Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

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trans-Acting Inhibition of Genomic RNA Dimerization by Rous Sarcoma Virus Matrix Mutants

Journal of Virology ◽

10.1128/jvi.75.1.260-268.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 260-268 ◽

Cited By ~ 25

Author(s):

Rachel A. Garbitt ◽

Jessica A. Albert ◽

Michelle D. Kessler ◽

Leslie J. Parent

Keyword(s):

Rous Sarcoma Virus ◽

Single Amino Acid ◽

Genomic Rna ◽

Wild Type ◽

Protein Fusion ◽

Dimer Formation ◽

Rna Sequence ◽

Rna Dimerization ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E−). To distinguish between cis andtrans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E−.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E−, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG− was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that thesrc sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG−, Myr1E−, and Myr1E−.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.

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Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Journal of Virology ◽

10.1128/jvi.01628-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10371-10382 ◽

Cited By ~ 12

Author(s):

Robert A. Dick ◽

Siddhartha A. K. Datta ◽

Hirsh Nanda ◽

Xianyang Fang ◽

Yi Wen ◽

...

Keyword(s):

Plasma Membrane ◽

Rous Sarcoma Virus ◽

Acyl Chain ◽

Membrane Binding ◽

Membrane Association ◽

Gag Protein ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Hiv 1

ABSTRACTPreviously, 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 interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin 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 interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.IMPORTANCERetroviruses 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.

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The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

Journal of Virology ◽

10.1128/jvi.01502-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10294-10302 ◽

Cited By ~ 44

Author(s):

Florian K. M. Schur ◽

Robert A. Dick ◽

Wim J. H. Hagen ◽

Volker M. Vogt ◽

John A. G. Briggs

Keyword(s):

Rous Sarcoma Virus ◽

Electron Tomography ◽

Virus Particles ◽

Terminal Domain ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Subtomogram Averaging ◽

Immature Virus ◽

Infectious Form ◽

Hiv 1

ABSTRACTThe polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to studyin vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation.IMPORTANCERetroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.

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Link between Genome Packaging and Rate of Budding for Rous Sarcoma Virus

Journal of Virology ◽

10.1128/jvi.77.17.9388-9398.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9388-9398 ◽

Cited By ~ 27

Author(s):

Eric M. Callahan ◽

John W. Wills

Keyword(s):

Plasma Membrane ◽

Rous Sarcoma Virus ◽

Nuclear Export ◽

Nuclear Accumulation ◽

Membrane Targeting ◽

Genomic Rna ◽

Wild Type ◽

Genome Packaging ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT The subcellular location at which genomic RNA is packaged by Gag proteins during retrovirus assembly remains unknown. Since the membrane-binding (M) domain is most critical for targeting Gag to the plasma membrane, changes to this determinant might alter the path taken through the cell and reduce the efficiency of genome packaging. In this report, a Rous sarcoma virus (RSV) mutant having two acidic-to-basic substitutions in the M domain is described. This mutant, designated Super M, produced particles much faster than the wild type, but the mutant virions were noninfectious and contained only 1/10 the amount of genomic RNA found in wild-type particles. To identify the cause(s) of these defects, we considered data that suggest that RSV Gag traffics through the nucleus to package the viral genome. Although inhibition of the CRM-1 pathway of nuclear export caused the accumulation of wild-type Gag in the nucleus, nuclear accumulation did not occur with Super M. The importance of the nucleocapsid (NC) domain in membrane targeting was also determined, and, importantly, deletion of the NC sequence prevented plasma membrane localization by wild-type Gag but not by Super M Gag. Based on these results, we reasoned that the enhanced membrane-targeting properties of Super M inhibit genome packaging. Consistent with this interpretation, substitutions that reestablished the wild-type number of basic and acidic residues in the Super M Gag M domain reduced the budding efficiency and restored genome packaging and infectivity. Therefore, these data suggest that Gag targeting and genome packaging are normally linked to ensure that RSV particles contain viral RNA.

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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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Single-molecule imaging of HIV-1 envelope glycoprotein dynamics and Gag lattice association exposes determinants responsible for virus incorporation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910008116 ◽

2019 ◽

Vol 116 (50) ◽

pp. 25269-25277 ◽

Cited By ~ 10

Author(s):

Nairi Pezeshkian ◽

Nicholas S. Groves ◽

Schuyler B. van Engelenburg

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Envelope Glycoprotein ◽

Virus Assembly ◽

Single Molecule Tracking ◽

Virus Particles ◽

Cell Plasma ◽

The Matrix ◽

Resolution Single ◽

Hiv 1

The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.

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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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Characterization of the genomic RNA from a Rous sarcoma virus mutant temperature sensitive for cell transformation

Nucleic Acids Research ◽

10.1093/nar/6.2.471 ◽

1979 ◽

Vol 6 (2) ◽

pp. 471-486 ◽

Cited By ~ 8

Author(s):

Jean-Luc Darlix ◽

Mireille Levray ◽

Peter A. Bromley ◽

Pierre-François Spahr

Keyword(s):

Rous Sarcoma Virus ◽

Cell Transformation ◽

Temperature Sensitive ◽

Genomic Rna ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Virus Mutant

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Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein

Molecular and Cellular Biology ◽

10.1128/mcb.4.12.2697-2704.1984 ◽

1984 ◽

Vol 4 (12) ◽

pp. 2697-2704

Author(s):

J E Buss ◽

M P Kamps ◽

B M Sefton

Keyword(s):

Plasma Membrane ◽

Myristic Acid ◽

Rous Sarcoma Virus ◽

Minor Component ◽

Rous Sarcoma ◽

Cytoplasmic Face ◽

Sarcoma Virus ◽

Stable Part ◽

Membrane Bound ◽

A Minor

Myristic acid, a minor component of cellular fatty acids, has been shown previously to be covalently bound to most molecules of p60src, the transforming protein of Rous sarcoma virus. We have now determined at what time during the life cycle of p60src, and where within the cell, this lipid becomes attached to the protein. p60src was found to acquire myristic acid at only one time, during or immediately after its synthesis. p60src is known to be synthesized on free polysomes and appears at the cytoplasmic face of the plasma membrane after a lag of 10 min. The addition of myristic acid to p60src therefore precedes the binding of the protein to the plasma membrane. The lipid attached to p60src is a permanent, metabolically stable part of the protein; we found no evidence for turnover of the myristyl moiety. However, we did find myristate attached to various soluble forms of p60src and to a large number of cytosolic cellular proteins as well. This demonstrates that the attachment of myristic acid to a protein is not in itself sufficient to convert a soluble protein into a membrane-bound protein.

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Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules.

The Journal of Cell Biology ◽

10.1083/jcb.100.2.409 ◽

1985 ◽

Vol 100 (2) ◽

pp. 409-417 ◽

Cited By ~ 55

Author(s):

M D Resh ◽

R L Erikson

Keyword(s):

Plasma Membrane ◽

Specific Antibody ◽

Rous Sarcoma Virus ◽

Distinct Pattern ◽

Cell Periphery ◽

Equal Distribution ◽

Rous Sarcoma ◽

Src Gene ◽

Sarcoma Virus ◽

Infected Cells

Antiserum to the Rous sarcoma virus (RSV)-transforming protein, pp60v-src, was produced in rabbits immunized with p60 expressed in Escherichia coli. alpha p60 serum immunoprecipitated quantitatively more pp60v-src than did tumor-bearing rabbit (TBR) sera. When RSV-transformed cell lysates were preadsorbed with TBR serum, the remaining lysate contained additional pp60v-src, which was recognized only by reimmunoprecipitation with alpha p60 serum and not by TBR serum. In subcellular fractions of RSV-infected chicken embryo fibroblasts (RSV-CEFs) and field vole cells probed with TBR serum, the majority of the pp60v-src was associated with the plasma membrane-enriched P100 fraction. However, alpha p60 serum revealed equal distribution of pp60v-src and its kinase activity between the P1 (nuclear) and P100 fractions. The same results were obtained for pp60c-src in uninfected CEFs. On discontinuous sucrose gradients nearly 50% of the P1-pp60v-src sedimented with nuclei, in fractions where no plasma membrane was detected. Indirect immunofluorescence microscopy of RSV-CEFs with alpha p60 serum revealed a distinct pattern of perinuclear fluorescence, in addition to staining at the cell periphery. Thus the use of a highly specific antibody reveals that enzymatically active pp60v-src and pp60c-src molecules are present in other intracellular structures, probably juxtareticular nuclear membranes, in addition to the plasma membrane in normal, uninfected, and wild-type RSV-infected cells.

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