scholarly journals Importance of Basic Residues in Binding of Rous Sarcoma Virus Nucleocapsid to the RNA Packaging Signal

2003 ◽  
Vol 77 (3) ◽  
pp. 2010-2020 ◽  
Author(s):  
Eun-gyung Lee ◽  
Annie Alidina ◽  
Cynthia May ◽  
Maxine L. Linial
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Rous Sarcoma ◽  
Charged Residues ◽  
Sarcoma Virus ◽  
Positively Charged

ABSTRACT In the context of the Rous sarcoma virus Gag polyprotein, only the nucleocapsid (NC) domain is required to mediate the specificity of genomic RNA packaging. We have previously showed that the Saccharomyces cerevisiae three-hybrid system provides a rapid genetic assay to analyze the RNA and protein components of the avian retroviral RNA-Gag interactions necessary for specific encapsidation. In this study, using both site-directed mutagenesis and in vivo random screening in the yeast three-hybrid binding assay, we have examined the amino acids in NC required for genomic RNA binding. We found that we could delete either of the two Cys-His boxes without greatly abrogating either RNA binding or packaging, although the two Cys-His boxes are likely to be required for efficient viral assembly and release. In contrast, substitutions for the Zn-coordinating residues within the boxes did prevent RNA binding, suggesting changes in the overall conformation of the protein. In the basic region between the two Cys-His boxes, three positively charged residues, as well as basic residues flanking the two boxes, were necessary for both binding and packaging. Our results suggest that the stretches of positively charged residues within NC that need to be in a proper conformation appear to be responsible for selective recognition and binding to the packaging signal (Ψ)-containing RNAs.

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 Yeast Three-Hybrid Screening of Rous Sarcoma Virus Mutants with Randomly Mutagenized Minimal Packaging Signals Reveals Regions Important for Gag Interactions

2000 ◽  
Vol 74 (19) ◽  
pp. 9167-9174 ◽  
Author(s):  
Eun-Gyung Lee ◽  
Maxine L. Linial
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Binding ◽  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Genetic Assay

ABSTRACT We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MΨ) made using PCR amplification after cotransformation with GagΔPR protein into yeast cells. Colonies with low β-galactosidase activity were analyzed to locate mutations in MΨ sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.

scholarly journals Importance of Basic Residues in Binding of Rous Sarcoma Virus Nucleocapsid to the RNA Packaging Signal

2003 ◽  
Vol 77 (7) ◽  
pp. 4468-4468
Author(s):  
Eun-gyung Lee ◽  
Annie Alidina ◽  
Cynthia May ◽  
Maxine L. Linial
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Rna Packaging Signal

scholarly journals Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 568 ◽  
Author(s):  
Shuohui Liu ◽  
Rebecca Kaddis Maldonado ◽  
Tiffiny Rye-McCurdy ◽  
Christiana Binkley ◽  
Aissatou Bah ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rous Sarcoma Virus ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Gag Polyprotein ◽  
Interaction Sites ◽  
Rous Sarcoma ◽  
Sarcoma Virus

Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions.

scholarly journals An MΨ-Containing Heterologous RNA, but Notenv mRNA, Is Efficiently Packaged into Avian Retroviral Particles

1999 ◽  
Vol 73 (11) ◽  
pp. 8926-8933 ◽  
Author(s):  
Jennifer D. Banks ◽  
Bonnie O. Kealoha ◽  
Maxine L. Linial
Keyword(s):  
Splice Site ◽  
Rous Sarcoma Virus ◽  
Full Length ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Subgenomic Rnas ◽  
Rous Sarcoma ◽  
Virus Rna ◽  
Sarcoma Virus ◽  
Packaging Efficiency

ABSTRACT Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MΨ, is located upstream of the 5′ splice site and therefore is present on both genomic and spliced RNAs. We now show that an MΨ-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MΨ. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MΨ is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3′ end of MΨ was found to reduce the packaging efficiency of heterologous RNAs.

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 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.

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