Spliced Spleen Necrosis Virus Vector RNA Is Not Encapsidated: Implications for Retroviral Replication and Vector Design

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.

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Mapping of receptor binding domains in the envelope protein of spleen necrosis virus.

Journal of Virology ◽

10.1128/jvi.69.7.4339-4346.1995 ◽

1995 ◽

Vol 69 (7) ◽

pp. 4339-4346 ◽

Cited By ~ 7

Author(s):

I Martinez ◽

R Dornburg

Keyword(s):

Receptor Binding ◽

Envelope Protein ◽

Necrosis Virus ◽

Binding Domains ◽

Spleen Necrosis Virus ◽

Spleen Necrosis

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Establishment of infection by spleen necrosis virus: inhibition in stationary cells and the role of secondary infection.

Journal of Virology ◽

10.1128/jvi.41.1.183-191.1982 ◽

1982 ◽

Vol 41 (1) ◽

pp. 183-191 ◽

Cited By ~ 23

Author(s):

I S Chen ◽

H M Temin

Keyword(s):

Secondary Infection ◽

Necrosis Virus ◽

Virus Inhibition ◽

Spleen Necrosis Virus ◽

Spleen Necrosis

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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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