640-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Breanna L. Rice ◽

Matthew S. Stake ◽

Leslie J. Parent

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Rous Sarcoma Virus ◽

Binding Domain ◽

Nuclear Entry ◽

Gag Protein ◽

Nuclear Localization Signals ◽

Rous Sarcoma ◽

Importin Α ◽

Sarcoma Virus

ABSTRACT Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS (TNPO3), which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), which are known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functions of importin-α/importin-β and importin-11 have been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 directly binds to Gag and mediates its nuclear entry. To our surprise, this interaction did not require the cargo-binding domain (CBD) of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3, including SR domain-containing splicing factors and tRNAs that reenter the nucleus. These results suggest that RSV hijacks this host nuclear import pathway using a unique mechanism, potentially allowing other cargo to simultaneously bind TNPO3. IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

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Characterization of Rous Sarcoma Virus Gag Particles Assembled In Vitro

Journal of Virology ◽

10.1128/jvi.75.6.2753-2764.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2753-2764 ◽

Cited By ~ 83

Author(s):

Fang Yu ◽

Swati M. Joshi ◽

Yu May Ma ◽

Richard L. Kingston ◽

Martha N. Simon ◽

...

Keyword(s):

Nucleic Acid ◽

Rous Sarcoma Virus ◽

Radial Distance ◽

Binding Motif ◽

Gag Protein ◽

Binding Motifs ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

The Mean

ABSTRACT Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 × 107 Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

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