VP3 is crucial for the stability of Nora virus virions

Sadanandan, Sajna Anand; Ekström, Jens-Ola; Venkateswara Rao, Jonna; Hultmark, Dan; Hofer, Anders
VP3 is crucial for the stability of Nora virus virions

Author: Sadanandan, Sajna Anand,Ekström, Jens-Ola,Venkateswara Rao, Jonna,Hultmark, Dan,Hofer, Anders
Year: 2016
Source: https://trepo.tuni.fi/bitstream/10024/99835/1/vp3_is_crucial_for_2016.pdf
Vi us Resea ch 223 (2016) 20–27
Con en s lis s a ailable a ScienceDi ec
Vi us Resea ch
jou nalhomepage: www.else ie .com/loca e/ i us es
VP3 is c ucial o he s abili y o No a i us i ions
Sajna Anand Sadanandana, Jens-Ola Eks öma,c, Venka eswa a Rao Jonnab,
Ande s Ho e b, Dan Hul ma ka,c,∗
aDepa men o Molecula Biology, Umeå Uni e si y, SE-901 87 Umeå, Sweden
bDepa men o Medical Biochemis y and Biophysics, Umeå Uni e si y, SE-901 87 Umeå, Sweden
cIns i u e o Biomedical Technology, Uni e si y o Tampe e, FI-33520 Tampe e, Finland
a i c l e i n o
A icle his o y:
Recei ed 15 Ma ch 2016
Recei ed in e ised o m 15 June 2016
Accep ed 17 June 2016
A ailable online 18 June 2016
Keywo ds:
RNA i uses
No a i us
Capsid s abili y
Vi us biology
a b s a c
No a i usisanen e ic i us ha causes pe sis en ,non-pa hologicalin ec ion inD osophila melanogas e .
I eplica es in he ﬂy gu and is ansmi ed ia he ecal-o al ou e. No a i us has a single-s anded
posi i e-senseRNAgenome,whichis ansla edin ou open eading ames.Reading ame h eeencodes
he VP3 p o ein, he s uc u e and unc ion o which we ha e in es iga ed in his wo k. We ha e shown
ha VP3 is a ime ha has an ␣-helical seconda y s uc u e, wi h a unc ionally impo an coiled-coil
domain. In o de o iden i y he ole o VP3 in he No a i us li e cycle, we cons uc ed VP3-mu an s using
he cDNA clone o he i us. Ou esul s show ha VP3 does no ha e a ole in he ac ual assembly o he
i us pa icles, bu i ions ha lack VP3 o ha bo VP3 wi h a dis up ed coiled coil domain a e incapable
o ansmission ia he ecal-o al ou e. Remo ing he egion downs eam o he pu a i e coiled coil
appea s o ha e an e ec on he ﬁ ness o he i us bu does no hampe i s eplica ion o ansmission.
We also ound ha he VP3 p o ein and pa icula ly he coiled coil domain a e c ucial o he s abili y o
No a i us i ions when exposed o hea o p o eases. Hence, we p opose ha VP3 is impe a i e o No a
i us i ions as i con e s s abili y o he i al capsid.
© 2016 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license
(h p://c ea i ecommons.o g/licenses/by/4.0/).
1. In oduc ion
No a i us is a small RNA i us ha causes pe sis en , non-
pa hological in ec ion in D osophila melanogas e (Habayeb e al.,
2006). I is an en e ic i us ha is ansmi ed ia he ecal-o al
ou e and high i us i e s ha e been obse ed in he eces o
in ec ed animals (Habayeb e al., 2009). I possibly deﬁnes a new
amily o pico na-like i uses. Based on sequence compa isons o
he eplica i e enzymes, he No a i us is mos closely ela ed
o i uses o he amilies Pico na i idae, which in ec e eb a es,
and Iﬂa i idae, which in ec insec s (Habayeb e al., 2006; Koonin
e al., 2008). Ye , he No a i us has a unique size and o gani-
za ion o i s genome. Pico na i idae includes impo an human
en e o i uses such as he polio i us, while honeybee pa hogens
like he de o med wing and sacb ood i uses belong o Iﬂa i idae.
Some membe s o hese amilies a e able o es ablish pe sis en
in ec ions in hei hos s, p obably con ibu ing o p oblems such
as he pos -polio synd ome in humans and he colony collapse dis-
∗Co esponding au ho a : Depa men o Molecula Biology, Umeå Uni e si y,
SE-901 87 Umeå, Sweden.
E-mail add ess: [email p o ec ed] (D. Hul ma k).
o de in honeybees (P isco e al., 2011; Baj e al., 2015). The No a
i us may be a use ul model o s udy he phenomenon o RNA i us
pe sis ence. Fo ha pu pose, we ound i impo an o cha ac e ize
he i us in mo e de ail.
No a i us has a 12333 nucleo ides long (Eks öm e al., 2011)
single-s anded RNA genome o posi i e pola i y (Habayeb e al.,
2006). Expe imen al wo k and sequence analysis ha e indica ed
ha , ou o he ou open eading ames in he No a i us genome,
ORF1 encodes an inhibi o o he RNAi de ense mechanism ( an
Mie lo e al., 2012), while ORF2 encodes a long polyp o ein wi h he
helicase, p o ease and polyme ase egions ha a e closely ela ed
o he co esponding p o eins ound in he eplica i e casse es o
pico na-like i uses (Habayeb e al., 2006; Koonin e al., 2008).
No sequence simila i y was de ec ed be ween ORF4 and o he
known i us p oduc s, bu expe imen al e idence has shown ha
i encodes a polyp o ein ha is clea ed in o h ee capsid p o eins,
VP4A, B and C (Eks öm e al., 2011). Howe e , he ORF3-encoded
p o ein, VP3, emains la gely uncha ac e ized.
Small amoun s o an ORF3-encoded p o ein, wi h an es ima ed
molecula mass o 35kDa, we e de ec ed in he i ions du ing mass
spec ome y analysis o he VP4 capsid p o eins (Eks öm e al.,
2011). This sugges ed ha he VP3 p o ein was associa ed wi h he
s uc u al p o eins. The ﬁ s 71 nucleo ides o ORF3 o e lap wi h
h p://dx.doi.o g/10.1016/j. i us es.2016.06.011
0168-1702/© 2016 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
S.A.
Sadanandan
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/
Vi us
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223
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21
he
C- e minus
o
ORF2
and
he e o e,
ansla ion
o
ORF3
is
likely
o
occu
wi h
a
ame
shi
mechanism.
The
esul ing
VP3
p o ein
is
281–304
amino
acids
long,
depending
on
he
exac
posi ion
o
he
ame
shi ,
and
he
p edic ed
molecula
mass
app oxima ely
31–34
kDa.
Based
on
he
sequence,
Eks öm
e
al.
(2011)
p edic ed
an
␣-helical
seconda y
s uc u e
o
he
N- e minal
200
amino
acids
o
VP3,
and
ha
his
pa
o
he
p o ein
is
likely
o
o m
a
coiled-coil
domain.
Howe e ,
expe imen al
e idence
is
s ill
lack-
ing
o
his
model.
Thus
a ,
he
ole
o
VP3
in
he
li e
cycle
o
No a
i us
has
no
been
in es iga ed.
We
ha e
now
used
he
cDNA
clone
o
No a
i us
(Eks öm
e
al.,
2011)
o
s udy
he
impo ance
o
VP3
in
i ion
assembly
and
s uc-
u al
s abili y,
by
gene a ing
VP3
mu an s.
Du ing
he
cou se
o
his
s udy,
we
obse ed
ha
VP3
was
impo an
o
he
ansmission
o
No a
i us
ia
he
ecal-o al
ou e.
Consequen ly,
we
hypo hesized
ha
he
p esumed
coiled-coil
domain
in
his
p o ein
was
impo an
o
he
s abili y
o
he
i us
pa icles.
To
add ess
his
hypo hesis,
we
compa ed
he
s abili y
o
wild- ype
i ions
o
ha
o
VP3
mu an
i ions.
We
ound
ha
mu an
i ions
lacking
VP3
o
ha bo ing
a
dis up ion
in
he
p edic ed
coiled
coil
domain
we e
labile
unde
hea
and
p o ease
ea men s,
while
he
wild
ype
i ions
su i ed
hese
condi ions.
2.
Ma e ials
and
me hods
2.1.
Cloning,
exp ession
and
pu iﬁca ion
o
No a
i us
VP3
No a
i us
ORF3
was
cloned
in o
he
mul iple
cloning
si e
o
he
pETM-MBP1a
exp ession
ec o
(P yo
and
Lei ing,
1997)
using
o wa d
(GCTTCCATGGCATTAAAAGAGGAGATTTTTGATCAA)
and
e e se
(GCTTGGTACCTTACATAGAGTCATAAATTACTGATGT-
GCT)
p ime s.
The
pETM-MBP1a
ec o
con ains
a
hexa-His idine
ag
ollowed
by
he
mal ose
binding
p o ein
(MBP)
and
a
Tobacco
e ch
i us
(TEV)
clea age
si e,
a e
which
he
ORF3
sequence
was
inse ed.
This
cons uc
was
ans o med
in o
Rose a
(DE3)
cells
(Fu
e
al.,
2007),
which
we e
hen
g own
o e nigh
in
an
au o-
induc ion
LB
medium
(Edwin
e
al.,
2014)
a
20◦C.
Since
i
is
agged
wi h
he
mal ose
binding
p o ein,
he
exp essed
VP3
p o-
ein
could
be
isola ed
using
an
amylose
esin
a ﬁni y
ma ix
(NEB),
acco ding
o
he
ins uc ions
p o ided
by
he
manu ac u e .
The
isola ed
p o ein
was
clea ed
om
i s
MBP
ag
using
he
TEV
p o-
ease
and
he ea e
pu iﬁed
by
NiNTA
aga ose
ch oma og aphy
(Qiagen)
in
o de
o
emo e
bo h
MBP
and
TEV
p o ease.
The
now-
un agged
VP3
p o ein
was
subjec ed
Q-Sepha ose
(GE
heal hca e)
ch oma og aphy
in
o de
o
ob ain
pu e
VP3
(Fig.
1A,
lane
2).
2.2.
Analysis
o
VP3
oligome s
by
GEMMA
Pu iﬁed
No a
i us
VP3
p o ein
was
dissol ed
in
20
mM
ammo-
nium
ace a e
pH
7.8
o
a
ﬁnal
concen a ion
o
0.05
mg/ml.
GEMMA
analysis
was
pe o med
as
desc ibed
in
Ro ouga an
e
al.
(2008).
2.3.
Analysis
o
VP3
oligome s
by
c oss-linking
Fo
c oss-linking,
pu iﬁed
VP3
(0.25
mg/ml,
ﬁnal
concen a ion)
was
ea ed
wi h
dime hyl
pimelimida e
(Sigma,
0.75
mg/ml,
ﬁnal
concen a ion),
bo h
dissol ed
in
phospha e
bu e ed
saline,
pH
8.0.
The
eac ion
was
incuba ed
a
oom
empe a u e
o
30
min,
and
he ea e
s opped
by
addi ion
o
T is-HCL
bu e ,
pH
8.0,
o
a
ﬁnal
concen a ion
o
20
mM.
Bo h
na i e
and
c oss-linked
VP3
we e
ea ed
wi h
2×
Laemmli
p o ein
sample
bu e
(100
mM
T is
pH
6.8,
2%
SDS,
0.001%
b omophenol
blue,
20%
Glyce ol
and
2%
␤-me cap oe hanol)
a
a
1:1
a io,
hea ed
a
95◦C
o
10
min,
and
hen
un
on
a
13.6%
SDS-polyac ylamide
gel
(29:1
ac ylamide/bis-
ac ylamide)
as
desc ibed
(Laemmli,
1970).
Fig.
1.
S uc u al
Cha ac e iza ion
o
No a
i us
VP3
(A)
SDS-polyac ylamide
gel
elec opho esis
o
VP3
monome
and
c oss-linked
VP3
ime .
Ma ke :
PageRule
p es ained
p o ein
ladde
(The mo
Scien iﬁc).
(B)
Nano-Elec osp ay
GEMMA
anal-
ysis
o
No a
i us
VP3
(0.05
mg/ml)
a
2
psi
capilla y
p essu e.
(C)
Fa -UV
Ci cula
dich oism
analysis
o
No a
i us
VP3,
p esen ed
as
an
a e age
o
5
scans
aken
a
50
nm/min,
showing
a
spec um
ha
is
ypical
o
␣-helical
p o eins.
22
S.A.
Sadanandan
e
al.
/
Vi us
Resea ch
223
(2016)
20–27
2.4.
Ci cula
dich oism
CD
spec a
o
No a
i us
VP3
we e
ob ained
on
a
Jasco
Spec-
opola ime e
model
J-715
(Jasco
Co po a ion).
The
spec a
we e
collec ed
a
25◦C
in
a
s anda d
phospha e
bu e ed
saline
(pH
7.4)
using
a
qua z
cu e e
o
0.1
cm
pa h
leng h.
Shown
spec a
ep e-
sen
he
a e age
o
ﬁ e
scans
a
a
speed
o
50
nm/min.
Only
he
a
UV
egion
om
190
nm
o
260
nm
was
analyzed.
2.5.
Sequence
da a
and
seconda y
s uc u e
p edic ion
Seconda y
s uc u es
we e
p edic ed
using
he
Ne Su P
1.1
se e
(Pe e sen
e
al.,
2009)
and
he
Ne Tu nP
1.0
se e
(Pe e sen
e
al.,
2010),
accessed
ia
he
CBS
P edic ion
Se e s
(h p://www.
cbs.d u.dk/se ices).
P edic ion
o
coiled
coil
was
pe o med
using
he
Ma coil
se e
(Delo enzi
and
Speed,
2002)
accessed
ia
he
Bioin o ma ics
Toolki
a
he
Max-Planck
Ins i u e
o
De elopmen-
al
Biology
(h p:// oolki . uebingen.mpg.de/ma coil),
he
Pai coil2
se e
(McDonnell
e
al.,
2006)
and
he
Mul iCoil
se e
(Wol
e
al.,
1997),
bo h
accessed
a
he
MIT
Compu e
Science
and
A iﬁcial
In elligence
Labo a o y
(h p://g oups.csail.mi .edu/pai coil2.h ml
and
h p://g oups.csail.mi .edu/cb/mul icoil/cgi-bin/mul icoil.cgi)
2.6.
Es ablishing
wild
ype
and
mu an
No a
i us
in ec ion
in
ﬂies
The
in ec ious
cDNA
clone
o
No a
i us
(Eks öm
e
al.,
2011)
was
used
o
cons uc
VP3
mu an s
using
s anda d
molecula
cloning
echniques.
Lis
o
p ime s
used
o
cons uc
he
di e -
en
mu an s
is
gi en
in
Table
S1
o
Supplemen a y
ma e ials.
All
expe imen s
we e
pe o med
using
RelishE23 (Hedeng en
e
al.,
1999)
ﬂies,
ea ed
a
25◦C
on
s anda d
mashed-po a o
ﬂy
ood
(Yang
e
al.,
2015).
The
cons uc ed
clones
we e
mic oinjec ed
in o
decho iona ed
syncy ial-s age
emb yos
(Sp adling,
1986).
To
induce
exp ession
he
la ae
we e
subsequen ly
ea ed
on
ﬂy
ood
con aining
0.5
mM
CuSO4,
un il
hey
eached
he
adul
s age.
2.7.
Vi us
pa icle
pu iﬁca ion
Vi us
pa icles
we e
pu iﬁed
om
whole
ﬂy
ex ac
and
om
he
eces
o
in ec ed
ﬂies.
To
ex ac
i us
pa icles
om
in ec ed
ﬂies,
10–15
ﬂies
we e
homogenized
wi h
a
homogenizing
pes le
in
NT-
bu e
(100
mM
NaCl
and
10
mM
T is-HCl,
pH
7.4).
To
ex ac
i us
pa icles
om
eces,
eces
was
collec ed
om
10
o
15
ﬂies
by
keep-
ing
hem
in
15-ml
cen i uge
ubes
o
2–4
h
and
washing
he
ube
walls
wi h
NT-bu e .
T i on
X-100
was
added
o
a
ﬁnal
concen a-
ion
o
0.5%
o
he
whole
ﬂy
ex ac
and
0.01%
o
he
eces.
Vi us
pu iﬁca ion
was
pe o med
as
desc ibed
p e iously
by
Ande son
e
al.
(1966).
B ieﬂy,
he
i us
pa icles
we e
pelle ed
by
cen i u-
ga ion
a
38000
pm
o
70
min
using
he
SW41Ti
o o
(Beckman
Coul e ).
Pelle ed
pa icles
we e
dissol ed
in
1
ml
NT-T
bu e
(NT
bu e ,
0.01%
T i on
X-100)
and
loaded
on
op
o
he
suc ose
g a-
dien s.
Ra e-zonal
sepa a ion
was
pe o med
on
a
35–12%
(w/ )
linea
suc ose
g adien
in
NT-T
bu e
a
38000
pm,
o
90
min
in
4◦C.
G adien
ac ions
we e
collec ed
by
punc u ing
he
bo om
o
he
ubes
and
collec ing
0.5
ml/ ac ion.
Quan i a i e
RT-PCR
was
used
o
de e mine
i us
peak
ac ions.
2.8.
Quan i a i e
RT-PCR
To al
RNA
was
ex ac ed
om
he
samples
using
Au um
To al
RNA
Mini
Ki
(BioRad).
Quan i a i e
RT-PCR
was
pe o med
as
desc ibed
p e iously
(Habayeb
e
al.,
2009;
Eks öm
e
al.,
2011)
using
o wa d
p ime :
5-TTTCACTTTACTGTTGGTCTCC-3,
e e se
P ime :
5-ATTCCATTTGTGACTGATTTTATTTC-3and
Taq-
man
p obe:
5-FAM-AGAGTTAGTGGACAAGTTAGAGACTGGCAT-
TAMRA-3.
2.9.
P o ease
clea age
analysis
Wild- ype
and
mu an
i us
pa icles
pu iﬁed
by
a e-zonal
sepa a ion,
we e
subjec ed
o
p o ease
clea age.
They
we e
ea ed
wi h,
ei he
2000
U/ml
ypsin
(#T8003,
Sigma-Ald ich)
o
0.05
U/ml
panc ea ic
p o ease
(#P4630,
Sigma-Ald ich),
o
2
h
a
37◦C.
Bo h
ypsin
and
p o ease
we e
dissol ed
in
phospha e
saline
bu e ,
pH
7.4.
Following
his,
he
ea ed
pa icles
we e
incuba ed
wi h
a
p o ease
inhibi o
cock ail
(#1697498,
Sigma-Ald ich)
and
RNase
A
(The mo
Scien iﬁc),
o
30
min
a
37◦C.
To al
RNA
was
ex ac ed
and
i us
i e
was
de e mined
by
quan i a i e
RT-PCR
(as
desc ibed
in
Sec ion
2.6).
3.
Resul s
3.1.
VP3
o ms
ime s
In
o de
o
cha ac e ize
VP3
biochemically,
we
cloned
and
exp essed
he
p o ein
in
a
bac e ial
exp ession
ec o .
The
pu iﬁed
VP3
p o ein
uns
like
a
29–31
kDa
p o ein
on
an
SDS
gel
(Fig.
1A).
This
is
in
ag eemen
wi h
he
molecula
mass
p edic ed
om
he
sequence,
31–34
kDa,
depending
on
he
posi ion
o
he
amino
e -
minus.
I
is
also
consis en
wi h
he
p e iously
es ima ed
35
kDa
o
VP3
om
in ac
i ions
(Eks öm
e
al.,
2011).
Nano-Elec osp ay
GEMMA
de ec s
he
elec opho e ic
mobil-
i y
o
pa icles
in
ai ,
which
is
used
o
calcula e
molecula
diame e
and
consequen ly,
he
molecula
mass
o
he
pa icles.
We
ha e
used
his
me hod
o
ﬁnd
he
na i e
oligome ic
na u e
o
pu i-
ﬁed
VP3
p o ein.
GEMMA
shows
a
s ong
peak
a
97
kDa
(Fig.
1B),
which
co esponds
wi h
he
ime ic
o m
o
VP3.
Mino
peaks
a e
obse ed
a
33,
68,
and
190
kDa,
sugges ing
he
p esence
o
minimal
amoun s
o
in e media y
monome ic,
dime ic
and
hex-
ame ic
o ms
o
he
p o ein.
As
an
independen
es
o
he
esul s
ob ained
h ough
GEMMA,
we
c oss-linked
VP3
using
dime hyl
pimelimida e
and
analyzed
he
c oss-linked
p o ein
on
an
SDS-
polyac ylamide
gel
(Fig.
1A).
The
c oss-linked
p o ein
appea s
o
be
a ound
97
kDa,
which
would
co espond
o
a
VP3
ime .
Taken
oge he ,
ou
esul s
show
ha
No a
i us
VP3
has
a
p opensi y
o
o m
ime s.
3.2.
VP3
is
p edominan ly
˛-helical
Seconda y
s uc u e
p edic ion
o
VP3
had
indica ed
ha
he
N- e minal
egion
is
␣-helical
and
ha
he
egion
downs eam
o
his
showed
g ea e
likelihood
o
o ming
␤-s ands
(Eks öm
e
al.,
2011).
Ci cula
dich oism
was
pe o med
on
pu iﬁed
VP3
o
expe imen ally
alida e
he
p edic ed
seconda y
s uc u e.
The
CD
spec um
o
VP3
(Fig.
1C)
shows
a
posi i e
band
a
195
nm
and
wo
nega i e
bands
a
209
nm
and
220
nm.
Such
a
spec um
is
cha ac-
e is ic
o
␣-helical
p o eins
(G eenﬁeld,
2006).
On
calcula ing
he
pe cen ages
o
di e en
s uc u al
elemen s
in
he
p o ein,
using
he
CDNN
CD
decon olu ion
so wa e
(Böhm
e
al.,
1992),
i
was
ound
o
be
app oxima ely
77%
␣-helical,
10%
␤- u n,
8%
andom
coil
and
he
emaining
an ipa allel/pa allel
shee s.
These
esul s
pa allel
he
seconda y
s uc u e
p edic ion
o
No a
i us
VP3
pe -
o med
p e iously
by
Eks öm
e
al.
(2011)
3.3.
A
ime ic
coiled-coil
s uc u e
is
p edic ed
o
VP3
homologs
in
No a-like
i uses
The
simila i y
o
No a
i us
o
p e iously
desc ibed
i uses
is
la gely
es ic ed
o
he
eplica i e
casse e
encoded
by
ORF2,
which
shows
homology
o
pico na-like
i uses
(Habayeb
e
al.,
S.A.
Sadanandan
e
al.
/
Vi us
Resea ch
223
(2016)
20–27
23
Fig.
2.
Sequence
analysis
and
seconda y
s uc u e
p edic ion
o
VP3.
(A)
P obabili ies
o
␣-helix
( ed),
␤-s and
(blue)
and
coiled
coil
acco ding
o
Ma coil
(solid
black)
o
Pai coil2
(dashed
black),
and
ela i e
p obabili ies
o
coiled
coil
dime s
(pu ple)
o
ime s
(g een)
a e
shown
as
sliding
a e ages
o
13
amino
acids.
(B)
Schema ic
ep esen a ion
o
p edic ed
s uc u al
elemen s
in
VP3.
The
p obabili y
o
each
amino
acid
o
eside
in
any
o
he
se en
possible
posi ions,
a-g,
o
a
coiled
coil
is
indica ed
by
di e en
shades
o
ed.
P edic ed
␤- u ns
a e
shown
in
g ey
and
helix-b eaking
p oline
esidues
a e
black.
The
p edic ed
coiled
coil
segmen s,
CC1-CC3,
a e
delinea ed
by
blue
lines.
(C)
Sequence
conse a ion
be ween
h ee
D osophila
No a
i uses,
D.
melanogas e ,
D.
immig ans
and
D.
subobscu a;
3/3
indica es
ull
iden i y.
The
egion
ha
o e laps
wi h
ORF2
is
shown
g ey.
The
pe cen
o
esidues
ha
a e
ully
conse ed
is
indica ed
o
he
di e en
sub- egions
below
he
g aph.
Also
shown
is
he
egion
ha
is
conse ed
in
i uses
om
he
eph i id
ﬂies.
2006;
Koonin
e
al.,
2008).
TBLASTN
sea ch
in
he
EST
da abase
had
ea lie
iden iﬁed
ORF3-
and
ORF4- ela ed
EST
sequences
om
he
pa asi oid
wasp,
Nasonia
i ipennis
(Oli ei a
e
al.,
2010;
Eks öm
e
al.,
2011),
showing
ha
his
wasp,
o
possibly
i s
hos ,
ha bo ed
a
i us
ha
is
ela ed
o
he
D osophila
No a
i us.
Seconda y
s uc-
u e
es ima ion
showed
ha
he
VP3
homolog
in
his
Nasonia
No a
i us
also
had
a
pu a i e
coiled
coil
domain
in
he
N- e minal
pa
o
he
p o ein
(Eks öm
e
al.,
2011).
Addi ional
No a-like
sequences
a e
now
a ailable,
allowing
a
mo e
de ailed
compa a i e
s udy
o
VP3- ela ed
sequences.
Th ee
no el
No a-like
i uses
ha e
ecen ly
been
desc ibed,
wo
om
he
D osophila
species
D.
immig ans
and
D.
subobscu a
and
one
om
he
mo h
Spodop e a
exigua
(Jakubowska
e
al.,
2014;
an
Mie lo
e
al.,
2014).
Fu he mo e,
h ough
TBLASTN
sea ches
in
he
T ansc ip ome
Sho gun
Assembly
da abase,
we
could
e ie e
addi ional
No a-like
i us
sequence
assemblies
om
h ee
eph i id
ﬂies,
wo
addi ional
mo hs,
one
an
and
one
pa a-
si oid
wasp
(Table
S2),
as
well
as
sho e
sequence
agmen s
om
se e al
o he
insec
species.
These
sequences
we e
deﬁned
as
No a-
like
by
wo
c i e ia:
a)
hey
encode
ORF4
capsid
p o eins
o
he
unique
No a
i us
ype
and
b)
hei
eplica ion
casse es
a e
mo e
closely
ela ed
o
he
D.
melanogas e
No a
i us
ORF2
p o eins
han
o
o he
pico na-like
homologs.
We
will
he e
e e
o
all
No a
i us
homologs
as
No a
i uses.
No iceably,
he
ela ionship
be ween
he
di e en
No a
i uses
closely
mi o s
ha
o
hei
insec
hos s,
sug-
ges ing
a
long
his o y
o
co-e olu ion.
ORF1
and
ORF3
sequences
we e
poo ly
conse ed
be ween
he
di e en
No a
i uses,
bu
an
ORF3
homolog
could
always
be
iden iﬁed
as
a
sepa a e
open
ead-
ing
ame
be ween
he
be e
conse ed
ORF2
and
ORF4- ela ed
sequences.
Fig.
2
illus a es
p edic ed
seconda y
s uc u e
elemen s
in
he
VP3
p o ein
o
D.
melanogas e
No a
i us.
A
egion
spanning
he
ﬁ s
app oxima ely
200
amino
acids
displays
a
high
p obabili y
o
an
␣-helical
con o ma ion
( ed
cu e
in
Fig.
2A),
wi h
a
high
p ob-
abili y
o
coiled-coil
o ma ion
be ween
esidues
36–200
(black
cu e),
while
he
egion
downs eam
is
p edic ed
o
ha e
s e ches
o
␤-s ands
(blue
cu e)
in e up ed
by
␤- u ns
(g ey-shaded
in
Fig.
2B).
This
p edic ion
o
a
seconda y
s uc u e
is
ema kably
con-
se ed
among
he
No a
i uses,
in
pa icula
o
he
dip e an
and
lepidop e an
i uses
(Fig.
S1).
Howe e ,
he
p ima y
amino
acid
sequences
a e
poo ly
conse ed,
allowing
a
consis en
alignmen
only
o
he
i uses
om
he
h ee
D osophila
species
(Fig.
S2).
Only
when
we
used
an
algo i hm
ha
akes
seconda y
s uc u es
in o
conside a ion
was
i
possible
o
align
VP3
sequences
om
all
No a-
like
i uses
(Fig.
S3).
A
bes ,
we
see
a
modes
ex en
o
sequence
conse a ion
o
amino
acids
1–200,
while
he
egion
downs eam
is
e y
poo ly
conse ed,
ba ing
a
ew
amino
acid
posi ions.
A
sequence
simila i y
wi h
a
consis ency
sco e
o
5
o
abo e
was
seen
only
a
19
amino
acid
posi ions,
all
o
which
all
wi hin
he
␣-helical
egion.
Hence,
seconda y
s uc u e
p edic ion
is
conse ed,
despi e
he
modes
sequence
simila i y,
sugges ing
a
i al
unc ion
o
he
␣-helical
s uc u al
domain.
A
close
inspec ion
o
he
p edic ed
coiled
coil
egion
shows
u -
he
in e es ing
ea u es
in
VP3
o
he
D.
melanogas e
No a
i us.
Unlike
he
Ma coil
p edic ion
(solid
black
line
in
Fig.
2A),
he
Pai -
coil
algo i hm
p edic s
ha
he
coiled
coil
is
spli
in o
h ee
sepa a e
segmen s,
labeled
CC1-3
in
Fig.
2A,
wi h
a
ela i ely
low
coiled
coil
p obabili y
o
he
middle
segmen
(dashed
line
in
Fig.
2).
The
epea
uni
o
a
coiled
coil
is
se en
amino
acids,
o
abou
wo
u ns
o
he
␣-helix.
Fig.
2B
illus a es
he
colo -coded
p obabili y
o
each
amino
acid
in
VP3
o
be
in
any
o
hese
se en
posi ions,
labeled
a-g.
Fo
he
p edic ed
coiled
coil
segmen
CC3
his
gene a es
a
eg-
ula
pa e n
whe e
each
7-amino
acid
epea
is
ep esen ed
as
a
ed
diagonal.
The
phase
o
his
pa e n
shi s
by
one
posi ion
a
he
bo de
be ween
CC2
and
CC3,
sugges ing
a
dis up ion
o
he
coiled
coil
s uc u e
a
his
poin .
A
second
se
o
diagonals
in
CC1
and
CC2,
shi ed
by
h ee
posi ions
om
he
majo
diagonals,
indica es
wo
possible
o ien a ions
o
hese
segmen s.
In
he
D.
immig ans
No a
i us
he e
a e
p edic ed
phase
shi s
be ween
CC1
and
CC2,
as
well
as
be ween
CC2
and
CC3
(Fig.
S1),
sugges ing
discon inu-
i ies
be ween
all
h ee
segmen s.
Finally,
while
no
phase
shi s
a e
p edic ed
in
he
co esponding
coiled
coil
egion
o
he
No a
i us
om
D.
subobscu a,
a
helix-b eaking
p oline
esidue
a
he
bo de
be ween
CC2
and
CC3
(black
line
in
Fig.
2C)
indica es
ha
he
egu-
la
␣-helical
con o ma ion
is
in e up ed
in
his
place.
We
conclude
ha
he
mos
likely
con o ma ion
o
VP3
in
all
h ee
D osophila
i uses
include
wo
o
h ee
sepa a e
coiled
coil
segmen s.
O
hese,
24
S.A.
Sadanandan
e
al.
/
Vi us
Resea ch
223
(2016)
20–27
a
Mul icoil
p edic ion
sugges s
ha
CC1
is
mos
likely
engaged
in
coiled
coil
ime s,
while
he
p edic ion
o
CC2
is
mo e
unce ain
(g een
cu es
in
Fig.
2
and
Fig.
S1).
The
p edic ion
o
a
coiled
coil
ime
is
consis en
wi h
ou
obse a ion
wi h
GEMMA
ha
VP3
has
a
endency
o
o m
ime s
(Fig.
1A).
Howe e ,
CC3
is
p edic ed
o
o m
a
dime
in
he
D.
immig ans
and
D.
subobscu a
i uses
(pu -
ple
cu es)
and
he e
is
no
p edic ion
o
i s
highe
o de
s uc u e
in
he
D.
melanogas e
No a
i us.
A
sequence
compa ison
be ween
he
h ee
D osophila
No a
i uses
(Fig.
2C,
Fig.
S2)
shows
ha
he
CC1
segmen
is
highly
con-
se ed;
76%
o
he
posi ions
a e
iden ical
in
all
h ee
species.
CC2
is
less
well
conse ed
(23%
iden ical)
and
CC3
is
e en
mo e
a iable
(12%
iden ical)
and
he
la e
segmen
has
long
indels
a
he
N-
e minus
(Fig.
S2).
Ou side
he
coiled
coil
domains,
he
N- e minal
pa
is
highly
conse ed
(78%),
bu
ha
egion
pa ially
o e laps
wi h
he
polyme ase
o
ORF2
(g ey
egion
in
Fig.
2C
and
Fig.
S2)
and
i
is
unce ain
how
much
o
ha
sequence
is
included
in
he
ma u e
VP3.
Finally,
he
C- e minal
1/3
o
VP3,
which
is
domina ed
by
p edic ed
␤-s ands
and
u ns,
is
poo ly
conse ed.
Only
16%
o
he
esidues
a e
iden ical
in
he
h ee
D osophila
i uses,
mos
o
hem
in
he
C- e minal
50
amino
acids.
The
CC1
segmen
is
also
well
conse ed
in
he
No a
i uses
om
he
eph i id
ﬂies,
B.
la i ons,
B.
do salis
and
C.
capi a a.
A
egion
o
68
esidues,
ex ending
20
esidues
ups eam
o
CC1
and
downs eam
in o
he
bo de
egion
o
CC2,
is
38–40%
iden ical
o
he
co esponding
D.
melanogas e
sequence
(Fig.
2C).
Like
D.
sub-
obscu a,
he
eph i id
i uses
ha e
a
phase
shi
in
he
p edic ed
coiled
coil
a
he
CC1-CC2
bo de
(Fig.
S1).
Remaining
pa s
o
he
eph i id
i uses
and
he
en i e
sequences
o
he
lepidop e an
and
hymenop e an
i uses
a e
poo ly
conse ed
(Fig.
S2)
and
hey
can-
no
be
unambiguously
aligned
wi h
he
D osophila
i us
sequences,
bu
he
p edic ed
seconda y
s uc u es
a e
simila .
All
No a
i uses
ha e
a
egion
wi h
po en ial
o
o m
coiled
coil
ime s.
3.4.
VP3
is
c i ical
o
he
ecal-o al
ansmission
o
No a
i us
In
o de
o
cha ac e ize
he
unc ion
o
VP3
and
he
p edic ed
coiled-coil
mo i ,
we
cons uc ed
h ee
mu an s
(Fig.
3A)
using
he
cDNA
clone
o
No a
i us.
In
he
ﬁ s
mu an
(NV-VP3),
h ee
consecu i e
s op
codons
we e
in oduced
97
nucleo ides
down-
s eam
o
he
5end
o
ORF3,
o
24
nucleo ides
downs eam
o
he
o e lap
wi h
ORF2.
The
second
mu an
(NV-VP3CC)
is
a
dele ion
mu an ,
whe ein
54
nucleo ides
co esponding
o
ﬁ e
u ns
o
he
␣-helix,
we e
dele ed
om
ORF3,
dele ing
pa s
o
CC1
and
CC2.
In
he
hi d
mu an
(NV- uncVP3),
h ee
consecu i e
s op
codons
we e
in oduced
downs eam
o
he
posi ed
coiled-coil.
The
gene -
a ed
clones
we e
mic oinjec ed
in o
ui
ﬂy
emb yos
o
es ablish
i al
in ec ion.
To al
RNA
was
ex ac ed
om
injec ed
animals
and
hei
o sp ing
o
quan i a i e
RT-PCR
analysis,
in
o de
o
e alu-
a e
i us
i e s
(Fig.
3B,
no e
he
loga i hmic
alues).
The
wild- ype
cDNA
clone
o
No a
i us
(NV-W )
was
used
as
a
con ol
in
all
expe imen s.
In
p e ious
expe imen s
we
injec ed
ﬂy
emb yos
wi h
No a
i us
cDNA
clones
wi h
non- eplica ing
No a
i us
genome
sequences.
A e
he
injec ed
emb yos
g ew
in o
adul
we
nei he
de ec ed
emains
o
he
injec ed
cDNA
no
ansc ibed
i al
RNA.
In
con as ,
we
de ec ed
i us
i e s
in
all
h ee
i us
mu an s
es ed
in
his
wo k,
indica ing
ha
he
genomes
we e
eplica ing.
The
i us
i e
is
educed
in
he
NV-VP3
and
NV-VP3CC
mu an s,
bu
i
is
nea
he
wild- ype
le el
in
NV- uncVP3.
In
addi ion,
NV-W
and
he
NV- uncVP3
mu an s
a e
also
de ec ed
in
he
o sp ing
o
he
injec ed
animals.
Howe e ,
NV-VP3
and
NV-VP3CC
mu an s
a e
no
de ec able
in
he
o sp ing,
abo e
he
le el
whe e
non-speciﬁc
PCR
p oduc s
can
be
ound
(dashed
line
in
Fig.
3B).
This
indica es
ha ,
al hough
he
NV-VP3
and
NV-VP3CC
mu an s
ha e
epli-
ca ing
RNA
genomes,
hey
a e
no
ansmi ed
o
he
o sp ing.
I
is
in e es ing
o
no e
ha
dis up ion
o
he
coiled
coil
egion
leads
o
almos
he
same
pheno ype
as
emo ing
he
en i e
VP3
p o ein.
Dele ing
he
egion
downs eam
o
he
coiled
coil
appea s
o
ha e
a
sligh
e ec
on
he
ﬁ ness
o
he
i us,
bu
no
i s
eplica ion
o
ansmission,
since
he
NV- uncVP3
mu an
is
p esen
bo h
in
he
injec ed
animals
and
hei
o sp ing.
This
signiﬁes
ha
he
coiled
coil
egion
is
he
impe a i e
domain
o
VP3
and
ha
i
is
essen-
ial
o
he
ansmission
o
No a
i us
ia
he
ecal-o al
ou e.
This
opens
up
se e al
possibili ies,
one
o
which
is
a
ole
o
his
egion
in
he
assembly
o
i us
pa icles.
We
p oceeded
o
examine
whe he
hese
mu an s
we e
capa-
ble
o
assembling
i ions.
To
explo e
his
possibili y,
we
pu iﬁed
i us
pa icles
om
whole
ﬂy
ex ac
and
eces,
collec ed
om
ﬂies
in ec ed
wi h
NV-W ,
NV-VP3,
NV-VP3CC
o
NV- uncVP3.
Ra e-zonal
sepa a ion
was
used
o
pu i y
he
i us
pa icles
and
quan i a i e
RT-PCR
was
pe o med
on
o al
RNA
isola ed
om
he
suc ose-g adien
ac ions
(Fig.
3C).
In
his
scena io,
any
sig-
nal
ob ained
o
he
i us
will
ha e
o igina ed
om
assembled,
in ac
i ions.
Also,
unassembled
capsid
p o eins
will
sedimen
e y
di e en ly
as
compa ed
o
assembled
i us
pa icles.
Hence,
his
me hod
would
gi e
us
an
indica ion
as
o
whe he
i ions
ha e
been
assembled
o
he
di e en
mu an s.
Assembled
i us
pa icles
we e
de ec ed
in
in ec ed
ﬂies
o
all
h ee
mu an s,
sig-
ni ying
ha
mu a ion
o
VP3
did
no
a ec
he
ac ual
assembly
o
he
i ions.
Pa icles
we e
no
de ec ed
in
eces
o
he
NV-VP3
and
NV-VP3CC
mu an s
in
con as
o
NV-W
and
NV- uncVP3
mu an
pa icles,
which
we e
p esen
in
he
eces
also.
This
implied
ha ,
al hough
i ions
a e
assembled
o
all
he
mu an s,
only
he
NV- uncVP3
pa icles
can
be
de ec ed
in
he
eces.
The
ac
ha
he
NV-VP3
and
NV-VP3CC
pa icles
a e
no
de ec ed
in
he
eces
explains
why
hey
a e
no
ho izon ally
ansmi ed
o
he
o sp ing.
Taken
oge he ,
hese
esul s
indica e
ha
he
pu a i e
coiled-
coil
mo i
o
VP3
is
no
in ol ed
in
he
eplica ion
o
he
i al
genome
o
in
he
assembly
o
he
i ions.
This
domain
is
ins ead
c i ical
o
he
success ul
anspo
o
he
assembled
pa icles
o
he
eces
o
o
he
s abili y
o
he
pa icles
du ing
o
a e
ans-
po a ion
o
he
eces.
3.5.
VP3-coiled
coil
domain
aids
ansmission
by
s abilizing
he
i us
pa icles
The e
a e
se e al
p o eases
and
enzymes
wi hin
in ec ed
cells
ha
can
cause
ha m
o
i ions.
Addi ionally,
since
No a
i us
epli-
ca es
in
he
ui
ﬂy
midgu
(Habayeb
e
al.,
2009),
i
a els
h ough
he
hos ile
en i onmen
o
he
ﬂy
gu ,
wi h
a ying
pH
condi ions,
diges i e
enzymes
and
p o eases
(Lemai e
and
Miguel-Aliaga,
2013),
be o e
i
eaches
he
eces.
Upon
a i al
in
he
eces,
he
i ions
may
once
again
ace
enzymes
and
p o eases,
sec e ed
by
de eca ed
gu
mic obes,
be o e
success ully
in ec ing
a
new
hos .
We
pos ula ed
ha
he
conse ed
coiled-coil
domain
was
c ucial
o
he
s abili y
o
he
i ions
agains
ad e se
condi ions
wi hin
in ec ed
cells,
ei he
while
hey
unde ook
he
pe ilous
passage
h ough
he
ﬂy
gu ,
o
upon
de eca ion
along
wi h
he
eces.
Fi s ,
we
compa ed
he
s abili y
o
hea ed
mu an
pa icles
o
ha
o
wild
ype
pa icles.
Fo
his
pu pose,
NV-W
and
mu an
i i-
ons
we e
pu iﬁed
om
in ec ed
ﬂies
by
a e-zonal
sepa a ion
and
hei
con en
o
i al
RNA
was
quan iﬁed
as
shown
in
Fig.
3C.
Only
in ac
assembled
i us
pa icles
will
sedimen
a
ac ions
close
o
he
sedimen a ion
a e
o
wild- ype
pa icles.
Hence,
RNA
de ec ed
by
quan i a i e
RT-PCR
would
ha e
o igina ed
om
assembled
i i-
ons
o
i ion-like
s uc u es.
The
2.5
ml
and
3.0
ml
sedimen a ion
ac ions
we e
pooled
and
aliquo ed
in o
wo
equal
olumes.
One
ba ch
was
subjec ed
o
hea
ea men
a
55◦C
o
5
min,
ollowed
by
RNase
A
ea men
o
30
min
a
37◦C.
The
second
ba ch
was
subjec ed
only
o
RNase
A
ea men .
To al
RNA
was
ex ac ed
om
bo h
se s
o
pa icles
o
each
mu an
and
analyzed
by
quan-
i a i e
RT-PCR.
Any
signal
ob ained
would
ha e
o igina ed
om

S.A.
Sadanandan
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/
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Resea ch
223
(2016)
20–27
25
0
10000
20000
1.0
1.5 2.
0
2.5 3.0
3.5 4.0
4.5
Vi us i e, a bi a y uni s
Suc ose g adien ac ions, ml
0
10000
20000
1.0 1.5
2.0
2.5
3.0
3.5
4.0
4.5
Suc ose g adien ac ions, ml
ORF 1
ORF 2
ORF 3
ORF 4
No a i us genome
Helicase P o ease Polyme ase
Capsid p o eins
ORF 3
S op
NV- VP3
ORF 3
P edic ed coiled coil
ORF 3
ORF 3
Dele ion
S op
P edic ed coiled coil
P edic ed coiled coil
NV-VP3 CC
NV- uncVP3
NV-w
...CGCCGTTT
AATGAAGA......GTTTAGAA
...GAATTTAC
NV-W
NV-
VP3
NV-VP
3
CC
NV- uncVP3
A
0
2
4
6
8
10
Log i us i e, a bi a y uni s
Injec ed animals
O sp ing
nd
B
C
Whole lies Feces
Vi us i e, a bi a y uni s
NV-ΔVP3
NV-W
NV- uncVP3
NV-VP3ΔCC
Fig.
3.
VP3
is
equi ed
o
he
ansmission
o
No a
i us
ia
he
ecal-o al
ou e.
(A)
Schema ic
ep esen a ion
o
No a
i us
VP3
mu an s
gene a ed.
(B)
No a
i us
was
quan iﬁed
by
quan i a i e
RT-PCR
on
o al
RNA
om
RelishE23 ﬂies
and
hei
o sp ing,
in ec ed
wi h
ei he
unmodiﬁed
No a
i us
(NV-W )
o
any
one
o
he
VP3
mu an s
(NV-VP3,
NV-VP3CC
and
NV- uncVP3).
The
do ed
line
ep esen s
he
poin
below
which
PCR
a i ac s
appea ,
a e
35
cycles
o
ampliﬁca ion.
Such
signals
a e
also
obse ed
in
nega i e
con ols
wi hou
any
empla e
RNA.
(C)
Vi us
pa icles
we e
pu iﬁed
om
whole
RelishE23 ﬂies
o
om
eces
o
such
ﬂies,
in ec ed
wi h
ei he
NV-W
o
any
one
o
he
VP3
mu an s,
by
a e-zonal
sepa a ion
on
a
suc ose
g adien .
Quan i a i e
RT-PCR
was
pe o med
on
o al
RNA
ex ac ed
om
each
collec ed
ac ion
o
he
suc ose
g adien .
Fo
(B)
and
(C),
he
esul s
shown
a e
an
a e age
o
a
leas
3
independen
expe imen s
and
e o
ba s
indica e
s anda d
de ia ion.
Be ween
5–30
ﬂies
we e
used
o
each
expe imen ,
and
he
same
numbe
o
ﬂies
we e
used
o
he
con ol
and
he
mu an
being
es ed
in
ha
pa icula
expe imen .
in ac
i us
pa icles,
as
ee
RNA
eleased
due
o
capsid
disin e-
g a ion
would
be
deg aded
du ing
RNase
A
ea men .
This
would
hence
gi e
us
an
es ima e
o
he
pa icles
ha
su i ed
hea
ea -
men
wi hou
deg ada ion.
As
shown
in
Fig.
4A,
he
NV-W
and
NV- uncVP3
pa icles
esis
hea
ea men
and
can
be
de ec ed
a
i e s
close
o
he
unhea ed
con ols.
On
he
o he
hand,
he
NV-VP3
and
NV-VP3CC
pa icles
a e
almos
en i ely
decima ed
(a
leas
1000- old
dec ease)
and
can
ba ely
be
de ec ed
a e
hea
ea men .
This
clea ly
shows
ha
he
abili y
o
No a
i us
pa icles
o
wi hs and
hea
ea men
is
dependen
on
he
coiled-coil
mo i
bu
no
he
C- e minal
hi d
o
he
VP3
p o ein.
We
nex
p oceeded
in
a
simila
way
o
examine
whe he
VP3
was
impo an
o
he
s abili y
o
he
i ions
when
ea ed
wi h
p o eases.
The
combined
2.5
ml
and
3.0
ml
ac ions
o
a e-zonally
pu iﬁed
NV-W
and
mu an
i us
pa icles
we e
di ided
in o
h ee
equal
olumes.
The
ﬁ s
se
was
ea ed
wi h
ypsin
and
he
second
se
was
ea ed
wi h
panc ea ic
p o ease,
bo h
ollowed
by
ea -
men
wi h
p o ease
inhibi o
cock ail
and
RNase
A.
The
hi d
se
used
as
con ol
was
ea ed
only
wi h
p o ease
inhibi o
and
RNase
A.
Quan i a i e
RT-PCR
was
pe o med
on
o al
RNA
om
he
pa -
icles.
Like
in
he
hea - ea men
expe imen ,
he
NV-W
pa icles
su i ed
bo h
ypsin
and
p o ease
ea men
wi hou
any
signi -
ican
loss
o
i ions
(Fig.
4B).
The
NV- uncVP3
i ions
appea
o
be
only
pa ially
esis an
o
ypsin
and
p o ease
ea men
as
a
en- old
loss
o
i ions
was
obse ed.
On
he
o he
hand,
NV-VP3
and
NV-VP3CC
mu an
pa icles
we e
almos
comple ely
disin-
eg a ed
as
e y
li le
i al
RNA
was
de ec ed
om
hese
pa icles.
This
clea ly
signiﬁes
he
impo ance
o
VP3
in
he
s abili y
o
he
capsid
s uc u e
unde
p o ease
ea men .
Based
on
he
esul s
om
he
hea - ea men
expe imen
and
he
p o ease
clea age
analysis,
i
is
appa en
ha
VP3
is
c i ical
o
he
s abili y
o
No a
i us
i ions.
Speciﬁcally,
he
coiled-coil
mo i
appea s
o
be
a
unc-
ionally
impo an
domain
in ol ed
in
con e ing
s abili y
o
he
capsid
s uc u e.
The
egion
downs eam
o
he
coiled-coil
also
26
S.A.
Sadanandan
e
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/
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Resea ch
223
(2016)
20–27
0
1
2
3
4
5
Log i us i e, a bi a y uni s
RNase ea men
Hea ➔ RNase ea men
Log i us i e, a bi a y uni s
0
1
2
3
4
5
P o ease inhibi o
➔
RNase ea men
P o ease
➔
P o ease inhibi o
➔
RNase ea men
T ypsin
➔
P o ease inhibi o
➔
RNase ea men
A
B
NV-ΔVP3
NV-W
NV- uncVP3
NV-VP3ΔCC
NV-ΔVP3
NV-W
NV- uncVP3
NV-VP3ΔCC
Fig.
4.
VP3
is
c ucial
o
he
s abili y
o
No a
i us
Vi ions.
(A)
Pu iﬁed
i us
pa icles
we e
hea - ea ed
a
55◦C
o
5
min,
ollowed
by
RNase
A
ea men .
Con ol
samples
we e
ea ed
only
wi h
RNase.
qRT-PCR
was
pe o med
on
RNA
om
bo h
hea - ea ed
and
unhea ed
pa icles
o
es ima e
i us
i e .
This
g aph
shows
he
loss
o
NV-VP3
and
NV-VP3CC
i ions
upon
hea - ea men .
(B)
Pu iﬁed
i us
pa icles
we e
1)
ea ed
wi h
ypsin,
ollowed
by
ea men
wi h
a
p o ease
inhibi o
cock ail
and
RNase,
2)
ea ed
wi h
p o ease,
ollowed
by
ea men
wi h
a
p o ease
inhibi o
cock ail
and
RNase,
3)
ea ed
wi h
only
he
p o ease
inhibi o
cock ail
and
RNase.
Quan i a i e
RT-PCR
was
pe o med
on
RNA
om
all
pa icles
o
calcula e
i us
i e .
This
g aph
shows
he
se e e
disin eg a ion
o
NV-VP3
and
NV-VP3CC
i ions
and
a
pa ial
loss
o
NV- uncVP3
i ions
upon
ea men
wi h
ypsin
and
p o ease.
Fo
(A)
and
(B),
he
esul s
shown
a e
an
a e age
o
3
expe imen s,
whe ein
he
e o
ba s
indica e
s anda d
de ia ion.
Be ween
10–20
ﬂies
we e
used
o
each
expe imen ,
bu
he
same
numbe
o
ﬂies
we e
used
o
he
con ol
and
he
mu an
being
es ed
in
ha
pa icula
expe imen .
The
do ed
line
ep esen s
he
poin
below
which
all
alues
a e
conside ed
o
be
PCR
a i ac s
since
hese
a e
ob ained
om
quan i a i e
RT-PCR
signals
ha
appea
a e
35
cycles
o
ampliﬁca ion.
Such
signals
a e
also
obse ed
in
nega i e
con ols
wi hou
any
empla e
RNA.
seems
o
ha e
a
mode a e
e ec
on
he
s abili y
o
capsid
p o eins,
bu
i
is
appa en ly
no
c ucial.
4.
Discussion
Ou
esul s
show
ha
No a
i us
VP3
is
a
p ima ily
␣-helical
p o ein
ha
o ms
ime s
in
i s
na i e
con o ma ion.
The
␣-helical
egion
is
p edic ed
o
o m
a
coiled-coil
domain,
spli
in o
h ee
pa s,
and
compa isons
wi h
sequences
om
o he
No a
i uses
ha e
shown
ha
his
a angemen
is
well
conse ed.
To
he
bes
o
ou
knowledge,
no
known
s uc u al
p o eins
o
he
pico na-
like
i uses
ha e
been
p edic ed
o
shown
o
ha e
his
mo i .
The
appea ance
o
VP3,
along
wi h
VP4
p o eins
in
i us
pa icles
(Eks öm
e
al.,
2011)
sugges ed
ha
i
should
be
associa ed
wi h
he
capsid
p o eins.
I
was
shown
ha
he
s uc u al
p o eins
o
No a
i us
a e
dis inc ly
di e en
om
ha
o
known
pico na-like
i uses
(Eks öm
e
al.,
2011),
bu
he
ole
o
VP3
o
i s
associa ion
wi h
he
capsid
has
no
been
s udied
be o e.
In
Pico na i idae,
each
capsid
p o ein,
VP1,
VP2,
VP3
and
VP4
(un ela ed
o
he
No a
i us
p o eins
wi h
simila
names),
has
a
ole
in
he
di e en
s ages
o
capsid
assembly,
ma u a ion
and
s a-
bili y
(A nold
e
al.,
1987;
Hellen
and
Wimme ,
1992a;
Hellen
and
Wimme ,
1992b;
Cu y
e
al.,
1997;
Lin
e
al.,
2009;
Jiang
e
al.,
2014).
Hence,
mu a ions
in
any
o
hese
p o eins
can
ad e sely
a ec
one
o
mo e
s ages
o
he
i us
li e
cycle.
Fo
example,
mu a-
ions
in
he
Polio i us
VP2
p o ein
can
dis up
he
comple ion
o
mo phogenesis
and
he
e en ual
o ma ion
o
ma u e
i al
pa i-
cles
(Comp on
e
al.,
1990).
Fu he ,
single
amino
acid
subs i u ions
in
he
Foo
and
Mou h
disease
i us
VP1
p o ein
can
ende
he
i us
acid-s able,
while
amino
acid
subs i u ions
in
he
VP3
p o-
ein
ende
he
i us
acid-labile
(Ca idi
e
al.,
2015).
Simila
e ec s
ha e
been
obse ed
in
o he
i us
amilies
such
as
he
Calici i idae
and
Toga i idae
also.
In
he
No walk
i us,
he
VP2
p o ein
asso-
cia ed
wi h
he
shell
domain
o
he
i al
capsid
(Vongpunsawad
e
al.,
2013)
is
known
o
be
c ucial
o
he
exp ession
and
s abili y
o
he
capsid
p o eins
(Be olo i-Cia le
e
al.,
2003).
An
Alpha i us
nucleocapsid
p o ein
con ains
a
p edic ed
␣-helical
coiled
coil
domain
ha
is
essen ial
o
he
assembly
o
he
nucleocapsid
co e
(Pe e a
e
al.,
2001).
Simila ly,
he
coiled
coil
domain
o
adeno i us
p o ein
pIX
is
s a ed
o
ha e
an
impo an
ole
in
sel -associa ion
and
ime
o ma ion
o
pIX
molecules
(Rosa-Cala a a
e
al.,
2001).
The
co ec
s uc u al
associa ion
o
pIX
is
c i ical
o
he
icosahe-
d al
capsid
s uc u e
o
adeno i uses
( an
Oos um
and
Bu ne ,
1985;
Fu cini i
e
al.,
1989;
S ewa
e
al.,
1993).
Thus,
he
ole
o
ce ain
capsid-associa ed
p o eins
in
he
assembly,
s abili y
and
ma u a ion
o
i ions
ha e
been
p o en
in
se e al
i uses.
Ou
wo k
on
No a
i us
VP3
has
e ealed
ha
his
capsid-associa ed
p o ein
is
indispensable
o
he
s abili y
o
he
i ion
s uc u e.
Dis up ing
he
coiled-coil
domain
o
No a
i us
VP3
has
se ious
implica ions
on
he
s abili y
o
he
i ions,
such
ha
i
a ec s
he
ho izon al
ansmission
o
he
i us.
This
u he
ein o ces
ou
p emise
ha
No a
i us
VP3
is
absolu ely
essen ial
o
he
s uc u al
s abili y
o
he
capsid
p o eins.
To
unde s and
he
molecula
mechanisms
in ol ed
in
capsid
s abili y
is
impo an
no
only
o
gain
knowledge
o
an
elemen a y
aspec
o
No a
i us
biology,
bu
also
o
cha ac e ize
he
hos -
pa hogen
ela ionship
be ween
a
small
RNA
i us
and
i s
hos .
This
ela ionship
be ween
No a
i us
and
he
ui
ﬂy
also
happens
o
be
an
excellen
model
o
s udy
no el
pa hways
in ol ed
in
immune
esponses
agains
small
RNA
i uses.
Unde s anding
he
biology
o
i uses
also
helps
us
unde s and
be e
he
di e en
echniques
hey
use
o
modi y
cellula
beha io
and
o
supp ess
an i- i al
esponses.
I
would
indeed
be
e y
in e es ing
o
u he
dissec
he
mechanism
behind
how
VP3
p o ides
s abili y
o
he
No a
i us
i ions.
Conﬂic
o
in e es s
The
au ho s
decla e
ha
he e
is
no
conﬂic
o
in e es s.
S.A.
Sadanandan
e
al.
/
Vi us
Resea ch
223
(2016)
20–27
27
Acknowledgemen s
The
pu iﬁca ion
o
VP3
was
planned
and
pe o med
by
he
Umeå
P o ein
Expe ise
Pla o m.
We
hank
Jö gen
Åden
(Depa men
o
Chemis y,
Umeå
Uni e si y)
o
helping
us
pe o m
Ci cula
Dich oism
on
VP3.
This
esea ch
was
suppo ed
by
g an s
om
he
Swedish
Resea ch
Council,
he
Academy
o
Finland
and
he
Sig id
Juselius
Founda ion.
Appendix
A.
Supplemen a y
da a
Supplemen a y
da a
associa ed
wi h
his
a icle
can
be
ound,
in
he
online
e sion,
a
h p://dx.doi.o g/10.1016/j. i us es.2016.06.
011.
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VP3 is crucial for the stability of Nora virus virions

Abstract

VP3 is crucial for the stability of Nora virus virions
