January 24th 2011
Kathrin Hüging
Introduction to Coronaviruses
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Coronaviruses: Coronaviridae family, order Nidovirales
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Gained prominence during the SARS (severe acute
respiratory syndrome) outbreak in 2002 – 2003
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Prototypical coronavirus: Mouse Hepatitis Virus (MHV)
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Enveloped viruses with positive single stranded RNA genome
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S= Spike
M= Membrane
E= Envelope
N= Nucleocapsid
(Perlman & Netland, Nature Reviews Microbiology, 2009)
Introduction to DMVs
(Perlman & Netland, Nature Reviews Microbiology, 2009)
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Early upon infection, coronaviruses induce
the formation of double-membrane vesicles
(DMV) in host cells
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Replication and transcription complexes
(RTC) are targeted to DMVs ( site of
replication)
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DMVs are derived from the endoplasmic
reticulum (ER)
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But: DMV biogenesis seems not to depend
on conventional routing of proteins through
this transport pathway:
• no ER protein markers
• No markers of ER-Golgi intermediate
compartment
• No Golgi protein markers
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Conflicting data regarding the role of
autophagy in CoV replication
Aim of the study:
Open questions:
• What is the precise origin of DMV lipid bilayers?
• What is the host protein content?
• What cellular factors are essential for DMV formation?
• Is autophagy involved?
AIM
To unveil the origin of virus-induced double membrane vesicles (DMVs)
and to indentify host cell factors essential for CoV replication
MHV infection does not require an intact autophagy machinery
• mouse embrionic fibroblasts (MEF) from wildtype and Atg7 knockout
mice were infected with MHV and further analyzed:
Atg7: essential for autophagy
Nsp2/3: viral non-structural
proteins, are components of RTC
(replication and transcription
complexes)
Puncta represent virus-induced
DMVs that contain viral dsRNA
 no differences in DMV morphology
between wildtype and Atg7 knockout
mice
MHV infection does not require an intact autophagy machinery
Infection of MEFs with MHV reporter virus:
 no differences in virus
entry and replication of
the viral RNA
Titraton of MHV virus stock on MEFs:
 no differences in production
of viral progeny
 autophagy is not required for viral replication and production of viral progeny
Nonlipidated LC3/ATG8 associates with CoV-induced DMVs
• LC3/ATG8:
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autophagosome protein marker (GFP-LC3 commonly used)
contradictory data available regarding its presence on DMVs
Analysis of LC3 in HEK293 cells:
 endogenous LC3 colocalizes with DMV protein markers
 ectopically GFP-LC3 does not colocalize
Nonlipidated LC3/ATG8 associates with CoV-induced DMVs
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upon autophagy induction, cytoplasmic form LC3-I is converted into an active lipidated form
LC3-II
LC3-II coats autophagosomes
both the lipidation and the formation of LC3-II coated autophagosomes depends on several
proteins, including Atg7
Infection of wt and Atg7 ko MEFs with MHV:
Analysis of LC3 lipidation in HeLa cells:
 colocalization of LC3 with DMVs does not depend on presence of Atg7
 lipidation of LC3-I not required for colocalization to DMVs
LC3 is required for MHV and for formation of DMV
siRNA against LC3 reduces expression of nucleocapsid protein and viral RNA replication:
ectopic expression of non-lipidable LC3
rescues phenotype
siRNA against LC3 inhibits formation of DMVs
 BUT: if viral RNA is not replicated, no/low
protein expression  no DMV marker
available! (EM pictures needed to confirm IF
data)
Analogies between MHV-induced DMVs and EDEMosomes
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MHV induced DMVs:
• ER origin
• no conventional ER markers
• are associated with LC3-I but not with GFP-LC3-I
 share similarities with EDEMosomes
What are EDEMosomes?
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newly synthesized polypeptides in the ER can either attract folding machinery or
degradation machinery (ERAD machinery, ER-associated degradation)
normal growth conditions:
• activity of ERAD machinery is maintained at a low level (avoid premature
interruption of folding programs)
ERAD-tuning: clearance of ERAD regulators by selective sorting of ERAD regulators
(e.g. EDEM1 or OS-9) into EDEMosomes
EDEMosomes are small vesicles that emerge from the ER in a COPII independent
mechanism and deliver their content to endosomal compartments
poorly characterized: role of autophagy?
Influence of active and inactive autophagy on components of the ERAD
tuning pathway
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comparison of intracellular levels of EDEM1 (ERAD regulator) and p62
(canonical substrate of autophagy) under active or inactive autophagy
Inactive autophagy: Atg7 ko mice
Westernblot analysis of MEFs:
 EDEM1 levels are not affected
 p62 levels are increased in the Atg7 ko mice
• deletion of Atg7 was published to inhibit p62 turnover
Pulse-chase radiolabeling of MEFs:
 similar rates of disposal of EDEM1 in presence and
absence of ATG7
 EDEM1 level is not affected (Atg7 and conventional
autophagy are dispensible for EDEM1 turnover)
Influence of active and inactive autophagy on components of the ERAD
tuning pathway
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comparison of intracellular levels of EDEM1 (ERAD regulator) and p62
(canonical substrate of autophagy) under active or inactive autophagy
Inactive autophagy: chloroquine (CQ) treatment
Active autophagy: rapamycine (rap) treatment
Westernblot analysis of wt MEFs:
 CQ inhibits p62 degradation and delayed EDEM1
turnover  EDEM1 accumulation
 rap reduced intracellular p62 levels but increased EDEM1
by delaying its turnover
Pulse-chase radiolabeling of wt MEFs:
 EDEM1 turnover is decreased after treatment
with CQ or rap
 the pathway regulating EDEM1 turnover is distinct from autophagy
(because both activation and inactivation of autophagy seem to have an influence?)
Components of the ERAD tuning pathway are associated with
DMVS
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Hypothesis:
• MHV hijacks the ERAD tuning machinery to co-opt cellular membranes
for DMV generation
Infection of HeLa cells with MHV:
EDEM1 and Os-9 (ERAD regulators)
localize to DMVs
MHV infection leads to a defective clearance of
EDEM1 and OS-9
However, EDEM1 and OS-9 are not required for
MHV infectivity (supplement figure)
summary
• CoV hijack the ERAD tuning machinery for the generation of
DMVs, which provide the membranous support for viral RTCs
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By this, MHV interferes with the degradation of EDEM1 and
OS-9 by trapping them in DMVs
 data are consistent with the model in which DMVS are
generated from the host ER
• Explain the absence of conventional ER resident chaperones in
DMVs
• LC3 is required for DMV formation, even though the
conventional autophagy pathway is not essential (Atg7 ko has
no influence)
BUT: Atg5/7-independent type of autophagy, does also not involve
lipidation of LC3-II (Nishida et al., 2009)