Efficiency and selectivity of RNA packaging by rous sarcoma virus gag deletion mutants

Michael Sakalian, John Wills, Volker M. Vogt

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Abstract

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called Ψ that is required for packaging the viral RNA genome. Since the pol and env genes are dispensable for formation of RNA- containing particles, the gag gene product must have an RNA binding domain(s) capable of recognizing Ψ. To gain information about which portion(s) of Gag is required for RNA packaging in the avian sarcoma and leukemia virus system, we utilized a series of gag deletion mutants that retain the ability to assemble virus-like particles. COS cells were cotransfected with these mutant DNAs plus a tester DNA containing Ψ, and incorporation of RNA into particles was measured by RNase protection. The efficiency of packaging was determined by normalization of the amount of Ψ+ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of Ψ+ and Ψ- RNA in particles and in cells. The results indicate that much of the MA domain, much of the p10 domain, half of the CA domain, and the entire PR domain of Gag are unnecessary for efficient packaging. In addition, none of these deleted regions is needed for specific selection of the Ψ RNA. Deletions within the NC domain, as expected, reduce or eliminate both the efficiency and the specificity of packaging. Among mutants that retain the ability to package, a deletion within the CA domain (which includes the major homology region) is the least efficient. We also examined particles of the well-known packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather is the result of the cumulative effect of many point mutations throughout the gag gene.

Original languageEnglish (US)
Pages (from-to)5969-5981
Number of pages13
JournalJournal of virology
Volume68
Issue number9
Publication statusPublished - Sep 1 1994

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All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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