scholarly journals Mutations of the Rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products.

1984 ◽  
Vol 99 (6) ◽  
pp. 2011-2023 ◽  
Author(s):  
J W Wills ◽  
R V Srinivas ◽  
E Hunter
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Nuclear Membrane ◽  
Rous Sarcoma Virus ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

The envelope glycoproteins of Rous sarcoma virus (RSV), gp85 and gp37, are anchored in the membrane by a 27-amino acid, hydrophobic domain that lies adjacent to a 22-amino acid, cytoplasmic domain at the carboxy terminus of gp37. We have altered these cytoplasmic and transmembrane domains by introducing deletion mutations into the molecularly cloned sequences of a proviral env gene. The effects of the mutations on the transport and subcellular localization of the Rous sarcoma virus glycoproteins were examined in monkey (CV-1) cells using an SV40 expression vector. We found, on the one hand, that replacement of the nonconserved region of the cytoplasmic domain with a longer, unrelated sequence of amino acids (mutant C1) did not alter the rate of transport to the Golgi apparatus nor the appearance of the glycoprotein on the cell surface. Larger deletions, extending into the conserved region of the cytoplasmic domain (mutant C2), resulted in a slower rate of transport to the Golgi apparatus, but did not prevent transport to the cell surface. On the other hand, removal of the entire cytoplasmic and transmembrane domains (mutant C3) did block transport and therefore did not result in secretion of the truncated protein. Our results demonstrate that the C3 polypeptide was not transported to the Golgi apparatus, although it apparently remained in a soluble, nonanchored form in the lumen of the rough endoplasmic reticulum; therefore, it appears that this mutant protein lacks a functional sorting signal. Surprisingly, subcellular localization by internal immunofluorescence revealed that the C3 protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm. This observation suggests that the wild-type glycoproteins (and perhaps other membrane-bound or secreted proteins) are specifically transported to the nuclear membrane after their biosynthesis elsewhere in the rough endoplasmic reticulum.

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.

Small deletion in v-src SH3 domain of a transformation defective mutant of rous sarcoma virus restores wild type transforming properties

Virology ◽  
1992 ◽  
Vol 189 (2) ◽  
pp. 556-567 ◽  
Author(s):  
Philippe Dezélée ◽  
Jean Vianney Barnier ◽  
Annie Hampe ◽  
Danielle Laugier ◽  
Maria Marx ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Sh3 Domain ◽  
Wild Type ◽  
Small Deletion ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

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 Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

1984 ◽  
Vol 4 (3) ◽  
pp. 454-467 ◽  
Author(s):  
J G Krueger ◽  
E A Garber ◽  
S S Chin ◽  
H Hanafusa ◽  
A R Goldberg
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Immunofluorescence Microscopy ◽  
Partial Transformation ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Size Variant ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Avian Sarcoma

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.

scholarly journals Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

1985 ◽  
Vol 5 (5) ◽  
pp. 1122-1129 ◽  
Author(s):  
S K Anderson ◽  
C P Gibbs ◽  
A Tanaka ◽  
H J Kung ◽  
D J Fujita
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Rous Sarcoma Virus ◽  
Cellular Growth ◽  
Tyrosine Kinase Domain ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus ◽  
Carboxy Terminal

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.

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

2001 ◽  
Vol 75 (1) ◽  
pp. 260-268 ◽  
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.

scholarly journals Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes.

1980 ◽  
Vol 85 (2) ◽  
pp. 481-488 ◽  
Author(s):  
Y Mikuni-Takagaki ◽  
B P Toole
Keyword(s):  
Cell Surface ◽  
Rous Sarcoma Virus ◽  
Solution Treatment ◽  
Cell Detachment ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Complete Cell ◽  
Two Populations ◽  
Testicular Hyaluronidase

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.

scholarly journals Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

2000 ◽  
Vol 74 (7) ◽  
pp. 3245-3252 ◽  
Author(s):  
Susanne Werner ◽  
Birgitta M. Wöhrl
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Rous Sarcoma Virus ◽  
Active Sites ◽  
Polymerase Activity ◽  
Rnase H ◽  
Wild Type ◽  
Α Subunit ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The genes encoding the α (63-kDa) and β (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV αβ and αPol RTs within which one subunit was selectively mutated. When αβ heterodimers contained the Asp 181→Asn mutation in their β subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their α subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in αβ heterodimers mutated in the β subunit but was lost when the mutation was present in the α subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the α subunit of the heterodimeric RSV RT αβ.

Sign in / Sign up

Export Citation Format

Share Document