Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

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.

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Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging

Journal of Virology ◽

10.1128/jvi.00101-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6790-6797 ◽

Cited By ~ 45

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.

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

Nucleic Acids Research ◽

10.1093/nar/6.2.471 ◽

1979 ◽

Vol 6 (2) ◽

pp. 471-486 ◽

Cited By ~ 8

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

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

Journal of Virology ◽

10.1128/jvi.75.1.260-268.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 260-268 ◽

Cited By ~ 25

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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