Investigation of the novel proteins of Influenza B
Author(s)
Elderfield, Ruth Alice
Type
Thesis
Abstract
Influenza B viruses encode two small proteins, NB and BM2. BM2 is translated from segment 7 mRNAs by a mechanism involving sequence complementarity to the 18S ribosomal subunit. The importance of these complementary sequences was tested in the context of infectious virus using a reverse genetic approach. A series of mutations were introduced into this region of segment 7. Recombinant viruses with disrupted 18S complementarity displayed deficiency in BM2 expression in infected cells.
The BM2 protein is essential for virus replication because its ion channel activity is required during virion entry to the cell. There is also evidence that the cytoplasmic tail of BM2 is involved in viral assembly. A series of amino acid truncations and substitutions in the BM2 cytoplasmic tail were engineered. Recombinant viruses that lacked more than 5 residues at the carboxyl terminus of the protein were not recovered and key residues in the region -5 to -10 were identified.
The influenza B virus RNA segment 6 encodes the neuraminidase protein, NA, as well as NB. NB is a 100 amino acid transmembrane protein with a glycosylated ectodomain. NB is conserved in all natural influenza B virus isolates. Influenza B viruses that lack the NB protein can replicate in cell culture to wild-type levels, However, the deletant viruses showed attenuated growth in complex airway cultures derived from humans and ferrets. In vivo, infected ferrets excreted infectious virus in the nasal wash one day later than for viruses that encode NB. Alterations in the expression of the NA protein were not responsible for the attenuated phenotype shown by NB deletant viruses.
The role of the host cell ESCRT pathway or of the interferon-induced tetherin protein in assembly and release of influenza viruses was assessed. No evidence was found for either host pathway in the replication of influenza viruses.
The BM2 protein is essential for virus replication because its ion channel activity is required during virion entry to the cell. There is also evidence that the cytoplasmic tail of BM2 is involved in viral assembly. A series of amino acid truncations and substitutions in the BM2 cytoplasmic tail were engineered. Recombinant viruses that lacked more than 5 residues at the carboxyl terminus of the protein were not recovered and key residues in the region -5 to -10 were identified.
The influenza B virus RNA segment 6 encodes the neuraminidase protein, NA, as well as NB. NB is a 100 amino acid transmembrane protein with a glycosylated ectodomain. NB is conserved in all natural influenza B virus isolates. Influenza B viruses that lack the NB protein can replicate in cell culture to wild-type levels, However, the deletant viruses showed attenuated growth in complex airway cultures derived from humans and ferrets. In vivo, infected ferrets excreted infectious virus in the nasal wash one day later than for viruses that encode NB. Alterations in the expression of the NA protein were not responsible for the attenuated phenotype shown by NB deletant viruses.
The role of the host cell ESCRT pathway or of the interferon-induced tetherin protein in assembly and release of influenza viruses was assessed. No evidence was found for either host pathway in the replication of influenza viruses.
Date Issued
2010
Date Awarded
2010-06
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Barclay, Wendy
Sponsor
BBSRC
Creator
Elderfield, Ruth Alice
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)