Identifying Human Host Proteins that Interact with Influenza A Polymerase
Proteins Using the Yeast Two-Hybrid System
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Drew ,
1,2,5
Faganel ,
2
Johnson ,
2
Kinzler ,
1,2
Madhan ,
2
Schmotter ,
1,4
Scully ,
Cassandra
Peyton
Bree
Carly
Nayasha
Caleb
Erin
2
2,3
2
1,2
1
1
2
3
4
5
Luke Tourville , Alec Rose , Kevin Marshall , Marc Busch , and Heidi Sleister ( Biology, BCMB, Neuroscience, Psychology, Chemistry)
Background
Methods
 A Brief History
o Humans most likely began to contract the influenza virus when animals
first became domesticated.
o Settlements provided a sufficient number of hosts for an epidemic.
o It wasn’t until 1930 that the influenza virus was at last isolated from a pig
by Shope and Lewis.
PB1/PB2/PA
Design gene specific
primers with homology
to pGBKT7 vector and
homology to 5’ and 3’
ends of PB1/PB2/PA
ORF
 Structure and Function
o The Influenza A genome consists of
eight negative-sense strands of viral
RNA. Attachment requires the viral
glycoprotein hemagglutinin (HA),
which binds to the cell receptor,
sialic acid.
InFusion cloning by
homologous recombination
"Influenza Figure." Web. 1 Apr. 2012.
<http://micro.magnet.fsu.edu/cells/viruses/images/influenzafigure1.jpg>
 Purpose
o To distinguish which human host proteins interact with Influenza A virus
polymerase complex proteins.
pSCRIPT
PB1/PB2/PA
Isolate pSCRIPT
vector from E. coli
bacteria
PCR amplification of
PB1/PB2/PA from
pSCRIPT with 5’ and 3’
extensions
homologous to vector
PB1/PB2/PA
Forward
%GC
%GC
primer name
Tm
Influenza virus strain
Forward primer sequence
Swine Influenza A- PB1,
H3N2, A/Sw/MN/593/33/99
CATGGAGGCCGAATTC atggatgtcaatccgactcta
MN-PB1F
42.9
Swine Influenza A- PB2,
H3N2, A/Sw/MN/593/33/99
CATGGAGGCCGAATTC atggagagaataaaagaactaagag
MN-PB2F
32
Swine Influenza A- PB1,
H1N1, A/Sw/NC/44173/00
CATGGAGGCCGAATTC atggatgtcaatccgactttac
NC-PB1F
Swine Influenza A- PB2,
H1N1, A/Sw/NC/44173/00
CATGGAGGCCGAATTC atggagagaataaaagaattaagagatc
Swine Influenza A- PA,
H1N1, A/Sw/NC/44173/00
CATGGAGGCCGAATTC atggaagacttcgtacgaca
59.2
Reverse
%GC
%GC
primer name
Tm
Reverse primer sequence
MN-PB1R
39.1
61.5
57.6 GCAGGTCGACGGATCC ctaattgatcgccatccgaattc
MN-PB2R
43.5
60.5
40.9
59.2
GCAGGTCGACGGATCC ttacttttgccgtctgagatc
NC-PB1R
42.9
58.9
NC-PB2F
28.6
58.9
GCAGGTCGACGGATCC ctaattgatggccatccgaat
NC-PB2R
42.9
59.2
NC-PAF
45
59.4
GCAGGTCGACGGATCC ctatcttagtgcatgtgtgagga
NC-PAR
43.5
60.5
GCAGGTCGACGGATCC ttatttttgccgtctgagtcctt
By constructing primers specific to the polymerase genes, the segment of the plasmid DNA to be replicated was
selected. Sequences in black denote the segments of the primers that are complementary to the PB1/PB2/PA
genes and will allow replication to take place, while red sequences denote segments of the primers that are
homologous to the pGBKT7 vector and are necessary for recombination cloning to take place.
Control
(-HindIII)
PB1
(MN)
PB2
(MN)
PB1
(NC)
PB2
(NC)
PA
(NC)
PCR results of pSCRIPT+PB1/PB2/PA. PCR
products were run through a 0.8% agarose gel
and compared to the λ-HindIII DNA marker to
determine the size of the fragments. PCR
products were expected to be ~2.2kb in length.
All reactions except PB2(NC) were repeated to
achieve greater yield.
~2.3 kb
PB1/PB2/PA
pGBKT7
 Importance
o The Influenza A virus has multiple physiological effects on humans,
including fever, cough, chills, body aches and fatigue. By discerning
which human host proteins interact with the Influenza A virus, it may be
possible to further the progress of treatments for the disease.
~2.0 kb
Transform cloning mixture
into E. coli
pGBKT7+PB1/
PB2/PA
This gel illustrates the results of a bacterial cell cracking procedure for PB2(NC) and PA(NC). The vector
lacking the insert will move further down the gel than the vector with an insert due to its smaller size.
Control
(pGBKT7
vector
without
insert)
Transformants 1-10 of pGBKT7+PB2(NC)
1
2
3
4
5
6
7
8
9
Control
Transformants 7-10 of
(pGBKT7
pGBKT7+PA(NC)
vector
without
10
8
9
10
insert) 7
Plate on LBKanamycin agar media
to select for pGBKT7containing cells
Experimental Question & Strategy
 Question
o Which human proteins bind to the Influenza A polymerase complex
proteins PB1-H3N2 (A/Sw/MN/593/33/99), PB2-H3N2
(A/Sw/MN/593/33/99), PB1-H1N1 (A/Sw/NC/44173/00), PB2-H1N1
(A/Sw/NC/44173/00), and PA-H1N1 (A/Sw/NC/44173/00)?
Human
Protein
Results
Screen for correct
plasmid constructpGBKT7+PB1/PB2/PA
by bacterial cell
cracking, PCR, and
restriction digest
Transform
pGBKT7+PB1/PB2/PA
(bait) into haploid
yeast strain Y2HGold
Vector
without
insert
Vector
with PB2
insert
Vector
with PA
insert
InFusion recombination cloning was successful for producing the desired plasmids (pGBKT7 vector +
polymerase gene). PB2(NC) transformants 2 and 6 and PA(NC) transformant 7 were selected for further
analysis Transformants for PB1(MN), PB1(NC), and PB2(MN) had similar results.
Binding of the bait and prey
causes the transcriptional
activation of GAL4 which leads
to expression of reporter genes
Conclusions & Future Work
Gal4(BD)-PB1/PB2/PA
bait
Test PB1/PB2/PA for
toxicity to yeast
Test PB1/PB2/PA for
auto-activation of
reporter genes in yeast
Influenza A
polymerase
complex protein
 Conclusions
o The open reading frames of PB1/PB2/PA were successfully amplified by PCR.
o While screening (cracking) various bacterial transformants, those that were most likely to
contain the pGBKT7 vector with insert PB1/PB2/PA were determined.
o Isolation of plasmids from InFusion cloning that are bigger than the vector alone
suggests the InFusion recombination cloning was successful.
o Transformant colony 2, from pGBKT7+PB2(NC) and transformant colony 7, from
pGBKT7+PA(NC), were selected for further screening.
 Future Work
 Experimental Strategy
o The Yeast Two-Hybrid System (Y2H) is being used for the identification
of protein-protein interactions. Binding of the bait with the prey produces
the transcriptional activator function of yeast Gal4. This leads to the
expression of reporter genes which is detected by cell growth on SDLEU-TRP-HIS/X-alpha-Gal/AbA agar plates.
Sources
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"EBSCO Publishing Service Selection Page." EBSCO Publishing Service Selection Page. N.p., n.d. Web. 9 Apr. 2013.
<http://ehis.ebscohost.com/eds/pdfviewer/pdfviewer?sid=701ad167-dfee-40fd-a8co-55f1cd713f55%40sessionmgr11&vid=10&hid=103>.
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"Influenza." Medical Ecology @ www.MedicalEcology.org. N.p., n.d. Web. 9 Apr. 2013.
http://www.medicalecology.org/diseases/influenza/influenza.htm#sect3.5>.Clontech. Matchmaker Gold Yeast Two-Hybrid System User Manual. Montainview, CA:
Clontech Laboratories, 2010. Print.
•
"Influenza Figure." Web. 1 Apr. 2012. <http://micro.magnet.fsu.edu/cells/viruses/images/influenzafigure1.jpg>.
•
Kao, R. et al. (2010). Identification of influenza A nucleoprotein as an antiviral target. Nature Biotechnology 28, 600–605.
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Portela, A. and P. Digard. (2002). The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J. General Virology 83, 723–734.
•
Qiagen. (2006). QIAprep Miniprep Handbook. Print.
•
“Yeast Two-Hybrid Figure.” Web. Apr 4. 2012. <http://cmbi.bjmu.edu.cn/cmbidata/proteome/method/hybrid02.jpg>.
o
o
o
o
Sequence the pGBKT7+ PB1/PB2/PA plasmids
Determine Influenza A PB1/PB2/PA toxicity to yeast
Test Influenza A PB1/PB2/PA for auto-activation of yeast two-hybrid reporter genes
Isolate human protein candidates (prey) that may interact with Influenza A PB1/PB2/PA
(bait) by performing a yeast two-hybrid screen
o Screen candidates to determine true positive interactions and identify prey proteins
o Confirm positive interactions in a mammalian system
o Compare identified protein interactions in normal versus microgravity conditions
 Acknowledgement
o NASA – Iowa Space Grant Consortium (Base program and curriculum grants)