Bruzzone, R

Chu, SL

Lam, CH

Lam, YM

Nal, B

Tang, DJ

BMC Proceedings

English

PubMed

DJ Tang

YM Lam

CH Lam

SL Chu

R Bruzzone

B Nal

Springer - Publisher Connector

POSTER PRESENTATION Open Access
Mutational analysis of H5N1 hemagglutinins:
identification of molecular determinants for
efficient packaging into pseudotyped lentiviral
particles
DJ Tang*, YM Lam, CH Lam, SL Chu, R Bruzzone, B Nal
From Institut Pasteur International Network Annual Scientific Meeting
Hong Kong. 22-23 November 2010
Due to the high pathogenicity of H5N1 viruses, we have
developed and characterized lentiviral particles pseudo-
typed with HA (H5pp) from a Cambodia H5N1 isolate,
which can be used as a safe tool for high-throughput
serological studies without the requirement of BSL-3
facilities. However, not all H5 HAs give rise to efficient
production of H5pp. The main objective of this study is
to understand the intrinsic properties of H5cam and
H5anh (derived from A/Cambodia/2005/40808 and A/
Anhui/2005/01 respectively) which exhibited a dramatic
difference in their abilities to generate H5pp; and to
identify molecular determinants that control the assem-
bly and release of H5 pseudotyped lentiviral particles.
H5cam and H5anh both exhibited high level protein
expression in 293T cells. Although the cleavage of
H5cam appeared to be slightly better, the level of
cleaved HA2 was comparable between H5cam and
H5anh. Next, flow cytometry analysis was used to com-
pare surface HA expression for H5cam and H5anh.
Indeed surface expression of H5cam was significantly
higher than that of H5anh.
A deletion of lysine residue was found at the cleavage
site of H5anh when compared with H5cam. Swapping of
HA2 domain (including the cleavage site) did not
improve H5anh-pp production. Of notice, 8 AA residues
were found different for H5cam and H5anh at the 130-
loop and franking region of receptor binding domain
(RBD). A series of H5anh mutants at the 130-loop
region were generated for mutational analysis. Strikingly,
all H5anh mutants with alanine to valine mutation at
position 134, despite other sequence differences at 130-
loop flanking region, largely restored the ability of
H5anh to pseudotype lentiviral vector.
In conclusion, H5cam and H5anh showed similar level
of protein expression in total cell lysates when trans-
fected into 293T cells. However surface expression of
H5cam was detected at a higher level than that of
H5anh. This may partially explain the inability of H5anh
to produce H5pp. Site-directed mutagenesis revealed
that a single valine residue at position 134 of the 130-
loop of RBD is critical for cell surface association of H5
HA and hence efficient H5pp production in 293T cells.
It is likely that H5 HAs with alanine134 also confer
higher receptor binding affinity; and consequently H5pp
release from the producer cells is less efficient.
Published: 10 January 2011
doi:10.1186/1753-6561-5-S1-P69
Cite this article as: Tang et al.: Mutational analysis of H5N1
hemagglutinins: identification of molecular determinants for efficient
packaging into pseudotyped lentiviral particles. BMC Proceedings 2011 5
(Suppl 1):P69.
HKU-Pasteur Research Centre, Hong Kong, Hong Kong SAR
Tang et al. BMC Proceedings 2011, 5(Suppl 1):P69
http://www.biomedcentral.com/1753-6561/5/S1/P69
© 2011 Tang et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.


Mutational analysis of H5N1 hemagglutinins: identification of molecular determinants for efficient packaging into pseudotyped lentiviral particles

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Mutational analysis of H5N1 hemagglutinins: identification of molecular determinants for efficient packaging into pseudotyped lentiviral particles

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