In this poster, we describe a set of mutations engineered into an H9 wild isolate strain of influenza from seals.

1. Investigating the effect of natural variation on an unusual H9 wild isolate strain’s viral fitness
Islam Hussein1
, Brandt Meixell, Nichola J. Hill1
, Eric J. Ma1
, Jonathan Runstadler1,2
1
Department of Biological Engineering, MIT; 2
Division of Comparative Medicine, MIT
Future Directions• Testing more combinations, such as D253N
• Testing the activity of PB2 mutants in avian cell line (DF-1)
• Testing the in vitro and in vivo profiles of PB2 viral mutants engineered
by reverse genetics
Results Synopsis• 8BM3470 (8BM)viruswasgeneratedasaresultofareassortmentevent
that involved H9N2, H5N2, H7N3 and probably other viruses.
• Phylogenetic analysis of all 8 segments of the 8BM virus revealed that
the PB2 segment was donated from an H5N2 virus (A/snow goose/
Montana/466771-4/2006).
• H5N2PB2segmentacquisitionslightlyincreased8BMH9N2polymerase
activity when compared with a non-reassortant H9N2 strain.
• Amino acid residue # 591 is an important determinant of H9N2 PB2
function.
• Having a basic residue (R or K) at this position significantly increases
polymerase activity; K591 has been recorded in naturally circulating
H9N2 viruses in China and Saudi Arabia.
• Homology modeling indicates that replacing Q (neutral) with a
positively charged R or K changes the electrostatic surface at those
residues significantly, and might increase the binding affinity to other
interacting partners.
• Combining Q591K with E627K doesn’t offer any synergistic effect to
H9N2 polymerase function.
8BM3470 is likely a triple reassortant between H5N2, H9N2 and H7N3
Certain basic amino acids greatly enhance polymerase activity
A/egret/Hunan/1/2012 (H9N2)
A/northern shoveler/Interior Alaska/8B3470/2009 (H9N2)
A/snow goose/Montana/46671-4/2006 (H9N2)
A/snow goose/Quebec/17416/2006 (H9N2)
Virus isolation sites
Northern shoveler
Snow goose
Legend
Global Information Systems AnalysisPhylogenetic Analysis and Affinity Propagation Clustering
MO/06/H5N2
KO/07/H5N2OH/02/H6N2
MD/02/H6N2
MN/99/H4N6
NP PA
BC/07/H7N3
AL/07/mixed
AL/08/H3N8
AL/07/H4N6
AL/07/H1N1
AL/08/__N6
CA/08/H7N3 AL/08/H4N1
CA/08/H1N1
AL/08/H1N1
AL/08/H4N6
AB/09/H3N8
PB1
CA/08/H5N3
AB/08/H1N3
CA/08/H7N3AL/07/H7N3
CA/09/H10N7
CA/08/H2N8
CA/07/H7N3 CA/08/H4N6
PB2
MO/06/H5N2
QC/06/H9N2
Reassortment between two or more viruses is an
important mechanism for generating new viral
subtypeswithnovelinternalandexternalcomponents.
Introduction
Wild birds are the natural reservoirs of
avian influenza viruses, and can transmit
them to mammalian species.
The influenza virus is composed of 8 RNA
genome segments encoding 11 proteins.
The polymerase complex (RNP) is comprised of 4 of the 11 proteins, namely,
Nucleoprotein (NP), Polymerase Basic 1 and 2 (PB1 and PB2), and Polymerase Acidic
(PA). The RNP is an important determinant in host specificity and viral replication rate.
Fig. 1: Affinity propagation clustering (top) and phylogenetic analysis (bottom) provided a clue to the origins of this virus. Top:
Center cluster circles represent 8BM’s amino acid sequence; outer circles represent co-clustered sequences; black colored circles
are the most genetically related as represented in the phylogenetic tree. Bottom: Maximum-likelihood tree of each protein’s
nucleotide sequence, with 8BM highlighted. Only branches corresponding to the clusters on top are shown.
Fig. 2: GIS analysis on the Northern Shoveler and the Snow Goose indicated
that they intersect, lending credence to the reassortment hypothesis.
Identification of amino acids to test Polymerase Assay Design and Results
T S I A D M R M G P
A D T T N I Q V S L
106 107 147 247 390 473 508 575 590 679
8BM3470
Consensus
Fig. 3: Based on a multiple sequence alignment with 4 other closely-related strains, 10 amino acid changes
away from consensus were identified. We hypothesized that the reassortment event conferred these changes.
Fig. 4: Yamada et. al. (2010) characterized the
electrostatic surface of the PB1 protein from
(A) H1N1 (PDB: 3KHW) and (B) H5N1 (PDB:
3KC6), finding that E627K compensates for the
change S590G and R591Q. Figure from original
paper.
590
...FQSLVPKAARSQYSGFVRTLF... 8BM3470
...FQSLVPKAARGQYSGFVRTLF... WT (majority H9 strains)
...FQSLVPKAARSQYSGFVRTLF... Polymorphism
...FQSLVPKAARSRYSGFVRTLF... Test
...FQSLVPKAARSKYSGFVRTLF... Polymorphism
...FQSLVPKAARCQYSGFVRTLF... Polymorphism
...FQSLVPKAARGLYSGFVRTLF... Polymorphism
...FQSLVPKAARRQYSGFVRTLF... Polymorphism
...FQSLVPKAARGRYSGFVRTLF... Test
...FQSLVPKAARGKYSGFVRTLF... Test
Fig. 5: Natural polymorphisms
exist at position 590 and
591. Based on this set of
polymorphisms, we made
mutations in the PB2
polymerase of 8BM and
introduced them back into
the 8BM polymerase complex.
8BM3470 PB2 WT
S590 Q591
8BM3470 PB2 GK
G590 K591
Fig. 6: Homology modelling
using SWISSMODEL of the
PB2 protein from 8BM. An
H1N1 sequence (PDB: 3KHW)
was used as the base model.
Coulombic surface coloring
was performed in UCSF
Chimera. The surface coloring
indicates that the charge at
the surface where GK replaces SQ changes dramatically. We hypothesize that this may change the binding
affinity of PB2 with its interacting partners.
Gaussia Luc
NP
PA
PB1
PB2
UTR UTR
Fig. 7: A Gaussia luciferase-based assay
for measuring polymerase activity was
used to assess the effect of mutations on
the polymerase complex. Five plasmids,
housing the RNP complex components and the reporter plasmid, were transiently transfected into HEK293T
cells, and luminescence was measured 48 hours later.
WT
1
2
3
Q591R
FoldChange
10BM
1
2
3
8BM
FoldChange
10BM vs 8BM Q591R mutation
Fig. 8: The 8BM PB2 polymerase has a two-fold activity over a related
10BM16764R0 (10BM) polymerase. The Q591R mutation in the 8BM PB2
polymerase increases the polymerase activity by 3 fold.
Q
R
K
G S
10BM
G S
8BM
10X
1X
E627K fold change vs. WT
Fig. 9: E627K enhances the fold change of 591Q
compared to WT when combined with 590G/S. No
synergism detected between Q591K and E627K.
8BM
GK4.5X
SK4.0X
GR3.0X
SR3.0X
SL1.3X
RQ1.0X
SQ1.0X
GQ0.87X
GL0.86X
CQ
10BM
5.6X
4.0X
2.9X
2.8X
0.7X
1.2X
1.3X
1.0X
0.7X
0.6X0.68X
*
*
Fig. 10: The mutations 591K or 591R have the greatest effect on the polymerase activity compared to WT (starred). 591K already
exists in H9N2 viruses circulating in China and the Middle East.
Acknowledgments