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 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 In Vivo Selection of Rous Sarcoma Virus Mutants with Randomized Sequences in the Packaging Signal

1998 ◽  
Vol 72 (10) ◽  
pp. 8073-8082 ◽  
Author(s):  
Nicole A. Doria-Rose ◽  
Volker M. Vogt
Keyword(s):  
Rous Sarcoma Virus ◽  
Secondary Structure Prediction ◽  
Selection Strategy ◽  
Single Mutation ◽  
Base Pairing ◽  
Packaging Signal ◽  
In Vivo Selection ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Retrovirus genomes contain a sequence at the 5′ end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Ψ, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Ψ and transfected each pool into avian cells. Resulting infectious virus was allowed to spread by multiple passages, so that sequences could compete and the best could be selected. This method provides information on the kinds of sequences allowed, as well as those that are most fit. Several predicted stem-loop structures in Ψ were tested. A stem at the base of element O3 was highly favored; only sequences which maintained base pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the randomized region in all selected L3 stem mutants; we interpret this to mean that it compensates for the defects in L3. Randomized mutations adjacent to G213 maintained the wild-type base composition but not its sequence. The kissing-loop sequence at end of L3, postulated to function in genome dimerization, was not required for infectivity but was selected for over time. Finally, a deletion of L3 was constructed and found to be poorly infectious.

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.

Sign in / Sign up

Export Citation Format

Share Document