scholarly journals Rous Sarcoma Virus Translation Revisited: Characterization of an Internal Ribosome Entry Segment in the 5′ Leader of the Genomic RNA

2000 ◽  
Vol 74 (24) ◽  
pp. 11581-11588 ◽  
Author(s):  
Clarence Deffaud ◽  
Jean-Luc Darlix
Keyword(s):  
Rous Sarcoma Virus ◽  
Leukemia Virus ◽  
Secondary Structures ◽  
Open Reading Frames ◽  
Start Codon ◽  
Genomic Rna ◽  
Internal Ribosome Entry ◽  
Rous Sarcoma ◽  
Internal Ribosome Entry Segment ◽  
Sarcoma Virus

ABSTRACT The 5′ leader of Rous sarcoma virus (RSV) genomic RNA and of retroviruses in general is long and contains stable secondary structures that are critical in the early and late steps of virus replication such as RNA dimerization and packaging and in the process of reverse transcription. The initiation of RSV Gag translation has been reported to be 5′ cap dependent and controlled by three short open reading frames located in the 380-nucleotide leader upstream of the Gag start codon. Translation of RSV Gag would thus differ from that prevailing in other retroviruses such as murine leukemia virus, reticuloendotheliosis virus type A, and simian immunodeficiency virus, in which an internal ribosome entry segment (IRES) in the 5′ end of the genomic RNA directs efficient Gag expression despite stable 5′ secondary structures. This prompted us to investigate whether RSV Gag translation might be controlled by an IRES-dependent mechanism. The results show that the 5′ leaders of RSV and v-Src RNA exhibit IRES properties, since these viral elements can promote efficient translation of monocistronic RNAs in conditions inhibiting 5′ cap-dependent translation. When inserted between two cistrons in a canonical bicistronic construct, both the RSV and v-Src leaders promote expression of the 3′ cistron. A genetic analysis of the RSV leader allowed the identification of two nonoverlapping 5′ and 3′ leader domains with IRES activity. In addition, the v-Src leader was found to contain unique 3′ sequences promoting an efficient reinitiation of translation. Taken together, these data lead us to propose a new model for RSV translation.

scholarly journals RNA Dimerization Defect in a Rous Sarcoma Virus Matrix Mutant

2000 ◽  
Vol 74 (1) ◽  
pp. 164-172 ◽  
Author(s):  
Leslie J. Parent ◽  
Tina M. Cairns ◽  
Jessica A. Albert ◽  
Carol B. Wilson ◽  
John W. Wills ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Myristic Acid ◽  
Rous Sarcoma Virus ◽  
Leukemia Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The retrovirus matrix (MA) sequence of the Gag polyprotein has been shown to contain functions required for membrane targeting and binding during particle assembly and budding. Additional functions for MA have been proposed based on the existence of MA mutants in Rous sarcoma virus (RSV), murine leukemia virus, human immunodeficiency virus type 1, and human T-cell leukemia virus type 1 that lack infectivity even though they release particles of normal composition. Here we describe an RSV MA mutant with a surprising and previously unreported phenotype. In the mutant known as Myr1E, the small membrane-binding domain of the Src oncoprotein has been added as an N-terminal extension of Gag. While Myr1E is not infectious, full infectivity can be reestablished by a single amino acid substitution in the Src sequence (G2E), which eliminates the addition of myristic acid and the membrane-binding capacity of this foreign sequence. The presence of myristic acid at the N terminus of the Myr1E Gag protein does not explain its replication defect, because other myristylated derivatives of RSV Gag are fully infectious (e.g., Myr2 [C. R. Erdie and J. W. Wills, J. Virol. 64:5204–5208, 1990]). Biochemical analyses of Myr1E particles reveal that they contain wild-type levels of the Gag cleavage products, Env glycoproteins, and reverse transcriptase activity when measured on an exogenous template. Genomic RNA incorporation appears to be mildly reduced compared to the wild-type level. Unexpectedly, RNA isolated from Myr1E particles is monomeric when analyzed on nondenaturing Northern blots. Importantly, the insertional mutation does not lie within previously identified dimer linkage sites. In spite of the dimerization defect, the genomic RNA from Myr1E particles serves efficiently as a template for reverse transcription as measured by an endogenous reverse transcriptase assay. In marked contrast, after infection of avian cells, the products of reverse transcription are nearly undetectable. These findings might be explained either by the loss of a normal function of MA needed in the formation or stabilization of RNA dimers or by the interference in such events by the mutant MA molecules. It is possible that Myr1E viruses package a single copy of viral RNA.

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

1979 ◽  
Vol 6 (2) ◽  
pp. 471-486 ◽  
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

scholarly journals The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

1991 ◽  
Vol 11 (12) ◽  
pp. 5848-5859 ◽  
Author(s):  
I R Ghattas ◽  
J R Sanes ◽  
J E Majors
Keyword(s):  
Rous Sarcoma Virus ◽  
Cultured Cells ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Internal Promoter ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Virus Internal Ribosome Entry

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo.

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 Mutations in the Spacer Peptide and Adjoining Sequences in Rous Sarcoma Virus Gag Lead to Tubular Budding

2008 ◽  
Vol 82 (14) ◽  
pp. 6788-6797 ◽  
Author(s):  
Paul W. Keller ◽  
Marc C. Johnson ◽  
Volker M. Vogt
Keyword(s):  
Rous Sarcoma Virus ◽  
Mutational Analysis ◽  
Leukemia Virus ◽  
Alpha Helix ◽  
Insertion Mutagenesis ◽  
Structural Protein ◽  
Enveloped Virus ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Hiv 1

ABSTRACT All orthoretroviruses encode a single structural protein, Gag, which is necessary and sufficient for the assembly and budding of enveloped virus-like particles from the cell. The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) contain a short spacer peptide (SP or SP1, respectively) separating the capsid (CA) and nucleocapsid (NC) domains. SP or SP1 and the residues immediately upstream are known to be critical for proper assembly. Using mutagenesis and electron microscopy analysis of insect cells or chicken cells overexpressing RSV Gag, we defined the SP assembly domain to include the last 8 residues of CA, all 12 residues of SP, and the first 4 residues of NC. Five- or two-amino acid glycine-rich insertions or substitutions in this critical region uniformly resulted in the budding of abnormal, long tubular particles. The equivalent SP1-containing HIV-1 Gag sequence was unable to functionally replace the RSV sequence in supporting normal RSV spherical assembly. According to secondary structure predictions, RSV and HIV-1 SP/SP1 and adjoining residues may form an alpha helix, and what is likely the functionally equivalent sequence in murine leukemia virus Gag has been inferred by mutational analysis to form an amphipathic alpha helix. However, our alanine insertion mutagenesis did not provide evidence for an amphipathic helix in RSV Gag. Taken together, these results define a short assembly domain between the folded portions of CA and NC, which is essential for formation of the immature Gag shell.

scholarly journals Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

1981 ◽  
Vol 1 (5) ◽  
pp. 394-407 ◽  
Author(s):  
J A Cooper ◽  
T Hunter
Keyword(s):  
Murine Leukemia Virus ◽  
Rous Sarcoma Virus ◽  
Leukemia Virus ◽  
Specific Protein ◽  
Cellular Proteins ◽  
3T3 Cells ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Abelson Murine Leukemia Virus ◽  
Murine Leukemia

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.

The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos

1991 ◽  
Vol 11 (12) ◽  
pp. 5848-5859
Author(s):  
I R Ghattas ◽  
J R Sanes ◽  
J E Majors
Keyword(s):  
Rous Sarcoma Virus ◽  
Cultured Cells ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Internal Promoter ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Virus Internal Ribosome Entry

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo.

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