scholarly journals Genetic Determinants of Rous Sarcoma Virus Particle Size

1998 ◽  
Vol 72 (1) ◽  
pp. 564-577 ◽  
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.

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

1991 ◽  
Vol 11 (5) ◽  
pp. 2760-2768 ◽  
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.

scholarly journals PR Domain of Rous Sarcoma Virus Gag Causes an Assembly/Budding Defect in Insect Cells

2001 ◽  
Vol 75 (9) ◽  
pp. 4407-4412 ◽  
Author(s):  
Marc C. Johnson ◽  
Heather M. Scobie ◽  
Volker M. Vogt
Keyword(s):  
Rous Sarcoma Virus ◽  
Gag Protein ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Immunodeficiency Virus ◽  
Electron Microscopy Analysis ◽  
Sarcoma Virus ◽  
Microscopy Analysis ◽  
Pr Domain

ABSTRACT While baculovirus expression of Gag proteins from numerous retroviruses has led reliably to production of virus-like particles (VLPs), we observed that expression of Rous sarcoma virus Gag failed to produce VLPs. Transmission and scanning electron microscopy analysis revealed that the Gag protein reached the plasma membrane but was unable to correctly form particles. Addition of a myristylation signal had no effect on the budding defect, but deletion of the PR domain of Gag restored normal budding. The resulting VLPs were morphologically distinct from human immunodeficiency virus type 1 VLPs expressed in parallel.

scholarly journals Repositioning Basic Residues in the M Domain of the Rous Sarcoma Virus Gag Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11222-11229 ◽  
Author(s):  
Eric M. Callahan ◽  
John W. Wills
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Electrostatic Interactions ◽  
Particle Production ◽  
Gag Protein ◽  
Particle Release ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Acidic Phospholipids

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.

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

1991 ◽  
Vol 11 (5) ◽  
pp. 2760-2768
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.

scholarly journals Role of the Rous Sarcoma Virus p10 Domain in Shape Determination of Gag Virus-Like Particles Assembled In Vitro and within Escherichia coli

2000 ◽  
Vol 74 (21) ◽  
pp. 10260-10268 ◽  
Author(s):  
Swati M. Joshi ◽  
Volker M. Vogt
Keyword(s):  
Escherichia Coli ◽  
Rous Sarcoma Virus ◽  
Gag Protein ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Rous Sarcoma ◽  
Crude Extracts ◽  
Sarcoma Virus ◽  
Shape Determination

ABSTRACT Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coliwere examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.

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

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
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.

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

2009 ◽  
Vol 83 (13) ◽  
pp. 6790-6797 ◽  
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.

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

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.

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

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.

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

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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