Because diagnostic tests for highly pathogenic avian influenza (HPAI) viruses require
the use of replication-competent viruses in a biosafety level 3 containment, numerous
studies have looked at ways to develop alternative tests. Lentiviral particles
pseudotyped with H5 hemagglutinin (HA), the surface glycoprotein of influenza virus,
have been described as useful and safe tools for research and serological surveillance
on the HPAI viruses. However, not all H5 HA give rise to efficient H5 pseudotyped
lentiviral particles (H5pp) production. HA from A/Cambodia/408008/05 H5N1
(H5Cam) and HA from A/Anhui/1/05 H5N1 (H5Anh) exhibit a dramatic difference in
their ability to pseudotype lentiviral particles. H5Cam gives the highest H5pp
production among all HAs tested, whereas the lowest has been observed with H5Anh.
The objective of this study was to investigate the molecular determinants that govern
efficient H5pp production.
Based on the amino acid differences between H5Cam and H5Anh, H5Anh mutants
were generated by site-directed mutagenesis. Strikingly, a single amino acid change,
A134V, in the 130-loop receptor-binding domain of HA, significantly increased H5pp
production with H5Anh. The finding that valine 134 is crucial for H5pp production was
confirmed by reciprocal H5Cam and H5Anh mutants, which displayed either a
dramatic decrease or increase in H5pp production, respectively.
Influenza virus and H5pp bud at the plasma membrane, therefore changes in HA cell
surface expression could affect the production of H5pp. Thus, cell surface expressions
of H5Cam and H5Anh were compared by flow cytometry. Intriguingly, H5Cam
displayed a higher plasma membrane expression than H5Anh, suggesting that transport
is important for H5pp production. Introduction of V134A mutation in H5Cam reduced
its surface expression to that of H5Anh; by contrast, H5Anh mutant harboring A134V
mutation largely restored its expression.
Next the effect of A134V mutation on the binding of HA to sialic acid receptors was
investigated. A cell-based Enzyme-linked Immunosorbent Assay was developed to
measure binding of wild-type and mutated HA. Soluble recombinant proteins were
produced by mammalian cells stably transfected with HA gene ectodomain and were
mostly trimeric as indicated by discontinuous native gel electrophoresis. Interestingly,
H5Anh proteins exhibited a stronger binding to MDCK cells than H5Cam proteins, and
introduction of A134V mutation in H5Anh proteins reduced the binding. By contrast,
as predicted, the reciprocal V134A mutation induced a major increase in binding to
cellular receptors. It is likely that stronger binding of H5Anh to sialic acids could
hinder the release of H5pp. Consistent with this notion, the ability of H5Anh to
generate H5pp was significantly increased in a sialylation deficient Lec2 cell, a CHO
mutant cell line.
In conclusion, H5Cam allows efficient H5pp production whereas H5Anh does not.
With several lines of evidence, it is likely that the behavior of H5Anh can be explained
by a stronger binding to sialic acid receptors that is dependent on a single amino acid
residue at position 134. Since A134V is a naturally occurring mutation observed
occasionally in human host, these results may have implications for the understanding
of human host adaptations of H5N1 viruses. / published_or_final_version / Biochemistry / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/146146 |
| Date | January 2011 |
| Creators | Lam, Yuen-man, 林婉雯 |
| Contributors | Bruzzone, R, Jin, D |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47251360 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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