Doctoral Dissertations

Date of Award

8-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David L. Hacker

Committee Members

Jeffrey Becker, David Brian, Barry Bruce, Brad Reddick

Abstract

Southern cowpea mosaic virus (SCPMV) is a spherical RNA virus with T=3 icosahedral symmetry. The particle is composed of 180 subunits of the CP and one copy of the viral RNA. The CP has two domains, the random (R) domain formed by the N-terminal 64 amino acids (aa) of the CP and the shell (8) domain (aa 65-260). The R domain is highly basic in charge and is localized to the interior of the particle where it is proposed to interact with the viral RNA. The R domain may also interact with membranes since it has been previously shown that the SCPMV CP binds liposomes in the presence but not in the absence of the R domain.

The RNA binding site of the R domain was identified by northwestern blot and by electrophoretic mobility shift assay (EMSA) using recombinant wild-type R domain protein (rWTR) that included aa 1-57 of the SCPMV CP and a C-terminal non-viral extension that contained two histidine tags and an S peptide tag (His-S tag domain). Deletions within the R domain revealed that the N-terminal 30 aa function in RNA binding. The R domain-RNA interaction was nonspecific with regard to RNA sequence and was sensitive to high salt concentrations suggesting that the interaction is predominantly electrostatic. The RNA binding site includes eleven basic residues, eight of which are located in the arginine-rich region between aa 22- 30. It was demonstrated using alanine substitution mutants that the basic residues of the arginine-rich region but not those present at positions 3, 4, and 7 are necessary for RNA binding. None of the basic residues within the arginine-rich region are specifically required for RNA binding, but the overall charge of the N-terminal 30 aa is important. Proline substitution mutations within the N-terminal 30 aa, and alanine substitutions for prolines at positions 18, 20, and 21 did not affect the RNA binding activity of the R domain. However, it was demonstrated by circular dichroism (CD) that the conformation of the N-terminal 30 aa of the R domain changes from a random coil to an a-helix in the presence of 50% trifluoroethanol (TFE).

The ability of rWTR to interact with artificial membrane was also analyzed in vitro using a dye release assay. The results demonstrated that the R domain interacts with negatively charged but not neutral liposomes. Deletion of the N-terminal 16 aa of the R domain nearly abolished the membrane interaction activity. In addition, aa 17-30 were also shown to be important for the activity. The interaction was dependent upon the presence of phosphatidic acid (PA) or phosphatidylethanolamine (PE). The interaction was sensitive to high salt and acidic pH indicating that electrostatic interactions are involved in the liposome-R domain interaction. CD analysis and dye release assays of mutants with proline to alanine or alanine to proline substitutions within the N-terminal 30 residues of the R domain demonstrated a positive correlation between the extent of a-helical content in this region and the ability to lyse liposomes. A synthetic peptide (CP peptidei.30) corresponding to the N-terminal 30 aa of the SCPMV CP was also able to induce lysis of liposomes. The peptide underwent a conformational change from a random coil to an a-helix in the presence of negatively charged liposomes suggesting a possible role of protein secondary structure in the membrane interaction. These experiments demonstrated that the R domain of the SCPMV CP can bind RNA and interact with membranes. The roles of these activities in the SCPMV life cycle are discussed.

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