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

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.

Download Full-text

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

Viruses ◽

10.3390/v12050568 ◽

2020 ◽

Vol 12 (5) ◽

pp. 568 ◽

Cited By ~ 2

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.77.3.2010-2020.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2010-2020 ◽

Cited By ~ 22

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.

Download Full-text

In vitro synthesis of full-length DNA transcripts of Rous sarcoma virus RNA by viral DNA polymerase.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.72.12.4895 ◽

1975 ◽

Vol 72 (12) ◽

pp. 4895-4899 ◽

Cited By ~ 24

Author(s):

R. P. Junghans ◽

P. H. Duesberg ◽

C. A. Knight

Keyword(s):

Dna Polymerase ◽

Rous Sarcoma Virus ◽

Full Length ◽

Viral Dna ◽

In Vitro Synthesis ◽

Rous Sarcoma ◽

Virus Rna ◽

Sarcoma Virus

Download Full-text

Retroviral Splicing Suppressor Requires Three Nonconsensus Uridines in a 5′ Splice Site-Like Sequence

Journal of Virology ◽

10.1128/jvi.75.16.7763-7768.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7763-7768 ◽

Cited By ~ 11

Author(s):

Robert E. Paca ◽

Catherine S. Hibbert ◽

Christopher T. O'Sullivan ◽

Karen L. Beemon

Keyword(s):

Splice Site ◽

Rous Sarcoma Virus ◽

Negative Regulator ◽

Rous Sarcoma ◽

Virus Rna ◽

U1 Snrna ◽

Sarcoma Virus ◽

Site Consensus

ABSTRACT Rous sarcoma virus RNA contains a negative regulator of splicing (NRS) element that aids in maintenance of unspliced RNA. The NRS binds U1 snRNA at a sequence that deviates from the 5′ splice site consensus by substitution of U's for A's at three positions: −2, +3, and +4. All three of these U's are important for NRS-mediated splicing suppression. Substitution of a single nonconsensus C or G at any of these sites diminished NRS activity, whereas substitution of a single A generated a preferred 5′ splice site within the NRS.

Download Full-text

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing

Molecular and Cellular Biology ◽

10.1128/mcb.5.9.2298-2306.1985 ◽

1985 ◽

Vol 5 (9) ◽

pp. 2298-2306

Author(s):

S E Kane ◽

K Beemon

Keyword(s):

Rous Sarcoma Virus ◽

Rna Splicing ◽

Paper Electrophoresis ◽

Virus Genome ◽

Rous Sarcoma ◽

Virus Rna ◽

Dna Restriction Fragments ◽

Sarcoma Virus ◽

Dna Restriction ◽

Layer Chromatography

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

Download Full-text

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.

Download Full-text