scholarly journals cis-Acting Elements Important for Retroviral RNA Packaging Specificity

2002 ◽  
Vol 76 (10) ◽  
pp. 4950-4960 ◽  
Author(s):  
Benjamin E. Beasley ◽  
Wei-Shau Hu
Keyword(s):  
Leukemia Virus ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Core Element ◽  
Viral Packaging ◽  
Cis Acting ◽  
Spleen Necrosis ◽  
Flanking Regions ◽  
Packaging Signals

ABSTRACT Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Ψ and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Ψ. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5′-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.

scholarly journals The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

1999 ◽  
Vol 73 (11) ◽  
pp. 9170-9177 ◽  
Author(s):  
Jeanine L. Certo ◽  
Timur O. Kabdulov ◽  
Michelle L. Paulson ◽  
Jeffrey A. Anderson ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Viral Vector ◽  
Leukemia Virus ◽  
Gene Products ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Necrosis Virus ◽  
Spleen Necrosis Virus ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimericgag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Ψ) and another viral vector RNA containing the SNV packaging signal (E). The chimericgag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Ψ. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLVpol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNVcis-acting elements are not ideal substrates for MLVpol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and othercis-acting elements.

scholarly journals Functional Replacement of Nucleocapsid Flanking Regions by Heterologous Counterparts with Divergent Primary Sequences: Effects of Chimeric Nucleocapsid on the Retroviral Replication Cycle

2003 ◽  
Vol 77 (1) ◽  
pp. 754-761 ◽  
Author(s):  
William Fu ◽  
Wei-Shau Hu
Keyword(s):  
Virus Assembly ◽  
Leukemia Virus ◽  
Replication Cycle ◽  
Viral Rna ◽  
Rna Packaging ◽  
Gag Polyprotein ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis ◽  
Flanking Regions

ABSTRACT Nucleocapsid (NC) proteins in most retroviruses have a well-conserved Cys-His box(es) as well as more divergent flanking regions that are rich in basic residues. Mutations in the flanking regions can affect RNA packaging, virus assembly, and reverse transcription of the viral RNA. To gain a further understanding of the roles of NC flanking regions in the retroviral replication cycle, we generated and characterized chimeric gag-pol expression constructs derived from murine leukemia virus and spleen necrosis virus by replacing an NC flanking region from one virus with the counterpart from the other virus. We found that all four chimeras were able to generate virions, package viral RNA, and complete the viral replication cycle. Two chimeras had mild defects in virus assembly that correlated with a decrease in the isoelectric points of NCs, suggesting that the basic nature of NC is important in virus assembly. This finding indicates that, although the primary sequences of these flanking regions have little homology, the heterologous sequences are functional both as part of the Gag polyprotein and as processed NC protein.

scholarly journals RNA Structures and Their Role in Selective Genome Packaging

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1788
Author(s):  
Liqing Ye ◽  
Uddhav B. Ambi ◽  
Marco Olguin-Nava ◽  
Anne-Sophie Gribling-Burrer ◽  
Shazeb Ahmad ◽  
...  
Keyword(s):  
Viral Evolution ◽  
Complex Mixture ◽  
Rna Structures ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Viral Genomes ◽  
Viral Particles ◽  
The Impact ◽  
Packaging Signals

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

scholarly journals Revealing the density of encoded functions in a viral RNA

2015 ◽  
Vol 112 (7) ◽  
pp. 2227-2232 ◽  
Author(s):  
Nikesh Patel ◽  
Eric C. Dykeman ◽  
Robert H. A. Coutts ◽  
George P. Lomonossoff ◽  
David J. Rowlands ◽  
...  
Keyword(s):  
Coat Protein ◽  
Self Assembly ◽  
Rna Virus ◽  
Viral Rna ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Viral Yield ◽  
Recognition Motifs ◽  
Packaging Signals

We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid self-assembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens.

scholarly journals Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

1998 ◽  
Vol 72 (7) ◽  
pp. 5408-5413 ◽  
Author(s):  
Jeanine L. Certo ◽  
Betsy F. Shook ◽  
Philip D. Yin ◽  
John T. Snider ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rna Structures ◽  
Packaging Signal ◽  
Necrosis Virus ◽  
Hairpin Rna ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

scholarly journals Effects of Gag Mutation and Processing on Retroviral Dimeric RNA Maturation

2006 ◽  
Vol 80 (3) ◽  
pp. 1242-1249 ◽  
Author(s):  
William Fu ◽  
Que Dang ◽  
Kunio Nagashima ◽  
Eric O. Freed ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Viral Protein ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Protein Cleavage ◽  
Rna Packaging ◽  
Virus Rna ◽  
Rna Dimer ◽  
Rna Maturation ◽  
Hiv 1

ABSTRACT After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.

Sign in / Sign up

Export Citation Format

Share Document