Journal
of General Virology (1999), 80, 1537–1540. Printed in Great Britain
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SHORT COMMUNICATION
Circular configuration of the genome of ascoviruses
Xiao-Wen Cheng,† Gerald R. Carner and Thomas M. Brown
Clemson University, Department of Entomology, 114 Long Hall, Box 340365, Clemson, SC 29634-0365, USA
A circular configuration of genomic DNA was
observed in ascoviruses isolated from two species
of insects of the family Noctuidae [fall armyworm
(Spodoptera frugiperda) and cotton bollworm
(Helicoverpa zea)] using restriction endonuclease
(REN) digestion, conventional gel electrophoresis,
pulsed-field gel electrophoresis and Southern
hybridization analysis. This circular configuration of
ascovirus genomic DNA was established based on
the difference between linear and circular DNA in
the numbers of fragments resolved on agarose gel
electrophoresis after single and double REN digestion. Genomic DNA of ascoviruses was found to
be sheared after purification.
Among viruses affecting insects, ascoviruses are the latest
group reported and are assigned to the family Ascoviridae
(Federici, 1983). Only a few ascoviruses have been isolated.
These include ascoviruses from Heliothis spp. (Hudson &
Carner, 1981 ; Carner & Hudson, 1983), Trichoplusia ni
(Browning et al., 1982), Scotogramma trifolii (Federici, 1982),
Autographa precationis (Hamm et al., 1986), Spodoptera frugiperda
(Hamm et al., 1986) and Diadromus pulchellus (Bigot et al.,
1997 a, b). Except for the ascovirus from D. pulchellus, all other
ascoviruses were isolated from hosts in the family Noctuidae.
The ascoviruses are distinguished from other viruses by
producing a milky-white discoloration of the haemolymph in
the infected host, which is caused by accumulation of a high
concentration of virion-containing vesicles. Virion-containing
vesicles are formed by the cleavage of the host cell membrane,
a unique symptom of ascovirus infection (Federici, 1983). The
enveloped virions of ascoviruses are allantoid to bacilliform in
shape and have a size of 130¬400 nm. They have complete
symmetry and a double-stranded DNA genome of about
130–180 kbp (Federici et al., 1991).
There is conflicting evidence regarding the physical
Author for correspondence : Gerald Carner.
Fax 1 864 656 5065. e-mail GCarner!Clemson.Edu
† Present address : Laboratory for Molecular Virology, Great Lakes
Forest Research Centre, 1219 Queen St E, Sault Ste Marie, Ontario,
Canada P6A 5M7.
0001-6205 # 1999 SGM
configuration of the genomic DNA molecule of ascoviruses.
Electron microscopy of isolates from T. ni ascovirus (TAV)
indicated a linear configuration (Federici, 1983). However, a
restriction endonuclease (REN) fragmentation map generated
from D. pulchellus ascovirus (DpAV) provided evidence of a
circular genome (Bigot et al., 1997 a). There was also some
suggestion that DpAV may be a polydnavirus, rather than an
ascovirus (Bigot et al., 1997 b). Because both DpAV and TAV
are reported as members of the family Ascoviridae, the
observation of both linear and circular forms would be difficult
to explain in a systematic context. These conflicting reports
suggested the need to resolve this question by examining the
physical configuration of the ascovirus genomes isolated from
species of Noctuidae. In this communication, we report the
results of our investigation of the genome configuration for the
previously described ascoviruses of Noctuidae.
Analysis of the configuration of the ascovirus genome was
based on determining the number of fragments resulting from
enzymatic digestion of the DNA catalysed by several RENs.
The genomic DNA molecule and large products of hydrolysis
could be clearly resolved by pulsed-field gel electrophoresis
(PFGE). S. frugiperda ascovirus (SAV) was kindly provided by
B. A. Federici (University of California at Riverside). Helicoverpa zea ascoviruses (HAV) were isolated from Heliothis
virescens and H. zea on cotton in Blackville, South Carolina.
SAV and HAV were propagated by using a mitten pin
contaminated with ascovirus to puncture the proleg of third- to
fourth-instar larvae of either S. exigua or H. virescens. Inoculated
host larvae were reared on an artificial diet. Seven days postinoculation, the haemolymph with vesicles was collected and
virions in the vesicles were purified as described by Federici
et al. (1990). Viral DNA was purified by centrifugation in a
CsCl–EthBr gradient for 15 h. Five RENs were used to catalyse
cleavage of the viral genomic DNAs prior to agarose gel
analysis ; all RENs used were known to yield fewer than 14
fragmentation products. The five restriction enzymes were
NotI, Bsu36I, SmaI, AscI and PstI (Promega and New England
Biolabs). The genomic DNAs were cut by single and double
REN sequential digestion following the conditions recommended by the manufacturers. Products were separated on a
0±7 % agarose gel in conventional electrophoresis and 1 %
agarose gel on PFGE using a Bio-Rad CHEF-DRII, with
λ–HindIII and Pulse Marker (Sigma). The conditions for PFGE
analysis were as follows : interval 3–12 s, 120 ° angle, 130 V,
BFDH
X.-W. Cheng, G. R. Carner and T. M. Brown
Fig. 1. PFGE analysis of genomic DNA
from two previously described Noctuidae
ascovirus isolates. Pulse marker (lane 1)
was used to estimate fragment size. (A)
SAV : lane 1, size markers ; lane 2,
Bsu36I, lane 3, SmaI ; lane 4,
Bsu36I/SmaI double digestion. (B) HAV :
lane 1, size markers ; lane 2, AscI ; lane 3,
PstI ; lane 4, AscI/PstI double digestion.
Dotted lines in (B) indicate that the
fragments were seen on conventional gel
electrophoresis (Fig. 2).
at 14 °C for 16 h. DNA fragments in the agarose gel were
transferred onto a Nytran membrane (Schleicher and Schuell).
Genomic DNAs of the two ascoviruses were digested with
Sau3AI and randomly labelled with digoxigenin according to
the manufacturer’s protocol (Genius I, Boehringer Mannheim).
These labelled DNAs were used to perform Southern analysis
(Southern, 1975). The method to determine the genome
BFDI
configuration followed Bigot et al. (1997 a). Briefly, if enzyme
A produces X fragments of the genomic DNA, and enzyme B
produces Y fragments, then double digestion with enzymes A
and B will produce XY fragments for a circular genome and
XY-1 for a linear genome.
Results of PFGE following single and double digestion of
DNA indicated that the two previously described ascoviruses
Ascoviruses have a circular genome
Fig. 2. Conventional agarose gel (0±7 %) electrophoresis of HAV REN
digestion to show the small fragments indicated by dotted lines in Fig.
1 (B). Lane 1, λ–HindIII DNA size marker ; lane 2, PstI digestion ; lane 3,
AscI/PstI double digestion.
possess circular genomic DNA. Of the six different combinations of double REN digestions, four confirmed the
presence of a circular genome. In three double-digestions of
SAV, only one confirmed the presence of a circular genome
form (Fig. 1 A). An explanation might be that in the double
digestions, the two enzymes may have cut at sites very close
together producing a small fragment which exited the gel.
However, Southern analysis did not reveal any additional
fragments which could not be detected on an agarose gel (data
not shown). HAV was confirmed with no exception to have a
circular genome by the REN digestion analysis (Fig. 1 B
and Fig. 2 ; analysis with RENs other than those in Fig. 1 B and
Fig. 2 was not shown).
Bsu36I and SmaI produced five and seven fragments,
respectively, from the SAV genomic DNA in the single REN
digestion. In the double sequential digestion of SAV genomic
DNA with Bsu36I and SmaI, 12 fragments were produced (Fig.
1 A). Fragments with lengths shorter than 15 kbp in the single
digestion remained uncut in the double sequential REN
digestion (fragment 5 in lane 2 and fragments 5, 6 and 7 in lane
3 in Fig. 1 A). Fragments longer than 20 kbp in the single REN
digestion were cut into shorter segments by the double
sequential REN digestion (Fig. 1 A). AscI single digestion of
HAV genomic DNA (lane 2, Fig. 1 B) produced one fragment
of 173 kbp, which was the same size as the entire genome of
HAV. PstI single REN digestion resulted in 13 fragments (lane
3, Fig. 1 B), and those fragments that were smaller than 5 kbp
were resolved by 0±7 % conventional agarose gel electrophoresis (Fig. 2). In the double sequential REN digestion, 14
fragments was formed (lane 4, Fig. 1 B, and Fig. 2). We found
that AscI cut fragment 1 of the PstI digestion (lane 3, Fig. 1 B)
into two fragments (1 and 4) in the AscI}PstI digestion (lane 4,
Fig. 1 B).
Results from the double REN sequential digestion of HAV
confirmed that the REN AscI was active and catalysed the
cleavage of the genomic DNA of HAV in the single AscI
digestion (lane 2, Fig. 1 B). Since only one fragment of the
HAV–PstI digestion (fragment 1 in lane 3, Fig. 1 B) was cut by
AscI, this indicated that there is only one AscI cutting site in the
HAV genome. A single cut on the genomic DNA of this
ascovirus formed only one fragment of 173 kbp (Fig. 1 B). This
indicated that a simple circular form was opened to a linear
molecule ; a linear DNA would have been cut into two
products.
During the course of purification, shearing of genomic
DNA occurred (lane 2, Fig. 1 A). The shearing resulted in a
larger fragment (2) not as brightly stained by ethidium bromide
as the smaller fragments (3 and 4) in the Bsu36I digestion of
SAV genomic DNA (lane 2, Fig. 1 A). A possible explanation
is that a certain region on the SAV genomic DNA was prone
to shear. In such a situation, the size of the genomic DNA for
the ascovirus must be considered to decide if a fragment should
be assigned or not.
Our finding that ascoviruses have circular DNA is not in
conflict with previous experimental observations if one
considers that previous observation of a linear molecule by
electron microscopy may have been due to opening of the
circular DNA during DNA purification. We have demonstrated
the sensitivity of ascovirus genomic DNA to shearing and it is
possible that a portion of genomic DNA would have been
linearized in preparation for electron microscopy. This shearing
may explain why the ascovirus genome was observed as linear
by electron microscopy (Federici, 1983). Observation of DNA
by electron microscopy is limited by the number of DNA
molecules that can be examined. However, in analysis of the
genome physical configuration with REN by means of PFGE,
hundreds of nanograms of DNA can be examined.
Federici (1983) demonstrated that ascovirus replication was
initiated in the nuclei of susceptible cells and virions were
formed in the cytoplasm. Most double-stranded DNA insect
viruses with linear genomes replicate in the cytoplasm of cells,
e.g. entomopoxviruses and iridoviruses (Bergoin et al., 1969 ;
Stoltz & Summers, 1972 ; Mathiesen & Lee 1981), whereas, the
double-stranded circular DNA insect viruses replicate in the
nuclei of cells, e.g. baculoviruses (Huger & Krieg, 1961). Thus,
a circular configuration for ascoviruses fits into the general
pattern. However, the formal relationship between the site of
replication in the cells and the virus genome configuration is
neither fully established nor understood.
BFDJ
X.-W. Cheng, G. R. Carner and T. M. Brown
We would like to thank Y.-H. Wang for help in the PFGE operation,
Drs B. A. Federici and J. J. Hamm for providing ascovirus isolates, M.-Z.
Luo for helpful advice and B. M. Arif for help in the final revision.
Financial support was provided by Hatch Project 66-1614 and by a
USAID Project, Integrated Pest Management Research, Development
and Training Activities for Palawija Crops in Indonesia. This paper is
technical contribution number 4467 of the South Carolina Agricultural
and Forestry Research System, Clemson University.
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Received 21 January 1999 ; Accepted 23 February 1999
Xiao-Wen Cheng