Human parainfluenza virus type 3 (HPIV3), a member of the family Paramyxoviridae, is an enveloped, negative-sense single stranded RNA virus, and an important cause of upper and lower respiratory tract disease in children. Infection by HPIV3 requires fusion of the viral envelope with the plasma membrane of the host cell. Fusion occurs via the cooperative actions of the viral hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. HN, the receptor-binding protein, binds sialic acid and undergoes a series of conformational changes that are believed to trigger characteristic conformational changes in the F protein that are necessary for membrane fusion. The specific objectives of this dissertation are to elucidate, in greater detail, features of HN-F interaction and F refolding during the entry process.
Mutations were introduced at position I474 of the F protein, in heptad repeat 2 (HR2), a carboxy-proximal sequence that forms a highly stable structure with the amino-proximal heptad repeat 1 (HR1), known as the 6-helix bundle (6-HB). Formation of the 6-HB is believed to release energy required for fusion of virion and host cell membranes. Mutation of I474 to small, polar residues produced F proteins that are uncleaved and non-fusogenic; 6-HB formed by peptides based these mutants were thermodynamically unstable. Mutation of I474 to larger polar or nonpolar side chains, or to smaller polar side chains, resulted in F proteins that were cleaved and fusogenic to wild-type levels, although 6-HB stability was variable. The ability of HR2 peptides to inhibit virus infection and glycoprotein-driven fusion mirrored 6-HB stability. Tagging of HN with FLAG and hexahistidine tags permitted Ni-affinity purification of HN. Cotransfection of HN and F, including F I474D, S, and E mutants, showed that stable HN-F complexes could be isolated when cell membranes were solubilized with octyl glucoside. A distinct downregulation of HN expression was observed when HN and F were coexpressed in cells, likely as a co-translational or very early post-translational step.
A series of three-part chimeric F sequences consisting of fragments of HPIV3 and bovine PIV3 F coding regions were constructed. When expressed in the presence of the HPIV3 HN protein, the chimeric F proteins showed no differences in level of expression or fusogenicity. Also, there were no differences in the stability of homologous and heterologous HR1/HR2 complexes, and inhibition of HPIV3 infection by HR2 peptides of the HPIV3 and BPIV3 F proteins was similar.
HPIV3 resistant to inhibition by HR2 was produced by sequential passage of virus in increasing concentrations of a GST-HR2 fusion protein. Statistically significant resistance of populations as well as plaque-purified clones was demonstrated, although sequencing of resistant clones did not reveal mutations that could be clearly associated with resistance.
A model for HN and F in the fusion process is presented, based on data produced in this dissertation and on previously published evidence.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/29547 |
| Date | January 2008 |
| Creators | McKenna, Neil |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 152 p. |
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