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 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 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.

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