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Expression of Aedes trypsin-modulating oostaticfactor on the
virion of TMV: A potential larvicideDov Borovsky*†, Shailaja
Rabindran‡§, William O. Dawson‡, Charles A. Powell¶, Donna A.
Iannotti*, Timothy J. Morris*,Jeffry Shabanowitz�, Donald F.
Hunt**, Hendrik L. DeBondt††‡‡, and Arnold DeLoof§§
*Florida Medical Entomology Laboratory, University of
Florida–Institute of Food and Agricultural Sciences (IFAS), 200
Ninth Street Southeast,Vero Beach, FL 32962-4699; ‡Citrus Research
and Education Center, University of Florida–IFAS, 700 Experiment
Station Road, Lake Alfred, FL 33850;¶Indian River Research and
Education Center, University of Florida–IFAS, 2199 South Rock Road,
Fort Pierce, FL 34945; Departments of �Chemistryand **Chemistry and
Pathology, University of Virginia, Charlottesville, VA 22904;
††Department of Pharmacology, Catholic University of Leuven,Van
Evenstraat 4, B-3000 Leuven, Belgium; and §§Zoological Institute,
Catholic University of Leuven, Naamsestraat 59, B-3000 Leuven,
Belgium
Communicated by Lonnie O. Ingram, University of Florida,
Gainesville, FL, July 21, 2006 (received for review October 9,
2005)
We report the engineering of the surface of the tobacco
mosaicvirus (TMV) virion with a mosquito decapeptide hormone,
trypsin-modulating oostatic factor (TMOF). The TMV coat protein
(CP) wasfused to TMOF at the C terminus by using a read-through,
leakystop codon that facilitated expression of CP and chimeric
CP-TMOF(20:1 ratio) that were coassembled into virus particles in
infectedNicotiana tabacum. Plants that were infected with the
hybrid TMVRNA accumulated TMOF to levels of 1.3% of total soluble
protein.Infected tobacco leaf discs that were fed to Heliothis
virescensfourth-instar larvae stunted their growth and inhibited
trypsin andchymotrypsin activity in their midgut. Purified CP-TMOF
virions fedto mosquito larvae stopped larval growth and caused
death.Because TMV has a wide host range, expressing TMV-TMOF
inplants can be used as a general method to protect them
againstagricultural insect pests and to control vector
mosquitoes.
genetic engineering � Helitothis virescens � mosquitoes � plants
� digestion
The major digestive enzymes of many insect species are
midgutserine proteases, including trypsin and chymotrypsin.
Thus,controlling digestion in insects by using trypsin inhibitors
hasreceived substantial attention because of their widespread
oc-currence and potential for insect control (1–4). In
anautogenousadult mosquitoes and their larvae, trypsin and
chymotrypsin arethe major gut digestive enzymes. These enzymes
digest either theprotein-rich blood meal the adult female uses for
egg develop-ment (5) or proteinacious food from the water for
larval growthand development (6). The sequence of physiological
events thatfollows the uptake of a blood meal in female mosquitoes
orprotein uptake in larvae is complicated, involving
osmoticpressure, juvenile hormone III, and other unknown factors
(5).Similar to mosquito larvae, protein digestion in the
tobaccobudworm (Heliothis virescens) is mediated by endo- and
exopep-tidases secreted from the midgut epithelium cells into
theluminal f luid of the midgut (7). The main digestive enzymes
inH. virescens midgut are the serine proteases (8). Because
mos-quitoes transmit many diseases, such as malaria, dengue,
andyellow fever, that exert social and economical burden in
tropicalcountries, and H. virescens causes extensive agricultural
damage,there is a considerable interest to control these
insects.
Although the factor(s) that directly stimulates trypsin
biosyn-thesis in mosquito and H. virescens are not known, a
decapeptide(YDPAPPPPPP) named trypsin-modulating oostatic
factor(TMOF) that terminates trypsin biosynthesis in mosquitoes
andH. virescens has been identified and characterized (9,
10).Mosquito ovaries synthesize and release TMOF into the
hemo-lymph after a blood meal (11), and the hormone modulates
thesynthesis of trypsin in the mosquito gut by binding a
TMOFreceptor (12). Cytoimmunochemical studies showed that TMOFis
also found in the brain and the neuroendocrine organs of adultand
larval Aedes aegypti (13). Thus, the hormone has a dual rolein
terminating serine-proteases biosynthesis not only in adultfemale
mosquitoes but also in larvae (13). Mosquito TMOF or
its analogues stop trypsin biosynthesis in the cat flea
Ctenoceph-alides felis, in the stable fly Stomoxys calcitrans, in
the house flyMusca domestica, in the midge Culicoides variipenis,
and in H.virescens (8–10). TMOF from the gray fleshfly
Neobellieriabullata (Neb-TMOF) has been sequenced and
characterized. Thehormone is an unblocked hexapeptide (NPTNLH),
that, like A.aegypti TMOF (Aea-TMOF), stops trypsin biosynthesis
and eggdevelopment in the fleshfly (14). In the fleshfly, it was
shownthat Neb-TMOF controls the translation but not the
transcrip-tion of the trypsin gene (15).
Because TMOF affects the synthesis of trypsin-like enzymesin the
larval gut of mosquito and H. virescens, we decided toexplore the
possibility of fusing TMOF with the coat protein(CP) of tobacco
mosaic virus (TMV), which produces largequantities of RNA and
protein in infected plant cells. TMVcauses very small degree of
mosaic symptoms and only slightreduction in plant growth after the
genetic modification to theCP (16). Although TMV is known to exist
on human skin, it doesnot infect humans. A hybrid viral RNA
containing the TMOFsequence fused to the coat protein ORF of TMVU1
(17) wasconstructed so that 5% of the CP subunits had TMOF fused
tothe carboxyl terminus. Virus particles isolated from
plantsinfected with this chimeric virus presented TMOF on
theirsurface. The capacity to engineer crops to synthesize
insectpeptide hormones could be used in the future to protect
cropsagainst agricultural pest insects and to harvest peptides that
canbe used to control mosquito larvae.
ResultsBioengineering of Chimeric TMV-Aea-TMOF. A hybrid TMV
RNAwas constructed containing a replicase (126/183 kDa protein)and
a cell to cell movement protein (30 kDa) ORF and the CPORF with a
read-through sequence (5�-TAGCAATTA-3�) fol-lowed by a trypsin
cleavage site (IGER) fused to the Aea-TMOF.The leaky stop signal
allowed a virion assembly with a predictedratio of 1:20
(CP-TMOF:CP) (Fig. 1) (18). A bioengineeredTMV without the leaky
stop signal transcribed TMOF on everyviral CP but did not assemble
into virus particles because ofsteric hindrance between every CP
caused by the left-handedhelical conformation of TMOF (Fig. 2)
(18).
Author contributions: D.B., W.O.D., C.A.P., D.F.H., and A.D.
designed research; D.B., S.R.,C.A.P., D.A.I., T.J.M., J.S., and
H.L.D. performed research; D.B., W.O.D., C.A.P., D.F.H., H.L.D.,and
A.D. analyzed data; and D.B. wrote the paper.
Conflict of interest statement: D.B. and A.D. have patents on
the TMOF technologythrough the University of Florida.
Abbreviations: CP, coat protein; HSD, honestly selectively
different; TMOF, trypsin-modu-lating oostatic factor; TMV, tobacco
mosaic virus.
†To whom correspondence should be addressed. E-mail:
[email protected].§Present address: Fraunhofer USA CMB, 9
Innovation Way, Suite 200, Newark, DE 19711.
‡‡Present address: Tibotec BVBA, Gen. De Wittelaan 11B 3, B-2800
Mechelen, Belgium.
© 2006 by The National Academy of Sciences of the USA
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2006 � vol. 103 � no. 50 � 18963–18968
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Characterization of CP-TMOF. Chimeric TMV-TMOF particleswere
purified from infected tobacco leaves 2 weeks after inoc-ulation.
Protein samples from 22 and 28 �g of isolated virions,respectively,
were fractionated by 12% SDS/PAGE and sub-jected to Western blot
analysis with antibody to the CP protein.CP antibody recognized
CP-TMOF protein band at 18.5 kDa(Fig. 3, lanes A and B) that
comigrated with purified wild-typeviral CP (17.5 kDa; Fig. 3, lane
C) and a faint band at 27.5 kDa,which is an aggregation of the CP
and the CP-TMOF that wasnot completely denatured (Fig. 3, lanes B
and C). Because themobility of the wild-type CP (17.5 kDa) and the
chimericCP-TMOF (18.5 kDa) band on 12% SDS/PAGE is the same,
theCP-TMOF was further analyzed. Treatment of the chimericTMV-TMOF
with trypsin-liberated TMOF that then was ana-lyzed by HPLC
interfaced to electrospray ionization (ESI) on atriple quadrapole
mass spectrometer. Abundant (M � H)� and(M � 2H)2� ions were
observed at m/z 1,047 and 524, respec-tively. A collision-activated
dissociation spectrum recorded on
the (M � 2H)2� ions confirmed the sequence YDPAPPPPPP, aswas
described in refs. 9 and 10. When synthetic TMOF (24 pmol)was added
to the trypsin digest, and the resulting sample wasanalyzed by HPLC
ESI/MS, signals observed previously at m/z1,047 and 524 increased
by a factor of 4. We conclude that theTMOF expressed in the
chimeric TMV-TMOF and releasedwith trypsin has an identical mass
spectra to synthetic TMOF andhas the sequence YDPAPPPPPP, as was
described in refs. 9 and10. Wild-type CP (control) did not liberate
TMOF after incu-bation with trypsin and mass spectra analysis. The
purifiedchimeric viral particles (396 �g) analyzed by ELISA showed
thatthe amount of TMOF presented on the surface of the chimericTMV
was 1.189 � 0.11 (micrograms � SEM; n � 3), which is ingood
agreement with the predicted value of 1.188 �g calculatedfor a
ratio 1:20 (CP-TMOF:CP).
Effect of Feeding CP-TMOF to Mosquito Larvae. Mosquito larvae
areaquatic animals in which the concentration of solute in
thehemolymph is higher than in water. As a consequence, they donot
drink, and factors that are dissolved in water will not enterthe
gut unless the larvae ingest solid food particles and at thesame
time swallow some water. Because mosquito larvae arefilter feeders
(6), we adsorbed the CP-TMOF virions on driedyeast to allow the
larvae to ingest it. Feeding A. aegypti larvae (36per group)
CP-TMOF virions (0–2.33 �g/�l) containing TMOF(0–26.4 �M) caused a
significant increase (n � 3; P � 0.05) inlarval mortality from 0%
to 4% in controls that were fed yeastor CP virions, to 35% and
87.5% in larvae that were fedCP-TMOF virions (23.7 and 26.4 �M
TMOF, respectively) (Fig.4a). Larvae that were fed on CP-TMOF
virions (26.4 �MTMOF) did not grow and starved to death, whereas
larvae thatwere fed CP or yeast grew normally (Fig. 4b). Larvae
that werefed CP-TMOF virions (26.4 �M TMOF) were 2.38-fold
shorter(P � 0.006; n � 20) than controls that were fed yeast (2.8 �
0.16and 6.66 � 0.084 mm � SEM, respectively), and trypsin
activityin the gut of these larvae was 90% inhibited (results not
shown).These results confirm an earlier report that TMOF
adsorbedonto yeast particles inhibits trypsin activity in A.
aegypti andCulex quinquefasciatus larvae (13). No difference in
length orinhibition of trypsin activity was found when CP virions
or yeastcells were fed to larvae. Thus, the inhibition of larval
growth wascaused by TMOF.
Feeding Tobacco Leaves Infected with TMV-TMOF to H.
virescens.Feeding 24 fourth-instar H. virescens larvae weighing
18–19 mgtobacco leaf discs infected with TMV-TMOF for 4 days
caused2.3-fold reduction in weight (n � 6; P � 0.009) as compared
withnoninfected controls (Fig. 5a). ANOVA showed that there
were
126 / 183K 30K CP
CP IGER TMOF COOHProtein N
RNA
Tryp
sin
Cle
avag
e Si
te
Rea
d Th
roug
h
Fig. 1. Genome organization of TMV-TMOF containing a
read-throughsequence. TMV-U1 ORF representing the replicase of 126
and 183 kDa (126K/183K), the cell-to-cell movement protein 30 kDa
(30K), and the viral coatprotein (CP) was fused to a read-through
sequence (5�-TAGCAATTA-3�), atrypsin cleavage site (IEGR), and an
A. aegypti TMOF (YDPAPPPPPP) sequencebehind a T7 promoter at the 5�
end of the construct. The TMV-TMOF RNA wastranscribed by using T7
RNA polymerase, and the transcribed RNA was me-chanically
inoculated onto Ni. tabacum (L) cv. xanthu. Gene distances are
notproportional to gene sizes.
Fig. 2. Ribbon drawing by means of MOLSCRIPT of TMV coat protein
(CP)fused to A. aegypti TMOF. The TMV-CP is shown in yellow, and
the N terminusis shown as NH2. TMOF exhibiting a left-handed helix
is shown in blue with atrypsin cleavage site at Arg165 in red. The
C terminus of the fusion protein isat the carboxyl end of TMOF
(COOH).
49.5 -
32.5 -
27.5 -
18.5 -
6.5 -
CP-TMOF
KDa
CBA
Fig. 3. Western blot analysis and SDS/PAGE of CP-TMOF. Purified
TMV-TMOFvirions (22 �g, lane A; 28 �g, lane B) were run on
SDS/PAGE, blotted to amembrane, and analyzed by antiserum against
TMV-CP. (Lane C) Wild-typeTMV-CP virions (28 �g) were run on
SDS/PAGE and stained with Coomassiebrilliant blue for comparison.
Arrow indicates the comigration of the CP (17.5kDa) and CP-TMOF
(18.5 kDa).
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significant differences as a result of TMOF, F(1, 37) � 8.68, P
�0.0055; larval age, F(3, 37) � 28.64, P � 0.0001; and
theinteraction of age, and TMOF treatment, F(3, 37) � 5.05, P
�0.005. Post hoc Tukey honestly selectively different (HSD)analyses
revealed that larvae that were fed TMV-TMOF hadsignificantly lower
weight gain on day 4, and larval weight did notchange significantly
during the first 3 days of feeding, indicatingthat TMV-TMOF
prevented the growth of H. virescens. Ana-lyzing larval midguts
showed that trypsin activity in larvae thatwere fed TMV-TMOF was
reduced 3.1-fold (n � 6; P � 0.001)at day 3 and 2.2-fold (n � 6; P
� 0.037) at day 4 (Fig. 5b).ANOVA showed that there were
significant differences as aresult of TMOF, F(1, 34) � 15.91, P �
0.0003; larval age, F(3,34) � 8.18, P � 0.0003; and the interaction
of age and TMOFtreatment, F(3, 34) � 6.69, P � 0.001. Post hoc
Tukey HSDanalyses revealed that larvae that were fed TMV-TMOF
hadsignificantly lower trypsin activity on days 3 and 4, and
trypsinactivity did not change over the 4-day period. Similarly,
chymo-trypsin activity in the midguts was reduced 1.5-fold (n � 6;
P �0.014) at day 3 and 2.6-fold (n � 4; P � 0.046) at day 4 (Fig.
5c).ANOVA showed that there were significant differences as aresult
of TMOF, F(1, 23) � 10.86, P � 0.0032; larval age, F(3,23) � 8.24,
P � 0.0007; and the interaction of age and TMOFtreatment, F(3, 23)
� 5.32, P � 0.0062. Post hoc Tukey HSDanalyses revealed that larvae
that were fed TMV-TMOF hadlower chymotrypsin activities on days 3
and 4, and the chymo-trypsin activity of the TMV-TMOF fed larvae
did not changeover the 4-day period. The experiment was repeated
two moretimes with similar results (data not shown). These results
indicate
that TMV-TMOF causes inhibition of trypsin and
chymotrypsinactivity in the midgut of H. virescens preventing
normal larvalgrowth.
Effect of TMOF on the Trypsin Gene. Larvae were fed
TMV-TMOFinfected or noninfected leaf discs for 3 and 4 days, and
their gutswere dissected and tested for H. virescens trypsin
transcript byNorthern blot analysis. Trypsin gene transcript levels
were equalin the guts of larvae that were fed noninfected (control)
orinfected TMV-TMOF leaf discs for 3 and 4 days (Fig. 6),although
trypsin activity was reduced 3.1- and 2.1-fold after 3and 4 days of
feeding the CP-TMOF virions to larvae, respec-tively (Fig. 5b).
These results indicate that TMOF affects thetranslation of the
trypsin message in the gut.
DiscussionWe constructed virions of TMVU1 (19) to present TMOF
on thesurface by using a read-through sequence so that the
TMOFpeptide was presented from the C-terminal of 5% of the viral
CPsubunits. This approach allowed efficient assembly of the viralCP
subunits despite the steric hindrance that the left-handedhelical
portion of TMOF probably exerted. When it was ex-pressed on each CP
subunit virion assembly was prevented
Fig. 4. Feeding A. aegypti larvae CP-TMOF virions. (a) Three
groups offirst-instar larvae were fed increasing concentrations of
CP-TMOF virions inthe presence of Brewer’s yeast for 6 days (solid
line). Control groups were fedCP virions without TMOF (dashed
line). Larval survival was followed daily andplotted against TMOF
concentrations (micromolar) calculated from the con-centrations of
CP-TMOF virions that were fed. The results are expressed asmeans of
three determinations � SEM, and the experiment was repeatedthree
times. Significant differences: *, P � 0.005 in larval mortality
betweenfeeding CP-TMOF virions and CP virions (control). (b) Effect
on A. aegypti larvalgrowth after feeding larvae CP-TMOF virions
(I), CP virions (II), and Brewer’syeast (III) for 6 days. (Scale
bar: 5 mm.)
Fig. 5. Effect of feeding H. virescens tobacco leaf discs that
were infectedwith TMV-TMOF. (a) Weight gain, after feeding for 4
days on noninfected(control). *, P � 0.009 versus TMV-TMOF infected
leaf discs. (b) Trypsin activityin the gut after feeding for 3 and
4 days on noninfected (control). *, P � 0.001and P � 0.037,
respectively, versus on TMV-TMOF-infected disc leaves.
(c)Chymotrypsin activity in the gut after feeding for 3 and 4 days
on noninfected.
*, P � 0.014 and P � 0.046, respectively, versus
TMV-TMOF-infected leaf discs.Dashed line, larvae fed on control
leaf discs; solid line, larvae fed on leaf discsinfected with
TMV-TMOF. Each point represents a mean � SEM of individuallarvae (n
� 4–6), and the results represent one of three
independentexperiments.
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(unpublished observations). The same strategy was used
suc-cessfully in producing malarial epitopes and
angiotensinI-converting enzyme inhibitor on the CP of TMV. In both
cases,the recombinant CP and the wild-type CP coassembled into
virusparticles only after a leaky stop signal was used with a ratio
of1:20 (chimeric CP:CP) (18, 20). Turpen et al. (18) reported
that62% of the coat protein is unavailable for substitutions
becauseof steric hindrance. Thus, these reports combined with
ourobservations suggest that expressing recombinant proteins oneach
CP prevents assembly of the viral particles by sterichindrance. The
release of TMOF in the larval gut from thevirions by trypsin
allowed a rapid transport of the free TMOFthrough the gut
epithelial cells into the hemolymph (21). In thehemolymph, the
hormone bound its gut receptor located at thehemolymph side of the
gut (12) and caused cessation of trypsinactivity in the gut,
starvation, and eventual death (13). This modeof action is
different from Bacillus thuringiensis toxins that bindto receptors
located in the larval midgut epithelial brush bordermembrane and
insert into the membrane, causing formation ofpores or ion channels
and osmotic pressure imbalance thateventually kills the insects
(22, 23).
The ability of the CP-TMOF chimera to starve and killmosquito
larvae was demonstrated when CP-TMOF virions wereadsorbed onto
yeast particles and fed to A. aegypti larvae for 5–6days. The
estimated lethal dose of TMOF that fused to the CPand caused 87.5%
mortality was �26.4 �M (equivalent to 140ng/�l TMOF or 2.33 �g/�l
CP-TMOF), this concentration is7.5-fold lower than when TMOF alone
is adsorbed onto yeastparticles and fed to larvae (13). It is
possible that the CP-TMOFvirions bind better to the yeast particles
or that the CP-TMOFvirions aggregate and then bind to the yeast
cells, making iteasier for the larvae to internalize (6). Lower
concentrations ofTMOF caused less mortality because at lower
concentrations,less CP-TMOF virions bound to the yeast particles
that themosquito larvae fed on; mosquito larvae are filter feeders
andswallow particles (6). When radioactively labeled TMOF
wasincubated with the same amount of yeast particles that were
fedto larval mosquito, �10% of the radioactively labeled TMOFbound
the yeast particles (D.B., unpublished observations).Using these
results, we estimate that �10% of the availableCP-TMOF virions also
would bind the yeast particles that were
added to each well; this amount of yeast is sufficient for
larvaldevelopment (13). Thus, the effective concentrations that
causedhigh mortality are probably 10-fold lower than the total
con-centrations that were used during the feeding trials.
Similarresults were observed when H. virescens larvae were fed leaf
discsthat were infected with TMV-TMOF. Larval weight gain
was2.3-fold lower than controls (P � 0.001), and trypsin
andchymotrypsins activities in the midgut were 2.2- and
2.6-foldlower, respectively, at day 4. On the other hand, control
groupsthat were fed uninfected leaf discs were not affected.
The inhibition of trypsin activity is due to the
down-regulationeffect of TMOF and not to the CP that was fused to
TMOF.Earlier observations showed that when TMOF was mixed
withartificial food and directly fed to H. virescens larvae,
trypsinactivity, and weight gain were reduced greatly (8). The
Northernblot analysis strengthens these observations and suggests
that thetrypsin gene is down-regulated by TMOF in H. virescens
througha translational control mechanism. Such a mechanism would
beexpected for a hormone that is released after the trypsin
messagealready has been transcribed. Thus, it is now possible to
suggestthat Aea-TMOF down-regulates trypsin mRNA in the gut by
atranslational control mechanism in H. virescens, as was shown
forNe. bullata (15).
The ability to express foreign genes in plants will allow
futureplant protection to change from using chemical insecticides
tobiological and environmental friendly natural proteins and
pep-tides that can be designed to exert specific control on
agriculturalpest insects. TMV-infected plants are ideal for high
productionof proteins (e.g., TMOF) that can be harvested, isolated,
andused against vector insects such as mosquitoes that are of
medicalimportance because they transmit diseases in many parts of
theworld. Plant RNA virus-based vector such as tobamoviruses havea
wide host range, they move easily between plant cells, theyrapidly
and systematically spread in the infected plant, the RNAreplicates
at high level, and the infection is maintained for thelifetime of
the plant (16). In addition, the virus can be selectedor
manipulated so it does not have an adverse effect on the plant.The
ease of manipulating the TMV genome as a DNA copybefore
transcribing it in vitro into infectious RNA makes it anideal
system for expressing foreign proteins fused to the CP. Theamount
of CP protein expressed in infected plants constitutes70% of total
plant protein (24) or up to 10% of plant dry weight(25). Malaria
epitopes of 15- and 12-mer peptides that were fusedto the CP with a
read-through sequence produced 3 and 15micromoles of these
peptides, respectively, from 1 kg of tobaccoleaf extract (18). Our
TMV-TMOF produced 4.8 micromolesTMOF per kilogram of leaf extract
(1.3% of total solubleproteins). On the other hand, expressing TMOF
in tobaccoplants as a fusion with tomato prosystemin produced
325-foldless TMOF (0.004% of total soluble protein) and low
inhibition(4%) of H. virescens larval growth (26).
Because TMV has a wide host range, this technique ofexpressing
TMOF on the coat protein of TMV can be used as ageneral method to
protect plants against agricultural insect peststhat use serine
proteases as their main digestive enzymes. Ourresults provide a
previously undescribed approach to controlagricultural pest insects
that cause extensive crop damage andmosquitoes, which are vectors
of medically important diseasessuch as malaria, dengue,
encephalitis, and yellow fever.
Materials and MethodsInsects. A. aegypti larvae were reared from
eggs at 26°C on a diet ofBrewer’s yeast, lactalbumin, and lab chow
(1:1:1) with a 16:8light:dark cycle. Adults were fed on 10% sucrose
solution or chickenblood to obtain eggs. Larvae of H. virescens
(Lepidoptera:Noctu-idae) were hatched from eggs (AgriPest, Zebulon,
NC) and raisedindividually in 24-well tissue culture plates
(Falcon, Franklin Lakes,NJ) on artificial diet (Southland Products,
Lake Village, AR) at
Fig. 6. Northern blot analysis of H. virescens larval
gut-trypsin transcriptafter feeding for 3 and 4 days on leaf discs
that were not infected (control) andinfected with TMV-TMOF. The
blot was probed with trypsin and actin probes.Arrow represents
expected trypsin transcript at �0.9 Kb. The Northern blotanalysis
was repeated twice.
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26°C with a 16:8 light:dark cycle. Under these conditions, the
first-and second-instar stages were 2 days each, and the
fourth-instarstage was 4–5 days.
Construction of TMV-TMOF with Read-Through Context Sequence.
Anintermediate cDNA clone of TMV having a direct fusion of theTMOF
peptide sequence (preceded by a trypsin cleavage site,IGER; Fig. 1)
with the coat protein at the carboxyl terminus firstwas constructed
and used as a template for further constructs.The read-through
context sequence in the TMV 126/183 kDasequence (TAGCAATTA) was
introduced at the 5� end of theIGER-TMOF sequence (18, 20) by using
the following primers:M88 (forward)
(5�-TGGACCTCTGGTCCTGCATCATAG-CAATTAATTGGTGAACGTTATGATCCTGCT-3�),
M89 (re-verse)
(5�-AGCAGGATCATAACGTTCACCAATTAAT-TGCTATGATGCAGGACCAGAGGTCCA-3�);
M28 (reverse):3� end of TMV,
(5�-TTCGAGCTCGGTACCTGGGCCCCTAC-CGGG-3�); and T4 (forward): nt
5154–5175 in TMV (5�-CAGAGGAGGTGTGAGCGTGTGT-3�). Letters in bold
denoteTMV coat protein sequence, letters in italics denote
IGER-TMOF sequence, and underlined letters denote the read-through
sequence that allowed the peptide to be inserted at theC terminus
of the CP at 5% of the rate of translation of CP. PCRamplification
was carried out by 10 cycles of 5 sec at 94°C, 5 secat 45°C, and 30
sec at 74°C, followed by 20 cycles of 5 sec at 94°C,5 sec at 50°C,
and 30 sec at 74°C and 1 cycle at 74°C for 4 min.The amplified
fragments were isolated by using a Geneclean kit(Qbiogene, Irvine,
CA) and used as templates for overlap-extension PCR with primers T4
and M28. The final product wasdigested with NcoI and KpnI, purified
by using a Geneclean kit(Qbiogene) and ligated into a similarly
digested TMV004 plas-mid (cDNA of TMVU1). Escherichia coli DH5�
cells weretransformed by electroporation (Bio-Rad, Hercules, CA)
withthe ligation mixture, and the cells were plated on LB agar
platescontaining ampicillin (100 �g/ml). After incubation,
colonieswere isolated and grown overnight in LB liquid medium
con-taining ampicillin and plasmid DNA was isolated (27).
Cloneswith the correct fragment were identified by agarose
electro-phoresis after digestion with NcoI and KpnI and regrown
forlarge-scale plasmid isolation and analyzed by PCR with
primerpair T77 (forward) (5�-GGTCCTGCATCATAGCAATTA-3�)and M28
(reverse).
In Vitro Transcription and Plant Inoculation. Plasmid cDNA of
TMVwas linearized with KpnI, extracted with
phenol:chloroform:i-soamylalcohol (25:24:1, by volume), and
precipitated withethanol. In vitro transcription was carried out by
using T7 RNApolymerase (19, 28). The transcribed RNA was
inoculatedmechanically into leaves of Nicotiana tabacum (L)
cv.Xanthi nn, a systemic host for TMV. Inoculated plants,
whichdeveloped mild mosaic symptoms (16), were maintained in
agrowth room at 25°C under light (10,000 lux) for 10 days atwhich
time virions were purified from the leaves.
Purification of CP-TMOF. TMV virions were extracted by using
amodified procedure described in ref. 29. Briefly, infected
tissue(169 g) was frozen in liquid nitrogen and extracted with
chlo-roform:butanol:phosphate buffer pH 7.2 (2:1:1 by volume),
theextract filtered through a cheese cloth and centrifuged (7,000
�g for 15 min at 4°C). Virions were precipitated with 4% PEG at4°C
and centrifuged at 10,000 � g for 15 min at 4°C. The pelletwas
redissolved in 25 mM sodium phosphate buffer (pH 7.2), andthe
solution was recentrifuged at 45,000 � g for 1 h at 4°C in
anultracentrifuge by using a Ti 45 fixed angle rotor. The pellet
wasredissolved overnight at 4°C in 5 ml of sodium phosphate
buffer(pH 7.2). The virions were denatured in glacial acetic acid
at 2°Cand incubated on ice for 30 min with occasional shaking.
Theviral RNA was precipitated by centrifugation in a Sorvall
centrifuge at 5,000 rpm and 4°C in a SS-34 rotor, and the
clearsupernatant was dialyzed against water in a dialysis bag at
4°C for48 h. The CP-TMOF formed a white cloudy precipitate,
whichwas centrifuged down at 45,000 � g for 4 h at 4°C. The
pelletcontaining CP-TMOF (purity of 98%) was resuspended insodium
phosphate buffer (pH 7.2), stored at 4°C, and theconcentration of
the recombinant TMOF was determined byELISA (30).
Purification of TMOF. Trypsin (25 �g), sequencing enzyme
grade(Sigma, St. Louis, MO), was incubated for 24 h at
roomtemperature with CP-TMOF (18.77 mg) in 20 mM Tris�HCl/0.2%
SDS/0.01% NaN2, pH 9.0. Liberated TMOF was purifiedby using C18
reversed-phase HPLC (9).
Mass Spectrometry. Mass spectra of recombinant TMOF andsynthetic
TMOF were recorded by using a combination ofmicrocapillary HPLC
interfaced to electrospray ionizations on atriple quadrupole mass
spectrometer (TSQ 700; Finnigan Corp,San Jose, CA) (14, 31). The
TMOF mass spectra ions have beenpublished in refs. 9 and 10.
Gel Electrophoresis and Western Blot Analysis. The protein
extractedfrom the purified virions was analyzed by 12% SDS/PAGE
(32)and electroblotted onto nitrocellulose paper (33). The
blottedproteins were probed with TMVU1 coat protein
antibodies(1:1,000), followed by goat anti-rabbit antibody alkaline
phos-phatase conjugate (Southern Biotechnology Associates,
Bir-mingham, AL). The proteins were visualized with
nitrobluetetrazolium (0.3 mg/ml) and 5-bromo-4-chloro-3-indolyl
phos-phate (0.15 mg/ml) dissolved in 100 mM Tris�HCl/0.5 mM
MgCl2buffer, pH 9.6. No difference in mobility is observed between
theCP-TMOF (18.5 kDa) and the wild-type CP (17.5 kDa) on
12%SDS/PAGE.
Bioassay of CP-TMOF by Using Mosquito Larvae. To determine
theeffect of the CP-TMOF virions on mosquito larvae, three groupsof
first-instar larvae (36 larvae per group) were grown individ-ually
in microtiter plates containing 188 �l of sterile distilledwater
and different concentrations of CP-TMOF virions (0.166to 2.33
�g/�l, equivalent 10 to 140 ng/�l TMOF) and Brewer’syeast (200 �g)
(13). Larval mortality was followed at 24-hintervals for 5–7 days.
Controls were fed yeast or CP virions(0.166 to 2.33 �g/�l) without
TMOF. At the end of the exper-iment, live larvae were rinsed in
saline, larval-length measured,and photographed under a Hitachi
dissecting microscope.
Bioassay of Leaves Infected with TMV-TMOF. To test the effect
oftobacco plants infected with TMV-TMOF on H. virescens,tobacco
leaves were removed from plants that were infected withTMV-TMOF,
and circular discs (12 mm in diameter) were cutfrom the leaves by
using a cork borer. The leaf discs then weretransferred with their
lower side down to 24-well tissue cultureplates containing 500 �l
of agar (2% wt/vol) per well. Fourgroups of H. virescens larvae
(six per group) of early fourth-instarwere weighed and transferred
to the leaf discs, and the tray thenwas covered with tissue paper
and a plastic lid. The larvae wereincubated at 27°C, and at 24-h
intervals, six larvae were removed,weighed individually, and their
guts were dissected out under adissecting microscope and washed in
saline. Each gut washomogenized in 50 mM Tris�HCl buffer (pH 7.9)
and assayed fortrypsin and chymotrypsin activities by using
N�-benzoyl-D-L-arginine 4-nitroanilide hydrochloride (BApNA) and
N-benzoyl-L-tyrosine-p-nitroanilide (BTpNA) (Sigma), respectively
(34).Larvae were monitored daily and provided with
additionaltobacco discs as needed. Control groups were fed tobacco
leafdiscs that were not infected with TMV-TMOF. Each experimentwas
repeated three times.
Borovsky et al. PNAS � December 12, 2006 � vol. 103 � no. 50 �
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Three-Dimensional Model. A ribbon model of TMV-TMOF wasbuilt by
using SYBYL molecular-modeling software (TRIPOS)and the program
MOLSCRIPT (35) described in refs. 15 and 36.The model is based on
the x-ray diffraction of TMV (2TMV) inthe protein structure data
bank (www.ncbi.nlm.nih.gov/Structure) and the NMR solution
structure of TMOF (37).
Northern Blot Analysis. Total RNA was extracted from H.
virescensmidguts by using TRIzol reagent (GIBCO BRL,
Gaithersburg,MD) according to the manufacturer’s instructions.
Denatured totalRNA (5 �g per lane) was run by electrophoresis on
1.5% dena-turing gel by using Northern MAX kit (Ambion, Austin,
TX),transferred to BrightStar� nylon membrane (Ambion), and
hybrid-ized with a 531-bp 32P-labeled cDNA probe (nt 255–786) that
wasamplified by PCR from H. virescens cDNA (AF237416) by using
primers DB788 (forward) (5�-AACAGATGGCGTATCCGTCT-TGGCTC-3�) and
DB789 (reverse) (5�-TTACGCGTTAGAT-GAAATCCAAGC-3�). The blot was
washed according to themanufacturer’s instructions and exposed to
x-ray film for 24 h at�80°C. The efficiency of the RNA transfer was
compared with H.virescens actin (AF368030).
Statistical Analysis. Data were analyzed by using the Student
ttest one-tail analysis followed by ANOVA and post hoc TukeyHSD
analyses, and P � 0.05 was considered to be
statisticallysignificant.
This work was partially supported by National Institutes of
Health (NIH)Grant AI 41254, NATO Grant CRG 940057, Insect
Biotechnology, Inc.and Bayer grants (to D.B.), and NIH Grant
GM37537 (to D.F.H.).
1. Hilder V, Gatehouse A, Sheerman S, Barker R, Boulter D (1987)
Nature330:160–163.
2. Johnson R, Narvaez J, An G, Ryan C (1989) Proc Natl Acad Sci
USA86:9871–9875.
3. Ryan CA (1990) Ann Rev Phytopathol 28:425–449.4. Broadway RM
(1995) J Insect Physiol 41:107–116.5. Borovsky D (2003) Life
55:435–441.6. Clements AN (1992) The Biology of Mosquitoes (Chapman
& Hall, New York),
Vol 1, pp 74–97.7. Terra WR (1990) Ann Rev Entomol 35:181–200.8.
Nauen R, Sorge D, Sterner A, Borovsky D (2001) Arch Insect Biochem
Physiol
47:169–180.9. Borovsky D, Carlson DA, Griffin PR, Shabanowitz J,
Hunt DF (1990) FASEB
J 4:3015–3020.10. Borovsky D, Carlson DA, Griffin PR,
Shabanowitz J, Hunt DF (1993) Insect
Biochem Mol Biol 23:703–712.11. Borovsky D, Song Q, Ma M,
Carlson DA (1994) Arch Insect Biochem Physiol
27:27–38.12. Borovsky D, Powell CA, Nayar JK, Blalock JF, Hayes
TK (1994) FASEB J
8:350–355.13. Borovsky D, Meola SM (2004) Arch Insect Biochem
Physiol 55:124–139.14. Bylemans D, Borovsky D, Hunt DF, Grauwels L,
De Loof A (1994) Regul Pept
50:61–72.15. Borovsky D, Janssen I, Vanden Broeck J, Huybrechts
R, Verhaert P, De Bondt
HL, Bylemans D, De Loof A (1996) Eur J Biochem 237:279–287.16.
Hull R (2002) Mathew’s Plant Virology (Academic, San Diego), 4th
Ed.17. Donson J, Kearney CM, Hilf ME, Dawson WO (1991) Proc Natl
Acad Sci USA
88:7204–7208.18. Turpen TH, Reini SJ, Charoenvit Y, Hoffman SL,
Fallarme V, Grill LK (1995)
Biotechnology 13:53–57.
19. Dawson WO, Beck DL, Knorr DA, Grantham GL (1986) Proc Natl
Acad SciUSA 83:1832–1836.
20. Hamamoto H, Sugiyama Y, Nakagawa N, Hashida E, Matsunaga Y,
TakemotoS, Watanabe Y, Okada Y (1993) Biotechnology 11:930–932.
21. Borovsky D, Mahmood F (1995) Regul Pept 57:273–281.22.
Knight PJK, Carroll J, Ellar DJ (2004) Insect Biochem Mol Biol
34:101–
112.23. Hua G, Jurat-Fuentes JL, Adang MJ (2004) Insect Biochem
Mol Biol 34:193–
202.24. Culver JN, Lehto C, Close SM, Hilf ME, Dawson WO (1993)
Proc Natl Acad
Sci USA 90:2055–2059.25. Shivprasad S, Pogue GP, Lewandowski DJ,
Hidalgo J, Donson J, Grill LK,
Dawson WO (1999) Virology 255:312–323.26. Tortiglione C,
Fogliano V, Ferracane R, Fanti P, Pennacchio F, Monti LM, Rao
R (2003) Plant Mol Biol 53:891–902.27. Zhou C, Yang Y, Jong AY
(1990) Biotechnology 8:172–173.28. Allison RF, Janda M, Ahlquist P
(1988) J Virol 62:3581–3588.29. Gooding GV, Herbert TT (1967)
Phytopath 57:1285.30. Borovsky D, Powell CA, Carlson DA (1992) Arch
Insect Biochem Physiol
21:13–21.31. Hunt DF, Henderson RA, Shabanowitz J, Sakaguchi K,
Michel H, Sevilir N,
Cox AL, Appella E, Engelhard VH (1992) Science 255:1261–1263.32.
Laemmli UK (1970) Nature 227:680–685.33. Dunn SD (1986) Anal
Biochem 157:144–153.34. Borovsky D, Schlein Y (1987) Med Vet
Entomol 1:235–242.35. Kraulis P (1991) J Appl Crystallogr
24:946–950.36. Xin-Hua Y, DeBond HL, Powell CA, Bullock RC,
Borovsky D (1999) Eur
J Biochem 262:627–636.37. Curto EV, Jarpe MA, Blalock JB,
Borovsky D, Krishna NR (1993) Biochem
Biophys Res Comm 193:688–693.
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al.
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