Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

ABSTRACT Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.

Download Full-text

Conditions for Copackaging Rous Sarcoma Virus and Murine Leukemia Virus Gag Proteins during Retroviral Budding

Journal of Virology ◽

10.1128/jvi.73.3.2045-2051.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2045-2051 ◽

Cited By ~ 16

Author(s):

Robert P. Bennett ◽

John W. Wills

Keyword(s):

Plasma Membrane ◽

Murine Leukemia Virus ◽

Rous Sarcoma Virus ◽

Sequence Similarity ◽

Leukemia Virus ◽

Particle Assembly ◽

Gag Proteins ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Murine Leukemia

ABSTRACT Rous sarcoma virus (RSV) and murine leukemia virus (MLV) are examples of distantly related retroviruses that normally do not encounter one another in nature. Their Gag proteins direct particle assembly at the plasma membrane but possess very little sequence similarity. As expected, coexpression of these two Gag proteins did not result in particles that contain both. However, when the N-terminal membrane-binding domain of each molecule was replaced with that of the Src oncoprotein, which is also targeted to the cytoplasmic face of the plasma membrane, efficient copackaging was observed in genetic complementation and coimmunoprecipitation assays. We hypothesize that the RSV and MLV Gag proteins normally use distinct locations on the plasma membrane for particle assembly but otherwise have assembly domains that are sufficiently similar in function (but not sequence) to allow heterologous interactions when these proteins are redirected to a common membrane location.

Download Full-text

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.

Download Full-text

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity

Molecular and Cellular Biology ◽

10.1128/mcb.2.4.355-360.1982 ◽

1982 ◽

Vol 2 (4) ◽

pp. 355-360

Author(s):

A Ziemiecki ◽

R R Friis ◽

H Bauer

Keyword(s):

Protein Synthesis ◽

Half Life ◽

Rous Sarcoma Virus ◽

Kinase Activity ◽

Rabbit Serum ◽

Temperature Sensitive ◽

Apparent Contradiction ◽

Gag Proteins ◽

Rous Sarcoma ◽

Sarcoma Virus

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.

Download Full-text

Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

Journal of Virology ◽

10.1128/jvi.02733-15 ◽

2015 ◽

Vol 90 (5) ◽

pp. 2473-2485 ◽

Cited By ~ 5

Author(s):

Robert A. Dick ◽

Marilia Barros ◽

Danni Jin ◽

Mathias Lösche ◽

Volker M. Vogt

Keyword(s):

Plasma Membrane ◽

Nucleic Acid ◽

Rous Sarcoma Virus ◽

Membrane Binding ◽

Membrane Interaction ◽

Gag Proteins ◽

Peptide Assembly ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACTThe principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assemblyin vitrodramatically reduced binding of Gag to liposomes.In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize.IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assemblyin vitrowith purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.

Download Full-text

Genetic Determinants of Rous Sarcoma Virus Particle Size

Journal of Virology ◽

10.1128/jvi.72.1.564-577.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 564-577 ◽

Cited By ~ 45

Author(s):

Neel K. Krishna ◽

Stephen Campbell ◽

Volker M. Vogt ◽

John W. Wills

Keyword(s):

Particle Size ◽

Rous Sarcoma Virus ◽

Proteolytic Processing ◽

Gag Protein ◽

Gag Proteins ◽

Rous Sarcoma ◽

Interaction Domains ◽

Sarcoma Virus ◽

Exhaustive Study ◽

Rna Interaction

ABSTRACT The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.

Download Full-text

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

Download Full-text

Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2760 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2760-2768 ◽

Cited By ~ 26

Author(s):

G F Barker ◽

K Beemon

Keyword(s):

Protein Function ◽

Rous Sarcoma Virus ◽

Wild Type ◽

Gag Protein ◽

Gag Proteins ◽

Cis Acting ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Nonsense Codons ◽

The Stability

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. In contrast, the levels of spliced viral RNAs were not affected in our transient expression assays in chicken cells. Experiments using the transcription inhibitor dactinomycin showed that mutant unspliced RNAs were degraded more rapidly than wild-type RNA. Furthermore, mutant RNAs could be partially stabilized by coexpression of wild-type gag proteins in trans; however, intact gag proteins were not required to maintain the stability of RNAs which did not contain premature termination codons. Thus, termination codons seemed to destabilize the RNA not because of their effect on gag protein function but instead because they disrupted the process of translating the gag region of the RNA. Analysis of double-mutant constructs containing both deletions and termination codons within the gag gene also suggested that the stability of the unspliced RNA was affected by a cis-acting interaction between the RNA and ribosomes.

Download Full-text