Transcript Hendra Virus

Hendra Virus V Protein Inhibits Interferon
Signaling by Preventing STAT1 and
STAT2 Nuclear Accumulation
Jason J. Rodriguez, Lin-Fa Wang, and Curt
M. Horvath
Presented by Morgan Wood
What is Hendra Virus?
• Also called equine morbillivirus.
• It was first discovered in horse populations in Australia,
with the only three human cases occuring during an
outbreak in 1994 in Hendra, a suburb of Brisbane,
Australia.
• Both Hendra And Nipah Viruses are members of the
family Paramyxoviridae. This family covers a large
group of Negative-strand RNA viruses that cause Human
and animal diseases. They are together the founding
members of a genus, Henipavirus.
• It is believed that both Hendra and Nipah viruses have
natural reservoirs in large bat species in the areas where
the viruses occur.
Hendra Virus Cont.
• The symptoms of Hendra virus include respiratory illness
with sever flu-like symptoms, and can progress to
encephalitis (swelling of the brain).
• Both Hendra virus and Nipah virus have the potential to
be fatal.
• Both of these viruses infect humans when they come
into contact with bodily fluids or secretions of infected
animals; horses in the case of Hendra, and pigs in the
case of Nipah.
• Of the three cases of Hendra virus that have been
reported, two died as a result of the infection and the
third had a lengthy recovery.
Interferon signaling
• Interferon signaling is
involved in the innate immune
response to viruses in the
body.
• The signal is transmitted by
proteins called STATs, that
dimerize, once activated, and
are then able to travel into the
nucleus and drive
transcription of immune
genes.
• Preventing nuclear
accumulation of STAT
proteins is a common way
that this signal pathway is
disrupted.
Questions
1. Are the V proteins of Hendra and Nipah
Viruses Similar?
2. Does the Hendra virus V protein interfere with
interferon signaling?
3. Does the Hendra virus V protein form
aggregates with the STAT1 & STAT2 proteins?
4. Does the Hendra virus V protein prevent the
nuclear accumulation of STAT1 & STAT2
proteins?
Methods
• Amino acid sequencing was used to determine protein
sequences.
• Human fibrosarcoma 2fTGH cells were used in the
Luciferase assay as well as in the immunostaining
assay.
• 293T cells were used in the V protein aggregation
experiments as well as in the Luciferase assay
experiments, but the data was not shown in the latter.
• Green flourescent protein, from the sigma corporation,
was used in an experiment as well.
• Western blot was done to determine formation of an
aggregate.
Methods Cont.
• Chromatography was used to separate out molecular
complexes of different sizes for fig. 2b.
• A solution is passed through a seperating column,
fractions of the solution are collected seperatly as they
come out of the column. These fractions contain
different sized complexes in them.
• Larger things move through column faster, while small
molecules are trapped in pores in side the filter media,
they end up being washed out in later fractions.
• The molecules in the fractions are then blown apart to
allow for easier electrophoresis, and are then run
through a page process.
Are the V proteins of Hendra and Nipah
Viruses Similar?
• In order to establish the
relationship between
Hendra and Nipah viruses,
the authors sequenced the
amino acids of the V
proteins for both species.
• The overall similarity was
58%. There was an 81%
similarity between aa 1140, 44% between 141405, and 83% between
406-457.
• Answer: Yes, they are
quite similar.
Does the Hendra virus V protein
interfere with interferon signaling?
• It has been established in previous
studies that Nipah virus V protein
interferes with interferon signaling,
so it will be used as the standard to
judge Hendra virus against.
• Two luciferase reporter construct
were assembled and transfected
into human fibrosarcoma 2fTGH
cells, and 293 T cells.
• Cells were either transfected with
HA-tagged Nipah virus V (NiV),
Hendra virus V (HeV), or HAtagged vector with no cDNA.
• They were then treated with (+) or
without (-) IFN 12 h prior to lysis.
• Answer: yes, it
interferes with signaling
Does the Hendra virus V protein form aggregates
with the STAT1 & STAT2 proteins?
• 293T cells were transfected with either
blank expression vectors (C), Flagtagged Hendra virus V expression
vectors (H), or Flag-tagged Nipah virus
V expression vectors (N).
• The left column shows basal levels of
STAT proteins as well as the Flag
protein in the two right lanes.
• The right column shows a
coimmunoprecipitation of the STAT & V
protein complexes.
Does the Hendra virus V protein form
aggregates with the STAT1 & STAT2
proteins?
• 293T cells were transfected with
either green flourescent protein
(GFP), NiV, or HeV.
• Cells were then lysed and the
solution run through a
chromatography seperator.
• Fractions from each type of lysate
were then subjected to a western
blot using either STAT1 or STAT2
antibodies.
• The lower the fraction number that
the STAT protein shows up in, the
larger the complex that went
through the column.
• The aggregates formed were about
500kDa in size.
• Answer: YES.
Does the Hendra virus V protein prevent the
nuclear accumulation of STAT1 & STAT2
proteins?
• Immunostaining was used to show
that Hendra virus V protein actually
inhibits the nuclear accumulation of
the STAT1 & STAT2 proteins.
• Human fibrosarcoma 2fTGH cells
were transfected with plasmid
encoding HA-tagged Hendra virus
V protein.
• Cells were either left untreated
(UNT) or treated with IFNg for 30
minutes prior to fixation.
• The cells were stained with a red
flourescent stain for the HA tag,
and a green flourescent stain for
either STAT1 or STAT 2.
• Cells without the HeV appear with
green stain but no red stain.
• Answer: Yes.
Questions
1.
2.
3.
4.
Are the V proteins of
Hendra and Nipah Viruses
Similar?
Does the Hendra virus V
protein interfere with
interferon signaling?
Does the Hendra virus V
protein form aggregates
with the STAT1 & STAT2
proteins?
Does the Hendra virus V
protein prevent the nuclear
accumulation of STAT1 &
STAT2 proteins?
• Yes, 58% overall.
• Yes, reducing activity
by 80%.
• Yes, about 500kDa in
size.
• Yes.
Conclusions
• Hendra virus and Nipah virus are very closely
related, and their V proteins inhibit interferon
signaling in very similar ways.
• While human infections of these diseases
remain fairly low, a good understanding of them
is helpful, since both are zoonotic diseases
(passed from animals to humans).
• Understanding of these diseases could lead to
breakthroughs in the treatment of a number of
illnesses that are common in the zoo field.
Refferences
• Rodriguez, J. J., Wang, L., Horvath, C.M.
Hendra virus V protein inhibits interferon
signaling by preventing STAT1 and STAT2
nuclear accumulation. Journal of Virology
77(21): Nov. 2003 p. 11842-11845.
• Center for Disease Control website
www.cdc.org
• Dr. Bulla’s website