Packaging of heterologous RNAs by a minimal bovine leukemia virus RNA packaging signal into virus particles

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.

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Inhibition of Hepadnavirus Reverse Transcriptase-ε RNA Interaction by Porphyrin Compounds

Journal of Virology ◽

10.1128/jvi.02147-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2305-2312 ◽

Cited By ~ 37

Author(s):

Li Lin ◽

Jianming Hu

Keyword(s):

Reverse Transcriptase ◽

Protoporphyrin Ix ◽

Rna Packaging ◽

Packaging Signal ◽

B Virus ◽

Protein Priming ◽

Rna Interaction ◽

Iron Protoporphyrin ◽

Rna Packaging Signal ◽

Viral Reverse Transcription

ABSTRACT The hepatitis B virus (HBV) reverse transcriptase (RT) plays a multitude of fundamental roles in the viral life cycle and is the key target in the development of anti-HBV chemotherapy. We report here that the endogenous small molecule iron protoporphyrin IX (hemin) and several related porphyrin compounds potently blocked a critical RT interaction with the viral RNA packaging signal/origin of replication, called ε. As RT-ε interaction is essential for the initiation of viral reverse transcription, which is primed by RT itself (protein priming), the porphyrin compounds dramatically suppressed the protein-priming reaction. Further studies demonstrated that these compounds could target the unique N-terminal domain of the RT protein, the so-called terminal protein. Hemin and related porphyrin compounds thus represent a novel class of agents that can block HBV RT functions through a mechanism and target that are completely distinct from those of existing anti-HBV drugs.

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Solution Structure of the Rous Sarcoma Virus Nucleocapsid Protein: μΨ RNA Packaging Signal Complex

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.10.013 ◽

2007 ◽

Vol 365 (2) ◽

pp. 453-467 ◽

Cited By ~ 33

Author(s):

Jing Zhou ◽

Rebecca L. Bean ◽

Volker M. Vogt ◽

Michael Summers

Keyword(s):

Rous Sarcoma Virus ◽

Nucleocapsid Protein ◽

Solution Structure ◽

Rna Packaging ◽

Packaging Signal ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Rna Packaging Signal

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Involvement of the Matrix and Nucleocapsid Domains of the Bovine Leukemia Virus Gag Polyprotein Precursor in Viral RNA Packaging

Journal of Virology ◽

10.1128/jvi.77.17.9431-9438.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9431-9438 ◽

Cited By ~ 37

Author(s):

Huating Wang ◽

Kendra M. Norris ◽

Louis M. Mansky

Keyword(s):

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Viral Rna ◽

Limited Information ◽

Rna Packaging ◽

Alanine Scanning Mutagenesis ◽

Basic Residue ◽

Gag Polyprotein ◽

Human T Cell ◽

Polyprotein Precursor

ABSTRACT The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.

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High Affinity Nucleocapsid Protein Binding to the μΨ RNA Packaging Signal of Rous Sarcoma Virus

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.04.046 ◽

2005 ◽

Vol 349 (5) ◽

pp. 976-988 ◽

Cited By ~ 26

Author(s):

Jing Zhou ◽

John K. McAllen ◽

Yogita Tailor ◽

Michael F. Summers

Keyword(s):

Protein Binding ◽

Rous Sarcoma Virus ◽

Nucleocapsid Protein ◽

Rna Packaging ◽

Packaging Signal ◽

High Affinity ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Rna Packaging Signal

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

Journal of Virology ◽

10.1128/jvi.73.11.9170-9177.1999 ◽

1999 ◽

Vol 73 (11) ◽

pp. 9170-9177 ◽

Cited By ~ 17

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.

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