Scientia
Activation of Host Kinase Vps34 by Murine Norovirus Non-structural Protein NS4 Introduction:
IN-DEPTH Jessica Oros Randall Lab, University of Chicago
that other viruses such as dengue virus (DENV),
Despite Vps34 being used in the host
and encephalomyocarditis virus (EMCV) cannot
for autophagy, MNV may be utilizing Vps34
Each year, millions of individuals
replicate when the TAG-dependent antiviral
to aid the process of viral replication. Thus,
are infected with human norovirus, securing
pathway is knocked down. If the TAG-dependent
we hypothesize that Vps34 may be a point of
norovirus as the number one worldwide cause
antiviral pathway was the only means through
control by MNV during infection as it may
of gastroenteritis and causing about 200,000
which viral replication was controlled, DENV
support viral replication in an unknown way.
deaths each year. Tens of thousands of those
and EMCV would be able to replicate in TAG-
Furthermore, we aim to characterize which
deaths occurring in children in developing
knockout conditions. Since this is not observed,
viral non-structural protein is responsible for
countries. There are presently no norovirus
it indicates that there is some other TAG-
the change in Vps34 activation level. Out of the
vaccines or cures, making it a pressing issue to
independent antiviral pathway present that is
6 non-structural proteins of MNV, the most
understand the biology of norovirus infection.
still blocking viral replication. Interestingly,
likely candidates are NS4, NS5, and NS6 because
Unfortunately, human norovirus cannot be
MNV can still replicate in this TAG-deficient
of their previously elucidated roles in immune
easily grown in laboratory cultures. However,
model, meaning there is some mechanism by
evasion. NS4 has been shown to localize to host
murine norovirus (MNV) can serve as a model.
which MNV is evading the immune response.
intracellular membranes and thus may play a
MNV is used in the lab to understand the
Evasion may be possible at the viral replication
role in replication complex formation, but its
mechanisms behind infection in murine cell
stage via the replication complex or through
function is largely uncharacterized. The Randall
lines. MNV is a positive-sense RNA virus. Like
virus-derived factors such as interactions
Lab has previously shown that NS5 can produce
other viruses within the same class, MNV creates
between viral and host proteins. The aim of this
an immune evasion phenotype upon infection
a replication complex upon entry and uncoating
project is to elucidate the mechanisms by which
with DEMV and EMCV of BV2 cells transduced
inside the cell using host-cell membranes . The
MNV presents a phenotype of immune evasion
with NS5. Upon entry and uncoating, the viral
purpose of this replication complex seems to be
through interactions with host proteins.
genome is translated into a polyprotein, and
protecting viral genome replication from the
One possible player in MNV immune
NS6 is the protease responsible for the cleavage
hostile environment of the host cell cytoplasm.
evasion and replication could be the cellular class
and release of individual MNV non-structural
Luckily for host organisms, these replication
III phosphatidylinositol-3 kinase (PI3K) vacuolar
proteins .
complexes can be targetted via an interferon-
protein sorting 34 (Vps34), which plays a role in
In this study, we have shown that
gamma (IFNG) mediated antiviral pathway,
cellular trafficking via initiation of autophagy.
infection with MNV results in a decrease in
allowing for lysosomal degradation of the
Vps34
phosphatidylinositol
inhibitory Vps34 acetylation, indicating an
replicating virus. This pathway uses proteins
to produce phosphatidylinositol-3-phosphate
increase in activity. Out of the viral non-
involved in autophagy, a cellular process by
(PI3P), which acts as a membrane marker to
structural proteins tested, only non-structural
which foreign particles or faulty proteins are
begin the formation of the autophagosome
protein 4 (NS4) was able to reproduce the
engulfed and degraded through lysosomal
. In healthy cells, Vps34 is maintained in an
phenotype, indicating that it is responsible for
fusion. This recognition by the host cell is done
inhibitory state by the nutrient-sensing kinase
the release of Vps34 from its inhibitory state.
by the Atg12-Atg5-Atg16L1 complex, which is
mTOR. Under cellular stress or starvation, mTOR
comprised of autophagy-related (Atg) proteins
phosphorylates UVRAG, a protein complex
Materials and Methods:
that interact with host membranes to form an
involved in endocytosis, which will activate
Tissue Culture:
autophagosome . Conjugation of the ubiquitin-
Vps34 to initiate autophagy. The Randall lab
Murine norovirus (MNV) was used as
like microtubule associated protein light-chain
has previously shown that knockdown of Vps34
a model to study norovirus activity. Cell culture
3 (LC3) with Atg-5 allows interferon-inducible
prevents MNV replication, indicating that there
was done with murine microglial BV2 cells using
GTPases to localize to LC3
phosphorylates
and target the
may be interactions between the virus and
standard tissue culture protocol, which involves
replication complex, inhibiting viral replication
the host lipid kinase. Vps34 activity is tightly
growing the adherent cells in flasks with Gibco
. This pathway does not operate via degradative
regulated by the host cell through acetylation
Dulbecco’s Modified Eagle Medium (DMEM)
canonical autophagy, but it uses autophagy
by the acetyltransferase p300, which itself
supplemented with 10% fetal bovine serum
proteins to prevent MNV from replicating inside
is activated by autoacetylation . Acetylation
(FBS) and 1% each of non-essential amino acids,
the cell . The pathway is called the Targeting by
of Vps34 is inhibitory, and activation by
hydroxyethyl
AutophaGy protein (TAG) – dependent antiviral
deacetylation contributes to the formation of
(HEPES), and PIS. Cells were seeded and used for
pathway. Prior work in the Randall lab shows
autophagosomes.
experiments. In the case of interferon gamma
12
piperazineethanesulfonic
acid
