Summer Undergraduate Research Program - Fred Hutchinson ...
Summer Undergraduate Research Program - Fred Hutchinson ...
Summer Undergraduate Research Program - Fred Hutchinson ...
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How does VP35 from the Ebola virus antagonize Protein Kinase R?<br />
Emily P. Baker [1] , Nels C. Elde [2] , Harmit S. Malik [2]<br />
Emily P. Baker [1] , Nels C. Elde [2] , Harmit S. Malik [2]<br />
ecpb@u.washington.edu<br />
University of Washington [1] and Basic Sciences [2] University of Washington , <strong>Fred</strong> <strong>Hutchinson</strong> Cancer <strong>Research</strong> center, Seattle WA<br />
[1] and Basic Sciences [2] , <strong>Fred</strong> <strong>Hutchinson</strong> Cancer <strong>Research</strong> center, Seattle WA<br />
One of the hallmarks of Ebola infection is the virus’s ability to disable the host immune response. VP35 is one of the Ebola proteins that have<br />
been implicated in its ability to do this. Among other things VP35 is known to be an inhibitor of the cellular anti‐viral Protein Kinase R (PKR),<br />
though the mechanism of this antagonism is unknown[1]. Our aim was to test whether VP35 acts directly on PKR or if it acts on another aspect<br />
of PKR’s activation.<br />
Results<br />
It is known that<br />
VP35 does not<br />
inhibit PKR through<br />
its ability to bind<br />
dsRNA [1]<br />
Question<br />
PKR Activation<br />
In Response to Viral Infection<br />
Immune<br />
System<br />
2009 Best Poster Award<br />
Is VP35 a direct inhibitor of PKR?<br />
Representative Plate<br />
positive<br />
control<br />
+<br />
virus<br />
K3L<br />
VP35<br />
dsRNA Interferon<br />
?<br />
eIF2α<br />
Active<br />
PKR dimer<br />
146<br />
VP35<br />
VP35 inhibits<br />
PKR’s ability to<br />
block protein<br />
translation<br />
Inactive<br />
PKR monomer<br />
blank<br />
vector<br />
No yeast growth with VP35<br />
regardless of the PKR<br />
transformed.<br />
VP35<br />
eIF2α<br />
phosphorylated<br />
eIF2α<br />
Active<br />
PKR dimer<br />
162<br />
Methods<br />
eIF2α<br />
phosphorolated<br />
A PKR Yeast assay was used to<br />
test the ability of VP35 to inhibit<br />
PKR directly.<br />
Translation is blocked<br />
preventing viral<br />
replication<br />
PKR<br />
PKR<br />
Ribosome<br />
Viruses have a number of ways of defeating PKR<br />
PKR Antagonists<br />
rescues<br />
growth<br />
?<br />
•Viral proteins may sequester viral dsRNA so that<br />
it can’t interact with PKR<br />
VP35 section<br />
Discussion<br />
blocks<br />
growth<br />
blocks<br />
growth<br />
•Direct inhibition by viral proteins<br />
(e.g. pseudosubstrate inhibition by poxvirus K3L)<br />
PKR yeast transfromants transformed with VP35 have<br />
so far been unable to rescue growth showing that<br />
VP35 does not appear to directly interact with PKR<br />
Antagonists<br />
VP35 From Ebola ‐Zaire<br />
•Viral proteins can interact with other cellular<br />
proteins to indirectly inhibit PKR (e.g. interfere<br />
with interferon’s activation of PKR)<br />
The expression of VP35 needs to be confirmed using an HA tagged gene<br />
and<br />
a more sensitive assay will be used to test for direct inhibition of PKR<br />
by VP35 (yeast dot assay with serial dilutions)<br />
PKR Sources<br />
Human<br />
Gibbon<br />
Orangutan<br />
Rhesus<br />
African Green<br />
Woolly Monkey<br />
Hominoids<br />
K3L From Poxvirus<br />
Vaccinia<br />
positive control<br />
pSB‐146 Blank Vector<br />
negative control<br />
Old<br />
World<br />
New<br />
World<br />
Monkeys<br />
[1] Schümann M, Gantke T, Mühlberger E (2009) J Virol, 83: 8993‐8997<br />
Thanks to all the members of Malik Lab for their help and support.
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