Unusual Baculovirus of the Parasitoid Wasp Apanteles melanoscelus: Isolation and Preliminary Characterization
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JOURNAL OF VIROLOGY, Mar. 1979, p. 1118-11830 Vol. 29, No. 3
0022-538X/79/03-1 1 18/13$02.00/0
Unusual Baculovirus of the Parasitoid Wasp Apanteles
melanoscelus: Isolation and Preliminary Characterization
PETER J. KRELL AND DONALD B. STOLTZ*
Department of Microbiology, Dalhousie Univ,ersity, Halifax, Nova Scotia, Canada
Received for publication 14 August 1978
A baculovirus present in the female reproductive tract of the parasitoid wasp
Apanteles melanoscelus has been isolated and partially characterized. Viral DNA
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is double stranded, circular, and of highly variable molecular weight ranging from
2 x 10 to 25 x 10"3; the DNA is of homogeneous density at p = 1.694 g/ml.
Acrylamide gels resolve 18 polypeptide bands in the case of purified virions; four
to five of these appear in a semipurified nucleocapsid preparation. The electro-
phoretic profiles obtained are compared with those of two other baculoviruses.
Enveloped particles bearing a close structural peared in abstract form [P. J. Krell and D. B.
resemblance to baculoviruses are known to be Stoltz, Proc. 1st Int. Colloq. Invertebr. Pathol.,
present, often in considerable concentrations, in p. 343, 1976].)
the ovaries of many parasitoid braconid wasps MATERIALS AND METHODS
(27, 32, 33, 40; D. B. Stoltz and S. B. Vinson,
Adv. Virus Res., in press). The "infection" is Rearing of parasitoid and host. Parasitoid A.
probably passed vertically, since every female melanoscelus (Hymenoptera: Braconidae) wasps were
reared as previously described (14, 24, 32) from labo-
wasp of any species known to harbor such par- ratory colonies of the white marked tussock moth
ticles is affected (Stoltz and Vinson, in press); no Orgyia leucostigma (Lepidoptera: Lymantriidae); the
obvious detrimental effects due to the presence latter were reared on an artificial diet (11, 21).
of these particles can be identified. Large num- Isolation of parasitoid baculovirus. The anat-
bers of particles are injected into caterpillars omy of female braconid wasp reproductive tracts is
during oviposition and, at least in some cases, given in Stoltz et al. (33). The portion containing virus
particle nucleocapsids subsequently interact particles (calyx and oviduct) is approximately 0.25 mm
specifically with the nuclear pores of host cells long.
(32). It has been proposed that parasitoid parti- Ovaries were dissected out from immobilized (0°C
for 5 min) adult wasps by means of jeweller's forceps,
cles may be involved, whether directly or indi- and were separated from each other by severing the
rectly, in the abrogation of host cellular immune lateral oviducts with fine dissecting needles (entomo-
responses to parasitoid eggs (38, 39; Stoltz and logical minuten pins). Fluid could subsequently be
Vinson, in press). Consequently, it is hoped that expressed into buffer solutions by applying light pres-
some understanding of host/parasitoid interre- sure on the calyx with dissecting instruments. Calyx
lationships may provide new insights into the fluid was routinely dissected out into small volumes
basis of immunity in insects. (20 to 200 1l) of Tris-EDTA buffer (10 mM Tris-1.0
On the basis of acceptable morphological cri- mM EDTA, pH 7.5) and then subjected to low-speed
teria (32, 33; Stoltz and Vinson, in press), the centrifugation at 1,000 x g for 15 min; the latter step
braconid particles can be provisionally assigned was included to sediment any parasitoid eggs that
were present. Difficulties were encountered during
to the baculovirus group (35, 43), which is pres- attempts to purify calyx particles by standard proce-
ently being more precisely defined by the Bac- dures (discussed in Results) so that, although further
ulovirus Study Group (International Committee attempts to do this continue, we decided, primarily in
on Taxonomy of Viruses). With the aim of pro- the interests of conserving valuable material, to ap-
viding confirmation of the viral nature of these proach the problem indirectly, using the following
particles, our laboratory has begun to examine rationale: if, as was known from previous experience
their biochemical composition in relation to (32), the only ultrastructurally recognizable entity in
what is known about other baculoviruses. Our crude calyx fluid is virus, then presumably the only
preliminary studies on one such particle found possible contaminants would be soluble protein and
nucleic acid in the ambient fluid. It was felt that such
in the wasp Apanteles melanoscelus represent could be removed by one or two cycles of centrifuga-
the subject of this report. The ultrastructure of tion onto a sucrose cushion, followed by pelleting, a
this virus has been previously described (32; procedure that should result as well in maximum
Stoltz and Vinson, in press). retention of starting material. In practice, then, the
(A preliminary report of this work has ap- following protocol was adopted: calyx fluid from 10 or
1118VOL. 29, 1979 CHARACTERIZATION OF APANTELES BACULOVIRUS 1119
more wasps was layered onto a discontinuous two-step was added a final concentration of 0.1 mg of DNase I
sucrose gradient made up in 10 mM Tris and 1 mM (Worthington) per ml. After 2 h at 20°C, both gels
EDTA (pH 7.5), consisting of a lower 1.7-ml layer of were again stained with 0.1% EB and examined.
60% sucrose and an upper 1.7-mi layer of 30% sucrose The remaining two gels were washed with RNase
in a 4-ml IEC polyallomer tube. The sample was reaction buffer (0.1 M acetate, pH 5) at 20°C for 2 h.
centrifuged at 35,000 x g for 30 min at 5°C. The virus RNase (Worthington) to a final concentration of 0.1
band was removed by side puncture with an 18-gauge mg/ml was added to one. After 2 h at 20°C, gels were
needle and then pelleted through Tris-EDTA (35,000 again stained with EB.
x g for 30 min). This cycle was repeated, and the final DNA markers. Phage OX174 RF DNA was pur-
pellet was resuspended and dialyzed extensively chased from Biolabs (Beverly, Maine). PM2 DNA was
against Tris-EDTA buffer. a gift from A. R. Morgan of the University of Alberta.
Purification of nucleocapsids. Calyx fluid from Relaxed circular plasmid markers (pCR1, pML21, and
40 or more wasps was expressed into 10 mM Tris-1 pSC101) were donated by R. Lau.
mM EDTA (pH 9.5) and triturated for 5 min prior to DNA purification. Calyx fluid DNA was isolated
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overlayering on a continuous 20 to 54% sucrose gra- essentially by the method of Summers and Anderson
dient made up in 10 mM Tris-1 mM EDTA, pH 9.5. (36). Calyx fluid was made to 2 to 4% Sarkosyl in
Centrifugation was for 30 min at 35,000 x g in an IEC SSCE and heated at 60°C for 30 min. The sample was
SB-405 rotor. The nucleocapsid band was removed by then mixed with a solution of CsCl (Calbiochem, A
side puncture and dialyzed against several changes of grade) at a density of 1.55 g/ml containing 100 ,tg of
Tris-EDTA. EB per ml. After centrifugation in an IEC SB-405
Purification of other baculoviruses. Orgyia leu- rotor for 36 h at 130,000 x g and 20°C, fluorescent
costigma nuclear polyhedrosis virus polyhedra were DNA bands were removed by side puncture with an
purified from infected cadavers according to the pro- 18-gauge needle. DNA preparations were extracted
tocol of Arif and Brown (1); virions (one nucleocapsid three times with isoamyl alcohol, dialyzed extensively
per envelope) and nucleocapsids were isolated and against SSC-EDTA, and subsequently concentrated
purified from polyhedra according to standard proce- with Aquacide. The concentrated DNA was either
dures (1, 3), except that 4 to 20% Ficoll gradients were used directly or stored at -70°C.
used to purify virions, exactly as described by Bell and In most experiments conducted to date, calyx fluid
Orlob (2). Nonoccluded Oryctes rhinoceros baculovi- particles used for DNA isolation were twice centri-
rus particles were purified from the midguts of infected fuged onto a ..ucrose cushion and finally pelleted. In
beetles by using Ficoll gradients. one experiment, material thus prepared was sus-
Agarose gel electrophoresis. Calyx fluid from pended in buffer (without EDTA) containing 0.005 M
five or more wasps was dissected out into small vol- MgSO4-0.1 M acetate (pH 5) and digested with 0.008
umes (50 pl) of SSC (0.15 M NaCl-0.015 M sodium mg of pancreatic DNase I (Calbiochem) per ml for 3
citrate)-10 mM EDTA, pH 7.5, to which 2 to 4% h at 25°C prior to centrifugation on a CsCl-EB gra-
Sarkosyl (K & K Laboratories) was then added. Sam- dient. Activity of the nuclease was calculated to be
ples were heated for 30 min at 60°C, after which 56,000 U/mg by the method of Kunitz (17), using
0.002% bromophenol blue and 15% sucrose in TEA salmon sperm DNA as a substrate.
buffer (0.05 M Tris-0.02 M sodium acetate-0.2 mM Electron microscopy. DNA, banded to equilib-
Na2 EDTA, pH 8.5 with acetic acid; 13) were added. rium in CsCI-EB gradients, was used directly in the
DNA samples were finally run in 0.7% agarose, in the basic protein monolayer technique of Kleinschmidt as
same buffer, cast on a horizontal slab gel apparatus. modified by Davis et al. (9). The spreading solution
DNA was run into gels for 30 min at 50 V, and contained 20 pd of formamide, 10 pl of 0.2 M Tris-0.02
electrophoresis was continued for another 16 to 24 h M EDTA buffer at pH 8.5, 10 ,l of a 0.05% solution of
at a constant 30 V. After electrophoresis, gels were cytochrome c (Sigma, type III, aqueous), and 10 ,l of
soaked in an aqueous solution of 1 ytg of ethidium undiluted DNA. Immediately after mixing, the entire
bromide (EB) per ml for 1 h and then viewed on a 50 ,l of DNA solution was spread onto a freshly
shortwave UV transilluminator (model C-61; U.V. prepared hypophase of 10% formamide in 0.01 M
Products, San Gabriel, Calif.). Photography of stained Tris-0.001 M EDTA (pH 8.5). Parlodion-coated grids
gels was through a Kodak 23A filter using high-con- were touched to the hypophase surface 1 min after
trast copy film. Protease (Pronase or proteinase K) DNA spreading. Grids were stained for 30 s in 50 ,uM
digestion of samples had no effect on gel profiles uranyl acetate-50 M HCI in 95% ethanol, dehydrated
obtained and was therefore not used routinely. Various for 10 s in isopentane, and then rotary shadowed with
open circular molecular weight markers (e.g., PM2, platinum-palladium (80:20). Micrographs were taken
4X174 RF, and plasmids pCR1, pML21, and pSC101) with a Philips EM300 calibrated with a diffraction
were routinely included in gels, but were run in differ- grating replica (Ladd). Circular double-stranded (ds)
ent tracks from the samples. DNAs could not be used as internal standards with
To confirm the presence of DNA in agarose gel our material and were instead placed on separate grids.
profiles, enzyme digestions of gels were carried out. Micrographs of standards were taken at the beginning
Four cylindrical gels, each containing material from and end of each load of 16 plates; comparable data
six A. melanoscelus calyces, were obtained. Two gels were obtained from many such sittings. Negatives
were rinsed with 500 ml of DNase reaction buffer (0.1 were enlarged 10 times by projection so that the DNA
M acetate buffer, pH 5, containing 0.01 M Mg2SO4) at molecules could be accurately traced; their contour
20°C for 2 h to remove EB from the stained bands. lengths were measured with a Keuffel and Esser model
One of these remained as a control, while to the other 62-0300 planimeter.1120 KRELL AND STOLTZ J. X7IROL.
Buoyant density determination. Supercoiled putative soluble components of the calyx fluid;
and open circular calyx fluid DNA was isolated from the purity of the particulate fraction so obtained
50 wasps and extracted as described above. Each sam- is discussed below in relation to both agarose
ple was mixed with Micrococcus lysodeikticus DNA and polyacrylamide electrophoresis and Klein-
as a buoyant density marker (p = 1.731 g/ml; 8) and schmidt data.
centrifuged to equilibrium using a model E ultracen- Attempts were made to disrupt the virus
trifuge (22). Equivalent samples were run at three
different densities of CsCl. membrane with Nonidet P-40 and Triton X-100
Polyacrylamide gel electrophoresis. Virion and to release nucleocapsids for subsequent purifi-
nucleocapsid preparations and marker proteins (BDH, cation. Preliminary studies showed that the
molecular weight range 14,300 to 71,500) were solubi- structural integrity of nucleocapsids was at least
lized in 1% sodium dodecyl sulfate-1% ,B-mercaptoeth- partially disrupted upon treatment with these
anol in 10 mM Tris (pH 6.8) and heated at 80°C for 30 detergents. Advantage was subsequently taken
min and 100°C for 2 min. Electrophoresis in 11%
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of the observation that approximately 50% of
acrylamide discontinuous Tris-glycine-sodium dode- the nucleocapsids could be released from their
cyl sulfate gels was by the method of Weber and envelopes simply by dissecting calyx fluid out in
Osborn (41). After electrophoresis, gels were fixed in
12.5% trichloroacetic acid for 30 min; staining and Tris-EDTA buffer at pH 9.5 (Fig. 2). Sucrose
destaining procedures were carried out at 60°C. gradient centrifugation of such material concen-
trated nucleocapsids into a relatively broad,
RESULTS light-blue band at a mean density of approxi-
Purification virus and nucleocapsids.
of mately 1.13 g/ml. This band, while consisting
Electron microscopy reveals that the lumen of predominantly of nucleocapsids (Fig. 3), also
the calyx is replete with enveloped baculoviruses contained a small number of membrane frag-
(Fig. 1); this calyx fluid is therefore a good source ments derived from the envelope protrusion.
of highly concentrated virions. Light pressure on Nucleocapsids were found to be of constant di-
the calyx (see Materials and Methods) forces ameter (approximately 40 nm) but variable
the virus-containing fluid to emerge as a thick length (28 to 100 nm). To determine if there
paste, which slowly disperses into the buffer. were distinct size classes, the lengths of 186
Both Tris-EDTA and SSC-EDTA (both at pH nucleocapsids were measured and a histogram
7.5) were found to be suitable for preserving was plotted (Fig. 4). There appear to be two
virus morphology; Tris-EDTA was used for virus broad nucleocapsid length categories, one
purification, and SSC-EDTA was used for DNA around 51 nm and the other around 66 nm. In
purification. addition, there may be several other minor
Initial attempts to purify the particulate com- length classes (e.g., at 30, 40, and 100 nm), as
ponent (virus) of the calyx fluid included tech- well as several nucleocapsids with lengths inter-
niques, such as rate zonal and equilibrium gra- mediate between 45 and 90 nm. It is realized
dient centrifugation, now commonly employed that such data are subject to a degree of error,
in virus purification. However, given the amount due to preparation artifact, even though nega-
of difficulty and time required to produce suffi- tively stained material was subject to fixation
cient material for such experiments, standard (2% buffered glutaraldehyde for 20 s) prior to air
purification protocols resulted in an unaccepta- drying.
ble loss of material; when bands were obtained, Isolation and characterization of viral
they were very broadly smeared. We suspect at DNA. Initial experiments aimed at elucidating
present that much of the difficulty experienced the nature of the viral nucleic acid were carried
derives from the presence of the envelope pro- out by means of agarose gel electrophoresis,
trusion (see Fig. 1), which is extremely fragile using standard procedures for the separation
and readily sheared off, resulting in release of and visualization of ds DNA. As expected, from
nucleocapsids from envelopes. Aside from that, the observed heterogeneity in nucleocapsid
particles are in any case heterogeneous with length, electrophoretic profiles of EB-stained
respect to size and number of nucleocapsids per bands were complex (Fig. 5). DNA from crude
envelope; additionally, as will be shown, nucleo- calyx fluid and from particles twice centrifuged
capsids are themselves variable in size. onto a sucrose cushion reproducibly separated
As expected, centrifugation onto a 60% sucrose into about 16 bands. The presence of DNA in
cushion effectively concentrates calyx fluid par- gel profiles was confirmed by enzyme digestion
ticles into a thin white band. Ultrastructurally, experiments (not shown): after DNase treatment
such material contains pure virus with no ob- no bands remained, whereas RNase digestion
vious particulate contaminants. The sucrose had no observable effect. Electrophoretic pro-
cushion/pelleting protocol was subsequently files of DNA from calyx particles treated with
used routinely to separate virus particles from pancreatic DNase are also shown in Fig. 5. The:4" ' 4% '¢ i,b... '}. .
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FIG. 1. (A) Electron micrograph of virus particles as observed in situ (A. melanoscelus calyx lumenJ.
Virions consist of several nucleocapsids enveloped by a single unit membrane. Each envelope bears a long
tubular protrusion (arrow); these appendages are more readily observed in negatively stained material (B).
Two nucleocapsids are encircled. Arrowheads indicate outer surface of virion envelope. Brar = 1.0 ,um. (C)
Virus band resulting from sucrose gradient centrifugation. Calyx fluid in SSCE buffer (pH 7.5) was layered
onto a 30 to 60% two-step sucrose gradient and centrifuged for 30 min at 35,000 x g at 5°C.
1121:'-.,t
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FIG. 2. Crude calyx fluid dissected into Tris-EDTA buffer at pH 9.5 and negatively stained with uranyl
acetate. Compare with Fig. 1B, and note disruption of some virion envelopes (arrows) resulting in release of
nucleocapsids (arrowheads). Bar = 0.7 ,um.
FIG. 3. Semipurified nucleocapsid preparation. Note variation in length of nucleocapsids and presence of
"tails" at one end. Two contaminating fragments of the envelope protrusion (see Fig. 1, arrows) can be seen
(double arrows). Bar = 0.5 Am. The inset illustrates three nucleocapsids at higher magnification. Two lie in
the same orientation and can be seen to differ in length (bars), while the third (on the right) is seen end-on
and therefore presents a circular profile.
1122VOL. 29, 1979 CHARACTERIZATION OF APANTELES BACULOVIRUS 1123
are also included so as to provide an estimate of
contour length variability to be expected from a
unique length species of DNA. The histogram
reveals a major class of viral DNA contour
length at 8.1 megadaltons (Mdal) and two others
at 9.4 and 11.6 Mdal. In addition, six minor
classes at 4.7, 6.4, 14.1, 16.3, 18.4, and 21.4 Mdal
appear to be present. A small number of rela-
tively large molecules (30 to 40 Mdal) were
f
ro
1, r- Flh7i
50 sO 79 80 T')
observed, but the majority of molecular weights
were calculated to lie within the range of 2 to 25
Mdal. Contour length variability of covalently
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NUCLEOCAPSID LE NGIHH n5
FIG. 4. Histogram of lengths of nucleocapsids closed circular DNA has not as yet been ascer-
from a nucleocapsid preparation. Electron micro- tained; we would expect larger molecules to be
graph negatives were projected onto a wall, and the underrepresented, due to increased chances of
end-to-end lengths (excluding the tails) were mea- shearing. Whereas molecular weight data given
sured. here are for open circular DNA extracted from
crude calyx fluid only, it is now clear that DNA
results show that, with one possible exception, isolated from particles that were purified and
there is band-for-band correspondence between digested with nuclease (as described in Materials
crude calyx fluid, DNase-treated crude fluid, and and Methods) is of equivalent heterogeneity
upper-band DNA (see below) that was extracted (data not shown), again confirming that DNA
from DNase-treated purified calyx particles. The associated with the particulate component of
possible exception is band 3, which appeared to calyx fluid is heterogeneous.
be more intense in the case of DNase-treated Since in CsCl-EB gradients most of the DNA
particles. At present, we lack a satisfactory ex- appeared in the upper band, we made the as-
planation for augmentation of band 3, if such sumption that the majority of bands observed
occurs; conceivably, this could be due to nu- by gel electrophoresis represent relaxed circular
clease-induced nicking of a covalently closed species; accordingly, an attempt was made to see
species (band 1?) as a result of limited leakage whether any correlation, in terms of molecular
of enzyme into virus particles. In any case, the weight, could be made between these and the
results strongly suggest that there are no major size classes identified by electron microscopy
contaminating species of DNA in the non- (Table 1); appropriate relaxed circular DNA
particulate fraction of calyx fluid, thus confirm- markers were used in both cases to determine
ing heterogeneity of encapsidated viral DNA. molecular weights (see Fig. 5 and 7; 31). As can
OPM2 DNA, included as a control, was com- be seen, there is fairly good correlation between
pletely digested by the nuclease. size classes identified by electron microscopy
When calyx fluid or purified virus in SSC- and many of the major bands seen in agarose
EDTA was treated with 2 to 4% Sarkosyl and gels. Relative band positions and intensities also
centrifuged to equilibrium on a CsCl-EB gra- compare well with the histogram data. Band 9
dient, two fluorescent bands resulted (Fig. 6A). (= peak C) appears to represent the most com-
Crude calyx fluid from 20 wasps was sufficient mon molecular weight species (molecular weight
for visualization of these bands; about twice as = 7.9 x 10' by relative mobility or 8.1 x 106 by
much was required in the case of virus pelleted contour length measurement). Minor bands (1,
from sucrose cushions. The upper, more intense 2, 3, 5, 7 and 8) do not appear to correspond to
band was at p = 1.54 g/ml and consisted predom- any of the histogram peaks; presumably, elec-
inantly of open circular DNA (Fig. 6B), whereas tron microscopy of a larger sample of molecules
the lower, much less intense band was at p = would identify additional discrete size classes
1.58 g/ml and consisted almost entirely of su- corresponding to these peaks. In the absence of
perhelical DNA (Fig. 6C). markers in the higher-molecular-weight range,
As visualized by the Kleinschmidt procedure, we have not extrapolated for values above 9 x
relaxed circular DNA derived from crude calyx 106, so that correlations of bands 13 to 16 with
fluid material is highly variable in contour length histogram peaks E to H are made on the basis
(Fig. 6B). Measurements of well-spread circular of relative distribution and intensity/frequency
molecules were made, using OPM2 DNA (molec- only.
ular weight, 6.4 x 106; 26) and pX174 RF DNA Some linear single-stranded nucleic acid was
(molecular weight, 3.49 x 106; 29) as standards. occasionally observed in upper-band material
A histogram showing viral DNA contour lengths derived from crude calyx fluid, as were linear ds
is presented in Fig. 7; 4PM2 DNA measurements molecules (1124 KRELL AND STOLTZ J. VIROL.
BC-
,J
r e.
.'e
FIG. 5. Electrophoresis of A. melanoscelus DNA in 0. 761 agarose gels. (A) DNA from crude calyx fluid (four
Downloaded from http://jvi.asm.org/ on February 16, 2021 by guest
female wasps) collected in SSCE. (B) Upper band DNA isolated from a CsCl-EB gradient and dialyzed
against I x SSCE. Calyx particles from 50 female wasps uwere centrifuged tuice onto a sucrose gradient,
pelleted, and then suspended in 100 ,Il of 0.1 M acetate buffer (pH 5.0) containing 5 mM MgSO,1; this material
was then digested for 3 h at 25°C with 1 ,ug of DNase I. (C) PM2 DNA (I fig) in 20,il of 0.1 M acetate buffer and
5 mM MgSO4. (D) PM2 DNA, as above, but digested with 0.2 ,ig of DNase I for 3 h at 250C. (E,) DNA from
crude calyx fluid (eight A. melanoscelus females) in 40 ,il of 0.1 M acetate buffer containing 5 mM MgSO1. (F)
As in E, but digested for 3 h with 0.5 ,ug of DNase I at 250C. Track A uas run approximately 1 year prior to
tracks B to F (i.e., A is from a different gel). Other relaxed circular DNA markers (see the text) were subjected
to electrophoresis on the same gel as track A (but on separate tracks); their relative positions and molecular
weights are indicated on the left.
ing from shearing of circular forms. In a survey cesium chloride, using M. lysodeikticus DNA as
of 235 upper-band DNA molecules from purified, a marker (Fig. 8). This corresponds to a guanine
nuclease-treated particles, only 12.1% were lin- plus cytosine content of 35%, as calculated ac-
ear ds forms. cording to Schildkraut et al. (30).
Analytical buoyant density ultracentrifuga- Proteins. Polyacrylamide gel electrophoresis
tion of either lower- or upper-band DNA in each of purified A. melanoscelus baculovirus resolved
case resolved a single peak at p = 1.694 g/ml in at least 18 polypeptide bands (p16 to p105; Fig.qb
0
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.I
1. ..
-~
:~
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~:-
:~~~~~~~~~~- 0'
g ¼
FIG. 6. DNA from crude calyx fluid. (A) Bands appearing in CsCl-EB isopycnic gradient of DNA from
calyx fluid taken from 50 A. melanoscelus females. The calyx fluid in SSCE uas treated with 4% Sarcosvl at
60°C for 30 min, then centrifuged to equilibrium at 130,000 x g at 20°C for 36 h in CsCl (p = 1.58) and EB (100
ptg/ml). (B) Electron micrograph ofDNA taken directlv from the upper band shown in A. Note that the relaxed
circular molecules vary considerably in length. Bar = 1.0,m. (C) Supercoiled DNA from the lower band of the
11251126 KRELL AND STOLTZ J. VIROL.
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DNA CONTOUR LENGTH microns)
FIG. 7. (A) Histogram of lengths of relaxed circular DNA molecules. Molecular weights of the different size
classes were calculated using the mean size of (pPM2 and of CXl 74 RF DNA as standards. (B) Distribution of
lengths of circular PM2 molecules prepared in the same way and measured at the same magnification. md,
Mdal.
TABLE 1. Correlation of DNA molecular weight cleocapsids (Fig. 9B). Particles prepared by re-
species as determined by gel electrophoresis and peated (twice or three times) centrifugation onto
electron microscopy a sucrose cushion, followed by pelleting, exhibit
Possible corre- a polypeptide gel profile essentially identical to
Mol wt calculated
Agarose gel band from sponding peak that of crude calyx fluid. Thus, it does not appear
no.' mobility in aga-
rose gels (x O1)
from Klein- that crude fluid contains any major polypeptide
schmidt
histogram' not associated with virions; this is not to say
1_
that minor contaminants would not be detected
2 3.6 by methods with greater resolving power than
3 4.3 those employed in this investigation. The molec-
4 5.1 A ular weights of the polypeptides were calculated
5 5.9
6 6.1 B
7 6.6
8 6.9
9 7.9 C
10 8.6
11 8.9
12 9.1 D 694 1731 [
13 - z I
14 - E
15 - F
16 - G, H
b
Agarose gel band numbers are taken from Fig. 5.
Peaks of DNA molecular weights are taken from INCREASING DENSITY -
Fig. 7. FIG. 8. Equilibrium banding of superhelical calyx
fluid DNA and M. lysodeikticus marker DNA. DNA
was taken from the lower band of a CsCI-EB gra-
9), including approximately six major polypep- dient, EB was extracted with several changes of
tides: p16, p32, p38, p51, p56, and p68. Electro- isopropanol, and the DNA was then dialyzed against
phoresis of nucleocapsids resolved five: p16, p32, SSCE before being mixed with marker DNA. The
p38, p68, and p96, of which p32 and p38 are the DNA was centrifuged to equilibrium in CsCl (p =
major species. One or two of the minor "nucleo- 1.7198) at 44,777 rpm for 24 h in a Beckman model E
capsid" polypeptides could be derived from rem- ultracentrifuge equipped with UV optics. The posi-
nants of the envelope protrusion, which are tion of the bands was photographed, and a scan was
taken of the negative by a microdensitometer to show
found as contaminants in this material (see Fig. the relative position of the bands in the cell during
3). In Fig. 9A, the staining intensity of bands p32 centrifugation after equilibrium had been reached.
and p38 are roughly equivalent; often, however, Identical results were obtained with open circular
the p32 band was of lesser intensity, as in nu- DNA.VOL. 29, 1979 CHARACTERIZATION OF APANTELES BACULOVIRUS 1127
A B c D
- P 105
- P96
- P 87
- P 81
- P 77
.-
- P 68 um.4
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- P6 5
* - P56
- P 51
- P 46
mm~
P38
P36 -
P 331128 KRHELL AND STOLTZ.J. VIROL.
TABLE 2. Structural polNpeptides of A. known to occur in other DNA viruses; similarly,
melanoscellus tvirus and nucleocapsids" variation in the molecular weight of encapsi-
Virus polNpeptide Mol wt dated DNA molecules of other viruses also ap-
16,500
pears to be minimal (4, 42), or else involves only
P16" a small fraction of the virus particle population
P21 21,300
P28 28,000 (e.g., 6). The only known example of a cylindrical
P30 30,500 DNA-containing virus which exhibits variation
P32 32,000 in both nucleocapsid and genome length is that
P33 33,000 of fd bacteriophage; this, however, is a single-
P36 36,500 stranded DNA virus, and genomic variation
P38' 38,000 seems limited to a simple monomer/dimer rela-
P46 46,000 tionship (42). There are, however, several ex-
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P'51 51,000 amples of rod-shaped RNA viruses which ex-
P56" 56,000 hibit nucleocapsid and genomic RNA length var-
P65 65,000 iation (15, 16, 18, 20), although this generally
P68 68,000
P77 77,000 appears to be restricted to a small number (two
P81 81,000 to five) of discrete size classes. Examples of RNA
P87 87,000 virion heterogeneity are associated either with
P96 96,000 multipartite (16, 18) or defective interfering (15)
P105 105,000 systems. Which, if any, of these alternative strat-
"Molecular weights of virion polvpeptides were cal- egies may obtain in the case of Apanteles par-
culated with reference to the mobility of marker poly- ticles is presently unknown.
peptides in 11% acrylamide gels. Considerable heterogeneity in the lengths of
"Major virion polypeptide. non-encapsidated ds circular viral and plasmid
Major nucleocapsid polvpeptide. DNA molecules, on a scale approaching that
presented here, has occasionally been observed
agnostic for baculoviruses, since no other well- (19, 28, 44). What is known concerning the mode
defined type of virus shares these characteristics. of generation of circular DNA molecules of dif-
Also typical of baculoviruses is a genome con- ferent sizes in other such systems may provide
sisting of ds circular DNA, usually of high mo- some direction to future research on Apanteles
lecular weight (70 x 106 to 90 X 106; 5, 12, 36). viral DNAs. For example, smaller circles might
Previous investigations had suggested that on represent deletion forms of larger ones, as has
the basis of ultrastructural criteria particles ob- been shown in the case of HD and simian virus
served in the ovaries of many species of braconid 40 papovavirus DNAs (4, 7); alternatively, larger
wasps were almost certainly baculoviruses (32, molecules could be recombination products of
33). In the case of Apanteles particles, two other smaller circular forms as suggested for the origin
points of similarity can be mentioned: (i) inter- of simian virus 40 DNA dimers (10, 23). In any
action of particle nucleocapsids with the nuclear case, it seems likely that most Apanteles virus
pores of caterpillar cells (32) would appear to DNAs may share some degree of homology since
represent the functional equivalent of the un- purified DNA is of homogeneous density, as
coating of granulosis virus (34); and (ii) nucleo- determined by analytical ultracentrifugation.
capsid tail-like appendages are morphologically At least 18 polypeptide bands are resolved by
indistinguishable from those associated with the polyacrylamide gel electrophoresis of solubi-
nucleocapsids of Oryctes baculovirus (25; Stoltz lized, purified Apanteles virus. Two major nu-
and Vinson, in press). cleocapsid polypeptides with molecular weights
The identification in the present study of within the range of 25 x 10"3 to 40 x 10' are often
Apanteles calyx particle genomes consisting of observed, whereas only one is seen in the case of
ds circular DNA (or DNAs, depending on gen- nucleocapsids and capsids of other baculoviruses
omic complexity) supports our previous tenta- (5, 12, 37). Presumably, the main cylindrical
tive assignment of such particles to the baculo- portion of the baculovirus capsid is represented
virus group (35). Nevertheless, it is clear that in all baculovirus gel profiles by a polypeptide of
the Apanteles virus and those of other braconid a molecular weight in the range given above. Of
wasps (27, 33) must represent an extremely un- the two major nucleocapsid polypeptides iden-
usual type of baculovirus. Thus, whereas other tified, p38 seems predominant (especially in nu-
baculoviruses have nucleocapsids and encapsi- cleocapsid gel profiles) and probably represents
dated monomolecular genomes of uniform size, the capsid cylinder polypeptide. The additional
both these parameters have been shown to be major component observed in gels of Apanteles
extremely variable in the present instance. Ex- nucleocapsids presumably represents a putative
tensive variability in nucleocapsid size is not "tail" polypeptide. It is significant, in this regard,N OI,. 29, 1979 CHARACTERIZATION OF APANTELES BACULOVIRUS 1129
that a band of identical mobility (p32) also ap- toneura fumiferana. Can. J. Microbiol. 21:1224-1231.
pears in polyacrylamide gels of Oryctes baculo- 2. Bell, C. D., and G. B. Orlob. 1977. Serological studies on
virus polypeptides; as mentioned above, this vi- virions and polyhedron proteins of a nuclear polyhedro-
sis virus of the cabbage looper Trichoplusia ni. Virology
rus also possesses tail-like appendages which 78:162-172.
appear to be attached to the nucleocapsids (25). 3. Bergold, G. H. 1947. Die Isolierung des Polyedervirus
Common to all three baculoviruses examined is und die Natur der Polyeder. Z. Naturforsch. 2b: 122-
a polypeptide of relatively low molecular weight 143.
4. Bosslet, K., and G. Sauer. 1978. Biological properties
(range 16 to 20); it is of interest that what is and physical map of the genome of a new papovavirus,
probably a similar polypeptide has been ob- HD virus. J. Virol. 25:596-607.
served in all other baculoviruses examined to 5. Brown, D. A., H. M. Bud, and D. C. Kelly. 1977.
date (5, 12, 25, 37). Biophysical properties of the structural components of
a granulosis virus isolated from the cabbage white but-
In toto, Apanteles virus polypeptides com-
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terfly (Pieris brassicae). Virology 81:317-327.
prise a molecular weight in excess of 106, which 6. Burlingham, B. T., D. T. Brown, and W. Doerfler.
would require a minimum genome complexity of 1974. Incomplete particles of adenovirus. 1. Character-
approximately 20 x 106. This amount of infor- istics of the DNA associated with incomplete adeno-
virions of types 2 and 12. Virology 60:419-430.
mation could conceivably be coded for by the 7. Carroll, A., and F. J. O'Neill. 1978. Genome maps of
larger size classes of viral DNA or by a combi- simian virus 40 defectives propagated in human glio-
nation of several different species; we presently blastoma cells. Virology 87:120-129.
favor the latter alternative. (The possibility, 8. Cohen, J. C., M. J. Perdue, C. C. Randall, and D. J.
O'Callaghan. 1977. Herpesvirus transcription: altered
however remote, that some viral structural pro- regulation induced by FUdR. Virology 76:621-633.
teins could be coded for by the parasitoid ge- 9. Davis, R. W., M. Simon, and N. Davidson. 1971. Elec-
nome cannot at present be ruled out; were this tron microscope heteroduplex methods for mapping
the case, the smallest viral DNA molecules could regions of base sequence homology in nucleic acids.
Methods Enzymol. 21D:413-428.
well code for all information contributed by the 10. Goff, S. P., and P. Berg. 1977. Structure and formation
viral genome itself.) It is of interest to note that of circular dimers of simian virus 40 DNA. J. Virol. 24:
all DNA molecules purified from Cardiochiles 295-302.
nigriceps virus (31, 40) are of relatively low 11. Grisdale, D. 1973. Large volume preparation and proc-
essing of a synthetic diet for insect rearing. Can. Ento-
molecular weight (all1130 KRELL AND STOLTZ J. VIROL.
cells. Virology 7 1:28-40. 34. Summers, M. D. 1971. Electron microscopic observations
24. Morris, R. F. 1976. Influence of genetic changes and other on granulosis virus entry, uncoating and replication
variables on the encapsulation of parasites. Can. Ento- processes during infection of the midgut cells of Tricho-
mol. 108:673-684. plusia ni. J. Ultrastruct. Res. 35:606-625.
25. Payne, C. C., D. Compson, and S. M. de Looze. 1977. 35. Summers, M. D. 1975. Biophysical and biochemical prop-
Properties of the nucleocapsids of a virus isolated from erties of baculoviruses, p. 17-29. In M. Summers, R.
Oryctes rhinoceros. Virology 77:269-280. Engler, L. A. Falcon, and P. Vail (ed.), Baculoviruses
26. Pettersson, U., C. Mulder, H. Delius, and P. A. for insect pest control: safety considerations. American
Sharpe. 1973. Cleavage of adenovirus type 2 DNA into Society for Microbiology, Washington, D.C.
six unique fragments by endonuclease R.RI. Proc. Natl. 36. Summers, M. D., and D. L. Anderson. 1973. Character-
Acad. Sci. U.S.A. 70:200-204. ization of nuclear polyhedrosis virus DNAs. J. Virol.
27. Poinar, G. O., Jr., R. Hess, and L. E. Caltagirone. 12:1336-1346.
1976. Virus-like particles in the calyx of Phanerotoma 37. Summers, M. D., and G. E. Smith. 1978. Baculovirus
flatitestacea (Hymenoptera: Braconidae) and their structural polypeptides. Virology 84:390-402.
Downloaded from http://jvi.asm.org/ on February 16, 2021 by guest
transfer into host tissues. Acta Zool. (Stockholm) 57: 38. Vinson, S. B. 1972. Factors involved in the successful
161-165. attack on Heliothis tirescens by the parasitoid Cardi-
28. Roberts, T. M., and K. E. Koths. 1976. The blue-green ochiles nigriceps. J. Invertebr. Pathol. 20:118-123.
alga Agmenellum quadruplicatum contains covalentlv 39. Vinson, S. B. 1974. The role of the foreign surface and
closed DNA circles. Cell 9:551-557. female parasitoid secretions on the immune response of
29. Sanger, F., G. M. Air, B. G. Barrell, N. L. Brown, A. an insect. Parasitology 68:27-33.
R. Coulson, J. C. Fiddes, C. A. Hutchinson, P. M. 40. Vinson, S. B., and J. R. Scott. 1975. Particles containing
Slocombe, and M. Smith. 1977. Nucleotide sequence DNA associated with the oocyte of an insect parasitoid.
of bacteriophage 4X174 DNA. Nature (London) 265: J. Invertebr. Pathol. 25:375-378.
687-695. 41. Weber, K., and M. Osborn. 1975. Proteins and sodium
30. Schildkraut, C. L., J. Marmur, and P. Doty. 1962. dodecyl sulfate: molecular weight determination on
Determination of the base composition of deoxyribo- polyacrylamide gels and related procedures, p. 179-223.
nucleic acid and its buoyant density in CsCl. J. Mol. In H. Neurath, L. Hill, and C. L. Boeder (ed.), The
Biol. 4:430-443. proteins, 3rd ed., vol. 1. Academic Press, New York.
31. Sinsheimer, R. L. 1977. Recombinant DNA. Annu. Rev. 42. Wheeler, F. C., R. H. Benzinger, and H. Bujard. 1974.
Biochem. 46:415-438. Double length, circular, single-stranded DNA from fil-
32. Stoltz, D. B., and S. B. Vinson. 1977. Baculovirus-like amentous phage. J. Virol. 14:620-630.
particles in the reproductive tracts of female parasitoid 43. Wildy, P. 1971. The classification and nomenclature of
wasps. II. The genus Apanteles. Can. J. Microbiol. 23: viruses. Monogr. Virol. 5:81.
28-37. 44. Yagi, Y., and D. B. Clewell. 1976. Plasmid-determined
33. Stoltz, D. B., S. B. Vinson, and E. A. MacKinnon. resistance in Streptomyces faecalis: tandemly repeated
1976. Baculovirus-like particles in the reproductive resistance determinants in amplified forms of pAMal
tracts of female parasitoid wasps. Can. J. Microbiol. 22: DNA. J. Mol. Biol. 102:583-600.
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