Identification of a high affinity nucleocapsid protein binding element within the moloney murine leukemia virus Ψ-RNA packaging signal: implications for genome recognition

ABSTRACT Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops.

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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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