scholarly journals Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

2008 ◽  
Vol 83 (5) ◽  
pp. 2119-2129 ◽  
Author(s):  
Jason E. Weil ◽  
Michalis Hadjithomas ◽  
Karen L. Beemon
Keyword(s):  
Secondary Structure ◽  
Rous Sarcoma Virus ◽  
Translation Termination ◽  
Rna Stability ◽  
Premature Termination Codon ◽  
Termination Codon ◽  
Stem Loop ◽  
Future Design ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3′ untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3′ UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5′ end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

scholarly journals Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

1994 ◽  
Vol 14 (3) ◽  
pp. 1986-1996 ◽  
Author(s):  
G F Barker ◽  
K Beemon
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Stability ◽  
Subcellular Fractionation ◽  
Open Reading Frame ◽  
Termination Codon ◽  
Gag Protein ◽  
Reading Frame ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Nonsense Codons

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. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction

1994 ◽  
Vol 14 (3) ◽  
pp. 1986-1996
Author(s):  
G F Barker ◽  
K Beemon
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Stability ◽  
Subcellular Fractionation ◽  
Open Reading Frame ◽  
Termination Codon ◽  
Gag Protein ◽  
Reading Frame ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Nonsense Codons

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. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

scholarly journals Secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus 1 1Edited by D. E. Draper

1998 ◽  
Vol 284 (2) ◽  
pp. 205-225 ◽  
Author(s):  
Beate Marczinke ◽  
Rosamond Fisher ◽  
Marijana Vidakovic ◽  
Alison J Bloys ◽  
Ian Brierley
Keyword(s):  
Secondary Structure ◽  
Rous Sarcoma Virus ◽  
Mutational Analysis ◽  
Ribosomal Frameshift ◽  
Rous Sarcoma ◽  
Sarcoma Virus

Mutation of a termination codon affects src initiation

1984 ◽  
Vol 4 (9) ◽  
pp. 1738-1746
Author(s):  
S Hughes ◽  
K Mellstrom ◽  
E Kosik ◽  
F Tamanoi ◽  
J Brugge
Keyword(s):  
Rous Sarcoma Virus ◽  
Termination Codon ◽  
Initiation Codon ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Reading Frame ◽  
Amino Acid Peptide ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Rna Precursor

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.

scholarly journals Changes in Rous Sarcoma Virus RNA Secondary Structure near the Primer Binding Site upon tRNATrpPrimer Annealing

1999 ◽  
Vol 73 (8) ◽  
pp. 6307-6318 ◽  
Author(s):  
Shannon Morris ◽  
Jonathan Leis
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Primer Binding Site ◽  
Wild Type ◽  
Stem Loop ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Primer Annealing ◽  
Nuclease Mapping

ABSTRACT Predicted secondary-structure elements encompassing the primer binding site in the 5′ untranslated region of Rous sarcoma virus (RSV) RNA play an integral role in multiple viral replications steps including reverse transcription, DNA integration, and RNA packaging (A. Aiyar, D. Cobrinik, Z. Ge, H. J. Kung, and J. Leis, J. Virol. 66:2464–2472, 1992; D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864–3872, 1991; J. T. Miller, Z. Ge, S. Morris, K. Das, and J. Leis, J. Virol. 71:7648–7656, 1997). These elements include the U5-Leader stem, U5-IR stem-loop, and U5-TΨC interaction region. Limited digestion of the 5′ untranslated region of wild-type and mutant RSV RNAs with structure- and/or sequence-specific RNases detects the presence of the U5-Leader stem and the U5-IR stem-loop. When a tRNATrp primer is annealed to wild-type RNAs in vitro, limited nuclease mapping indicates that the U5-IR stem becomes partially unwound. This is not observed when mutant RNAs with altered U5-IR stem-loop structures are substituted for wild-type RNAs. The U5-Leader stem also becomes destabilized when the tRNA primer is annealed to either wild-type or mutant RNA fragments. Nuclease mapping studies of tRNATrp, as well as the viral RNA, indicate that the U5-TΨC helix does form in vitro upon primer annealing. Collectively, these data suggest that the various structural elements near the RSV primer binding site undergo significant changes during the process of primer annealing.

scholarly journals Quantifying antagonistic epistasis in a multifunctional RNA secondary structure of the Rous sarcoma virus

2006 ◽  
Vol 87 (6) ◽  
pp. 1595-1602 ◽  
Author(s):  
Rafael Sanjuán
Keyword(s):  
Secondary Structure ◽  
Rous Sarcoma Virus ◽  
Rna Secondary Structure ◽  
Rna Virus ◽  
Published Data ◽  
Fitness Effects ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Library Selection ◽  
Virus Genomes

Recent studies have suggested that antagonistic epistasis (i.e. mutations having smaller effects in combination than alone) may be common among RNA viruses, in contrast to other biological systems. Here, by re-analysing previously published data from a random viral library, selection and epistasis coefficients were estimated in the U5-IR stem and loop of the Rous sarcoma virus, a region that adopts a conserved secondary structure and is involved in various essential steps of viral infection. The estimated mutational fitness effects are extremely high and genetic interactions are antagonistic on average. This pattern might be representative of RNA virus genomes, which show high compaction and frequent secondary structures. The implications for RNA virus adaptability are explored.

scholarly journals Secondary Structure Analysis of a Minimal Avian Leukosis-Sarcoma Virus Packaging Signal

2000 ◽  
Vol 74 (1) ◽  
pp. 456-464 ◽  
Author(s):  
Jennifer D. Banks ◽  
Maxine L. Linial
Keyword(s):  
Secondary Structure ◽  
In Vivo Studies ◽  
Packaging Signal ◽  
Stem Loop ◽  
Reading Frame ◽  
Rous Sarcoma ◽  
Secondary Structure Analysis ◽  
Short Open Reading Frame ◽  
Sarcoma Virus

ABSTRACT We previously identified a 160-nucleotide packaging signal, MΨ, from the 5′ end of the Rous sarcoma virus genome. In this study, we determine the secondary structure of MΨ by using phylogenetic analysis with computer modeling and heterologous packaging assays of point mutants. The results of the in vivo studies are in good agreement with the computer model. Additionally, the packaging studies indicate several structures which are important for efficient packaging, including a single-stranded bulge containing the initiation codon for the short open reading frame, uORF3, as well as adjacent stem structures. Finally, we show that the L3 stem-loop at the 3′ end of MΨ is dispensable for packaging, thus identifying an 82-nucleotide minimal packaging signal, μΨ, composed of the O3 stem-loop.

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