Dissertation

The protein determinants for RNA packaging in avian sarcoma and leukemia virus

Sakalian, Michael

1994

Overview

In all retrovirus systems studied, the leader region of the RNA contains a cis-acting sequence called $\\Psi$ 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 $\\Psi$. The nucleocapsid protein (NC) of avian sarcoma and leukemia viruses (ASLV) is found tightly associated with genomic RNA in virions. I examined the binding properties of the ASLV NC protein in a gel mobility-shift assay. NC binding to RNA produced a high molecular weight complex that could be progressively increased in size by further addition of NC. In agreement with previous reports, the NC-RNA interaction was nonspecific. Comparison of the gel mobility-shift with filter-binding showed that filter-binding is not a valid assay for NC-RNA interaction. NC is initially produced as a part of the Gag polyprotein precursor and is not released in its mature form until after virus assembly and proteolytic maturation. To gain information about which portion(s) of Gag, in addition to NC, are required for RNA packaging in the ASLV, I examined a series of partial gag deletion mutants that still can lead to assembly of virus-like particles. The incorporation of RNA into these particles was assayed by RNase protection. The efficiency of packaging was determined by normalization of the amount of $\\Psi\\sp+$ RNA to the amount of Gag protein released in virus-like particles. Specificity of packaging was determined by comparisons of $\\Psi\\sp+$ and $\\Psi\\sp-$ RNA in particles and in cells. The results indicate that much of Gag is unnecessary for both efficient and specific packaging. Deletions within the NC domain reduced both the efficiency and specificity of packaging or eliminated it entirely. Among mutants that retained the ability to package, a deletion within the CA domain (which includes the Major Homology Region) was the least efficient. I also examined particles of the packaging mutant SE21Q1b. The data suggest that the random RNA packaging behavior of this mutant is not due to a specific defect but rather results from the cumulative effect of point mutations throughout the gag gene.

• Publisher: ProQuest Dissertations & Theses
• Subject: Molecular biology
• ISBN: 9798535581910