Gag Protein

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Leslie J Parent - One of the best experts on this subject based on the ideXlab platform.

  • TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain.
    Journal of virology, 2020
    Co-Authors: Breanna L. Rice, Matthew S. Stake, Leslie J Parent
    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.

  • Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus
    mBio, 2020
    Co-Authors: Rebecca J. Kaddis Maldonado, Breanna L. Rice, Eunice C. Chen, Kevin M. Tuffy, Estelle F. Chiari, Kelly M. Fahrbach, Thomas J. Hope, Leslie J Parent
    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.

  • tnpo3 mediated nuclear entry of the rous sarcoma virus Gag Protein is independent of the cargo binding domain
    bioRxiv, 2020
    Co-Authors: Breanna L. Rice, Matthew S. Stake, Leslie J Parent
    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, 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.

  • Interplay between the alpharetroviral Gag Protein and SR Proteins SF2 and SC35 in the nucleus
    Frontiers in Microbiology, 2015
    Co-Authors: Breanna L. Rice, Matthew S. Stake, Rebecca J. Kaddis, Timothy L. Lochmann, Leslie J Parent
    Abstract:

    Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural Proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag Protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag Protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the Protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

  • nucleolar trafficking of the mouse mammary tumor virus Gag Protein induced by interaction with ribosomal Protein l9
    Journal of Virology, 2013
    Co-Authors: Andrea R Beyer, Breanna L. Rice, Stephen P. Goff, Darrin V Bann, Ingrid Swanson Pultz, Melissa Kane, Tatyana V Golovkina, Leslie J Parent
    Abstract:

    The mouse mammary tumor virus (MMTV) Gag Protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting Proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal Protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Forster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells.

Alan Rein - One of the best experts on this subject based on the ideXlab platform.

  • hiv 1 Gag Protein with or without p6 specifically dimerizes on the viral rna packaging signal
    Journal of Biological Chemistry, 2020
    Co-Authors: Samantha Sarni, Alan Rein, Banhi Biswas, Shuohui Liu, Erik D Olson, Jonathan P Kitzrow, Vicki H Wysocki, Karin Musierforsyth
    Abstract:

    The HIV-1 Gag Protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag–RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native MS to investigate RNA binding of wild-type (WT) Gag and Gag lacking the p6 domain (GagΔp6). Both Proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of GagGag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

  • hiv 1 Gag Protein with or without p6 specifically dimerizes on the viral rna packaging signal
    bioRxiv, 2020
    Co-Authors: Samantha Sarni, Alan Rein, Banhi Biswas, Shuohui Liu, Erik D Olson, Jonathan P Kitzrow, Vicki H Wysocki, Karin Musierforsyth
    Abstract:

    The HIV-1 Gag Protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. While the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native mass spectrometry to investigate RNA binding of wild-type Gag and Gag lacking the p6 domain (Gagp6). Both Proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. Gagp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically nucleates Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

  • hydrodynamic and membrane binding properties of purified rous sarcoma virus Gag Protein
    Journal of Virology, 2015
    Co-Authors: Robert A Dick, Siddhartha A. K. Datta, Alan Rein, Yun-xing Wang, Hirsh Nanda, Xianyang Fang, Yi Wen, Marilia Barros, Volker M Vogt
    Abstract:

    UNLABELLED Previously, no retroviral Gag Protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag Protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the Protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCE Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

  • Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag
    Journal of virology, 2011
    Co-Authors: Siddhartha A. K. Datta, Patrick K. Clark, Xiaobing Zuo, Stephen J. Campbell, Yun-xing Wang, Alan Rein
    Abstract:

    Immature retrovirus particles are assembled from the multidomain Gag Protein. In these particles, the Gag Proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interProtein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the Protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag Protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this Protein is very different from HIV-1 Gag: it has much weaker Protein-Protein interaction and is predominantly monomeric in solution. This has allowed us to study the Protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the Protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.

  • Preparation of recombinant HIV-1 Gag Protein and assembly of virus-like particles in vitro.
    Methods in molecular biology (Clifton N.J.), 2009
    Co-Authors: Siddhartha A. K. Datta, Alan Rein
    Abstract:

    The mechanism of assembly of retroviruses is not fully understood. Purification of retroviral Gag Protein and studying its solution state and assembly properties might provide insights into retroviral assembly mechanisms. Here we describe a rapid method for the purification of Gag and its subsequent assembly into virus-like particles in a defined system in vitro. The purification scheme does not use affinity tags, but purifies the native Protein by virtue of its high affinity for phosphocellulose, a property presumably related to the affinity of Gag Proteins for nucleic acids.

Breanna L. Rice - One of the best experts on this subject based on the ideXlab platform.

  • TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain.
    Journal of virology, 2020
    Co-Authors: Breanna L. Rice, Matthew S. Stake, Leslie J Parent
    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.

  • Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus
    mBio, 2020
    Co-Authors: Rebecca J. Kaddis Maldonado, Breanna L. Rice, Eunice C. Chen, Kevin M. Tuffy, Estelle F. Chiari, Kelly M. Fahrbach, Thomas J. Hope, Leslie J Parent
    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.

  • tnpo3 mediated nuclear entry of the rous sarcoma virus Gag Protein is independent of the cargo binding domain
    bioRxiv, 2020
    Co-Authors: Breanna L. Rice, Matthew S. Stake, Leslie J Parent
    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, 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.

  • Interplay between the alpharetroviral Gag Protein and SR Proteins SF2 and SC35 in the nucleus
    Frontiers in Microbiology, 2015
    Co-Authors: Breanna L. Rice, Matthew S. Stake, Rebecca J. Kaddis, Timothy L. Lochmann, Leslie J Parent
    Abstract:

    Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural Proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag Protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag Protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the Protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

  • nucleolar trafficking of the mouse mammary tumor virus Gag Protein induced by interaction with ribosomal Protein l9
    Journal of Virology, 2013
    Co-Authors: Andrea R Beyer, Breanna L. Rice, Stephen P. Goff, Darrin V Bann, Ingrid Swanson Pultz, Melissa Kane, Tatyana V Golovkina, Leslie J Parent
    Abstract:

    The mouse mammary tumor virus (MMTV) Gag Protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting Proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal Protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Forster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells.

John W Wills - One of the best experts on this subject based on the ideXlab platform.

  • Repositioning Basic Residues in the M Domain of the Rous Sarcoma Virus Gag Protein
    Journal of virology, 2000
    Co-Authors: Eric M. Callahan, John W Wills
    Abstract:

    The first 86 residues of the Rous sarcoma virus (RSV) Gag Protein form a membrane-binding (M) domain that directs Gag to the plasma membrane during budding. Unlike other retroviral Gag Proteins, RSV Gag is not myristylated; however, the RSV M domain does contain 11 basic residues that could potentially interact with acidic phospholipids in the plasma membrane. To investigate this possibility, we analyzed mutants in which basic residues in the M domain were replaced with asparagines or glutamines. The data show that neutralizing as few as two basic residues in the M domain blocked particle release and prevented Gag from localizing to the plasma membrane. Though not as severe, single neutralizations also diminished budding and, when expressed in the context of proviral clones, reduced the ability of RSV to spread in cell cultures. To further explore the role of basic residues in particle production, we added lysines to new positions in the M domain. Using this approach, we found that the budding efficiency of RSV Gag can be improved by adding pairs of lysines and that the basic residues in the M domain can be repositioned without affecting particle release. These data provide the first gain-of-function evidence for the importance of basic residues in a retroviral M domain and support a model in which RSV Gag binds to the plasma membrane via electrostatic interactions.

  • Particle Size Determinants in the Human Immunodeficiency Virus Type 1 Gag Protein
    Journal of virology, 1998
    Co-Authors: Laurence Garnier, Lee Ratner, Benjamin Rovinski, Shi-xian Cao, John W Wills
    Abstract:

    The retroviral Gag Protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag Protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag Protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.

  • Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag Protein.
    Journal of virology, 1996
    Co-Authors: Leslie J Parent, Carol B. Wilson, Marilyn D. Resh, John W Wills
    Abstract:

    During retrovirus assembly, Gag Proteins bind to the inner leaflet of the plasma membrane to initiate the budding process. The molecular basis of this Protein-lipid interaction is poorly understood. For the human, immunodeficiency virus type 1 Gag Protein, we recently reported that the membrane-binding domain resides within the N-terminal 31 amino acids and consists of two components: myristate and a cluster of basic residues, which together promote membrane binding in vitro and budding in vivo (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). The positively charged residues associate electrostatically with acidic phospholipids to stabilize membrane binding, while myristate provides membrane-binding energy via hydrophobic interactions. Here we demonstrate that the human immunodeficiency virus type 1 Gag membrane-binding domain can fully replace the membrane-targeting function of the N-terminal 100 residues of the non-myristylated Rous sarcoma virus (RSV) Gag Protein. To further explore the importance of myristate and basic residues in membrane binding, we developed a gain-of-function assay whereby budding was restored to defective mutants of RSV Gag. Detailed mutational analysis revealed that the position, number, and context of charged residues are crucial to budding. Myristate provides additional membrane-binding energy, which is critical when a Gag Protein is near the threshold of stable membrane association. Finally, viruses with altered matrix (MA) Proteins that are noninfectious, even though they produce particles with high efficiency, were identified. Thus, we present the first evidence that the RSV MA sequence plays two distinct roles, membrane binding during particle assembly and a second, as yet undefined function required for viral infectivity.

  • The membrane-binding domain of the Rous sarcoma virus Gag Protein.
    Journal of virology, 1996
    Co-Authors: Michael F. Verderame, T D Nelle, John W Wills
    Abstract:

    The Gag Protein of Rous sarcoma virus (RSV) can direct particle assembly and budding at the plasma membrane independently of the other virus-encoded products. A previous deletion analysis has suggested that the first 86 amino acids of RSV Gag constitute a large membrane-binding domain that is absolutely required for these processes. To test this hypothesis, we inserted these residues in place of the N-terminal membrane-binding domain of the pp60v-src, a transforming Protein whose biological activity requires plasma membrane localization. The ability of the Src chimera to induce cellular transformation suggests that the RSV sequence indeed contains an independent, functional domain.

  • Transport and processing of the Rous sarcoma virus Gag Protein in the endoplasmic reticulum.
    Journal of virology, 1996
    Co-Authors: Neel K. Krishna, R A Weldon, John W Wills
    Abstract:

    The Gag Proteins of replication-competent retroviruses direct budding at the plasma membrane and are cleaved by the viral protease (PR) just before or very soon after particle release. In contrast, defective retroviruses that bud into the endoplasmic reticulum (ER) have been found, and morphologically these appear to contain uncleaved Gag Proteins. From this, it has been proposed that activation of PR may depend upon a host factor found only at the plasma membrane. However, if Gag Proteins were cleaved by PR before the particle could pinch off the ER membrane, then the only particles that would remain visible are those that packaged smaller-than-normal amounts of PR, and these would have an immature morphology. To distinguish between these two hypotheses, we made use of the Rous sarcoma virus (RSV) Gag Protein, the PR of RSV IS included on each Gag molecule. To target Gag to the ER, a signal peptide was installed at its amino terminus in place of the plasma membrane-binding domain. An intervening, hydrophobic, transmembrane anchor was included to keep Gag extended into the cytoplasm. We found that PR-mediated processing occurred, although the cleavage products were rapidly degraded. When the anchor was removed, allowing the entire Protein to be inserted into the lumen of the ER, Gag processing occurred with a high level of efficiency, and the cleavage products were quite stable. Thus, PR activation does not require targeting of Gag molecules to the plasma membrane. Unexpectedly, molecules lacking the transmembrane anchor were rapidly secreted from the cell in a nonmembrane-enclosed form and in a manner that was very sensitive to brefeldin A and monensin. In contrast, the wild-type RSV and Moloney murine leukemia virus Gag Proteins were completely insensitive to these inhibitors, suggesting that the normal mechanism of transport to the plasma membrane does not require interactions with the secretory pathway.

Jef D. Boeke - One of the best experts on this subject based on the ideXlab platform.

  • Mapping the multimerization domains of the Gag Protein of yeast retrotransposon Ty1.
    Journal of virology, 1997
    Co-Authors: Carrie Baker Brachmann, Jef D. Boeke
    Abstract:

    The two-hybrid system was used to define regions of the Ty1 Gag Protein responsible for multimerization. Gag truncations lacking the first 146 or the last 97 amino acids (Gag is 440 amino acids in length) interact. A severely C-terminally truncated molecule (lacking the last 207 amino acids) was the smallest truncation to interact, suggesting that some Protein-Protein interactions between Gag molecules are mediated through the first 233 amino acids. However, an internal deletion of amino acids 147 to 233 does not abolish Gag-Gag interaction, indicating that more than one region can mediate Gag interaction. Surprisingly, we found that a truncation lacking the last 97 amino acids interacts with itself but not with full-length Gag. This is apparently due to an artifact of the two-hybrid assay, since these same molecules coassemble with wild-type Gag into Ty1 virus-like particles.

  • A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag Protein.
    Journal of virology, 1996
    Co-Authors: G V Merkulov, Carrie Baker Brachmann, K M Swiderek, Jef D. Boeke
    Abstract:

    Cleavage of the Gag and Gag-Pol polyProtein precursors is a critical step in proliferation of retroviruses and retroelements. The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag Protein. Ty1 Gag is not obviously homologous to the Gag Proteins of retroviruses. The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation. Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus. A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag. Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs. While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1. Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.

  • Novel gene expression mechanism in a fission yeast retroelement : Tf1 Proteins are derived from a single primary translation product
    The EMBO Journal, 1993
    Co-Authors: Henry L. Levin, D. C. Weaver, Jef D. Boeke
    Abstract:

    Abstract In sharp contrast to the single ORF of the Schizosaccharomyces pombe retrotransposon Tf1, retroviruses and most retrotransposons employ two different ORFs to separately encode the Gag and Pol Proteins. The different ORFs are thought to allow for overexpression of the Gag Protein relative to Pol Protein presumed necessary for the assembly of functional retrovirus particles and virus-like particles (VLPs). The results of in vivo experiments designed to detect the transposition of Tf1 show that Tf1 is indeed active and can insert itself into the host genome via a true retrotransposition process. Thus, a paradox emerged between the lack of any obvious means of overexpressing Tf1 Gag Protein and the demonstrated functionality of the element. Epitope tagging experiments described here confirm that the Tf1 large ORF is intact and that there is no translational or transcriptional mechanism used to overexpress the Tf1 Gag Protein. In addition, we used sucrose gradients and antisera specific for Tf1 capsid (CA) and integrase (IN) to show that the Tf1 Proteins do assemble into uniform populations of macromolecular particles that also cosediment with Tf1 reverse transcription products. This evidence suggests that Tf1 Proteins form VLPs without using the previously described mechanisms that retroviruses and retrotransposons require to overexpress Gag Proteins.