Structure and Assembly of the Sindbis Virus E1 and E2 Transmembrane Proteins

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Title: Structure and Assembly of the Sindbis Virus E1 and E2 Transmembrane Proteins
Author: Whitehurst, Christopher Benton
Advisors: Dennis Brown, Committee Chair
Robert Rose, Committee Member
John Cavanagh, Committee Member
John Mackenzie, Committee Member
Douglas Lyles, Committee Member
Abstract: Sindbis virus is composed of two nested T=4 icosahedral protein shells containing 240 copies each of three structural proteins: E1, E2, and Capsid in a 1:1:1 stoichiometric ratio. E2 is a 423 amino acid glycoprotein with a membrane spanning domain 26 amino acids in length. A previous study had determined that deletions in the transmembrane domain could affect virus assembly and infectivity (Hernandez et al, 2003 J.Virol 77(23), 12710-9). Unexpectedly, a single deletion mutant (from 26 to 25 amino acids) resulted in a 1000-fold decrease in infectious virus production while another deletion of eight amino acids had no affect on virus production. To further investigate the importance of these mutants, other single deletion mutants and another eight amino acid deletion mutant were constructed. We found that deletions located closer to the cytoplasmic (inner) leaflet of the membrane bilayer had a more detrimental effect on virus assembly and infectivity than those located closer to the luminal (outer) leaflet of the membrane bilayer. We also found that selective pressure can restore single amino acid deletions in the transmembrane domain but not necessarily to the wild type sequence. These results suggest the position of the deletion and the length of the C terminal region of the E2 transmembrane domain are vital for normal virus production. Deletion mutants resulting in decreased infectivity, produce particles that appear to be processed and transported correctly suggesting a role involved in virus entry. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of intermediates as virus maturation occurs. E1 and E2 cysteine residues were labeled with iodoacetamide in the native virus particle and analyzed by mass spectrometry. This analysis identified cysteines of E1 and E2 that were found to be free in the native virus particle as well as those that were either solvent inaccessible or blocked by their involvement in disulfide bonds. Native virus labeled with iodoacetamide yielded a four log decease in viral infectivity. This suggests either the bound iodoacetamide alone may be involved in the loss of infectivity by destabilizing the virus particle or a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the presence of iodoacetamide. In addition, cysteines that were determined to be free in the E1 glycoprotein were mutated to serine. Mutation of these cysteines greatly decreased the amount of infectious virus production suggesting a disulfide bond rearrangement may occur as virus maturation proceeds.
Date: 2007-05-17
Degree: PhD
Discipline: Biochemistry
URI: http://www.lib.ncsu.edu/resolver/1840.16/4756


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