J. gen. Virol. (1988), 69, 2303-2312. Printedin Great Britain
2303
Key words: baculovirus/polyhedrin/L,dispar
Restriction Mapping of Lymantria dispar Nuclear Polyhedrosis Virus
DNA: Localization of the Polyhedrin Gene and Identification of Four
Homologous Regions
By J. T H O M A S M c C L I N T O C K t AND E D W A R D M. D O U G H E R T Y *
Insect Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, Maryland 20705, U.S.A.
(Accepted 20 May 1988)
SUMMARY
The genome of the multiple-embedded nuclear polyhedrosis virus (MNPV) of
Lymantria dispar (LdMNPV) was partially characterized by restriction endonuclease
analysis and a physical map was constructed using cosmid cloning and Southern cross
blot hybridization. Using BamHI, BglII, EcoRI and HindlII, the size of the genome
was estimated to be 88.5 x 106 Mr or 134-04 kbp. LdMNPV DNA was also analysed
using methylation-sensitive restriction enzymes. The resulting restriction profiles
suggested that extensive methylation did not occur at the nucleotide sequence
recognized by HpalI and MspI. A BamHI restriction map was constructed by
comparing overlapping BamHI fragments between cosmid clones containing partial
digests of viral DNA. The positions of the BglII, EcoRI and HindlII sites were
determined by Southern cross blot hybridizations and aligned to the BamHI restriction
map. At least four homologous regions were identified by cross blot hybridizations of
BgllI-digested LdMNPV DNA and such regions were found to be interspersed along
the genome in a fashion similar to that reported for other baculoviruses. Using
recombinant plasmids containing the HindlII-V fragment of Autographa californica
MNPV to probe Southern blots of LdMNPV DNA, the restriction fragment(s) that
contain the polyhedrin gene were identified. Based on these findings the map was
oriented with the polyhedrin gene of LdMNPV as the zero point.
INTRODUCTION
The multiple-embedded nuclear polyhedrosis virus (MNPV) of the gypsy moth, Lymantria
dispar (LdMNPV), is composed of double-stranded, circular DNA packaged into enveloped
virions which are occluded in a crystalline protein matrix called polyhedrin. Unfortunately, the
molecular characterization of LdMNPV has lagged far behind that of the MNPV of Autographa
californica (AcMNPV), which serves as the model system for studies on the molecular biology of
baculoviruses.
Physical restriction maps have been reported for several genotypic variants of AcMNPV
(Miller & Dawes, 1978; Smith & Summers, 1979; Vlak & Smith, 1982). Subsequently,
transcriptional and translational maps were constructed (Smith et al., 1982; Esche et al., 1982;
Erlandson & Carstens, 1983; Rohel et al., 1984; Rohel & Faulkner, 1984; Friesen & Miller,
1985) and used to locate and identify several genes on the AcMNPV genome. The product of one
such gene is the 33K polyhedrin protein which is expressed at high levels during the
intermediate and late phase of the viral replicative cycle (Vlak et al., 1981; Adang & Miller,
1982; Smith et al., 1983). By in vitro translation of polyhedrin from specific RNA, the polyhedrin
gene was mapped to the EcoRI-I fragment and assigned as the zero or start point with regard to
the orientation of the restriction map.
t Present address: Digene Diagnostics Inc., Building 334, University of Maryland, College Park, Maryland
20742, U.S.A.
0000-8212
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2304
J . T . McCLINTOCK AND E. M. DOUGHERTY
To study the organization and expression of the genome of L d M N P V , and as a prelude to
transcriptional and translational mapping, the D N A from a plaque-purified isolate of
L d M N P V was analysed using restriction endonucleases and a physical m a p was constructed
using cosmid cloning and Southern cross blot hybridization. This study presents the first
restriction map of L d M N P V . In addition, we show that the viral genome contains at least four
homologous regions (HRs) (Cochran & Faulkner, 1983; G u a r i n o et al., 1986) interspersed along
the D N A and unlike A c M N P V , a distantly related NPV, not all of these regions contain
numerous E c o R I sites. Using the H i n d l I I - V fragment of A c M N P V to probe Southern blots of
L d M N P V , we also identified the restriction fragments containing the polyhedrin gene. Based
on these findings the L d M N P V map was oriented with the polyhedrin gene as the zero point.
METHODS
Preparation o f L d M N P V DNA. A plaque-purified isolate of LdMNPV (5-7D) (McClintock et al., 1986b) was
propagated in L. dispar larvae and the viral DNA was extracted from virions isolated from purified polyhedral
inclusion bodies (PIBs) as previously described (Miller & Dawes, 1978).
Restriction endonuclease analysis and construction of cosmid clones. Purified viral DNA was digested with
restriction endonucleases (Bethesda Research Laboratories) and analysed by agarose gel electrophoresis. The Mr
of each restriction fragment was determined by comparing their electrophoretic mobilities to EcoRI- and HindIIIdigested tambda phage DNA markers of known Mr.
Recombinant cosmids were constructed by ligating linearized pHC79 cosmid DNA (Bethesda Research
Laboratories) with LdMNPV DNA partially digested with BamHI. The constructs were packaged in vitro
(Promega Biotec, Madison, Wis., U.S.A.) and selected by spreading infected Escherichia coli HB101 cells on LB
agar plates containing ampicillin. Recombinant cosmid DNA was isolated by the alkaline lysis method (Birnboim
& Doly, 1979), digested with BamHI, and analysed by agarose gel electrophoresis to determine the LdMNPV
restriction fragment present in each clone.
Southern cross blot hybridization. The LdMNPV genome was analysed and homologousregions detected by the
Southern cross blot hybridization procedure of Potter & Dressier (1986). Forty-five ~tg of LdMNPV DNA was
digested with either BamHI, EcoRI or HindlII. For labelling, 30 btgof LdMNPV DNA was digested with BgllI or
EcoRI, dephosphorylated and 3' end-labelled with [ct32-P]dATP by replacement synthesis using T4 DNA
polymeraseas described by O'FarreU et al. (1980). The unlabelled and labelled digests were separated in parallel by
preparative agarose gel electrophoresis (0.8%) and transferred to GeneScreen and GeneScreen-Plus (New
England Nuclear), respectively, according to the manufacturer's procedure. The cross blot sandwich was formed
by placing the blot containing the labelled DNA face up on a glass plate with the unlabelled blots turned 90° and
stacked face down. The blots were then covered with a piece of Whatman 3MM paper presaturated in formamidecontaining hybridization buffer [5 x SSC, 25 mM-sodiumphosphate pH 6.5, 10 x Denhardt's solution (0.1%
Ficoll, 0-1% polyvinylpyrrolidoneand 0-1~obovine serum albumin), 0.1 ~ SDS, 200 ktg/mldenatured yeast tRNA
and 50~ deionized formamide]. The blots were saturated with hybridization buffer, sandwiched using a second
glass plate, allowedto equilibrate at room temperature for 2 h and then incubated at 37 °C or 42 °C overnight. For
less stringent conditions either the concentration of formamide was lowered to 25 ~ or the temperature changed to
25 °C. Following hybridization, the blots were washed three times at room temperature for 20 min each in 2 x
SSC, 0-1~ SDS, then twice at 50°C for 40min in 0-1 x SSC, 0.1~ SDS, air-dried, and processed for
autoradiography.
Recombinant plasmids. To locate the polyhedrin gene, the HindlII-V fragment of AcMNPV was isolated from
agarose gels by a modification of the 'freeze-squeeze' method (Thuring et al., 1975), cloned into pBR325 by
conventional methods (Maniatis et al., 1982), labelled with [32p]dCTPby nick translation (Rigby et al., 1977), and
hybridized to blots containing LdMNPV DNA. In some experiments the fragment of interest was isolated from
the gel, radiolabelled by nick translation, and used to probe Southern blots of viral DNA.
RESULTS
Restriction endonuclease analysis o f L d M N P V D N A
Digestion of L d M N P V D N A with B a m H I , BglII, E c o R I or H i n d l I I generated the smallest
n u m b e r of fragments with distinguishable profiles (Fig. 1). The restriction fragment patterns
generated by these enzymes were used to estimate the Mr of the viral D N A . W h e n compared to
molecular size standards, the viral D N A was determined to be approx. 88.5 x 106 Mr or
134-04 kbp (Table 1).
L d M N P V D N A was also analysed using the methylation-sensitive restriction enzymes HpaII
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L d M N P V restriction mapping
2
2305
3
4
B-~
Fig. 1. Restriction endonuclease profiles of LdMNPV DNA. Samples of viral DNA were digested
with (lane 1) BamHI, (lane 2) HindlII, (lane 3) EcoRI or (lane 4) BgllI, and analysed in a 0.7~ agarose
gel. Following electrophoresis, the gel was stained with ethidium bromide and photographed under u.v.
illumination. Individual fragments for each enzymic digest were assigned an alphabetical designation
on the basis of size such that the largest fragment was designated A. The size of each fragment was
determined by comparing its mobility with EcoRI- or HindlII-digested lambda phage DNA fragments
of known size.
and MspI which recognize the tetranucleotide sequence 5'-CCGG-3'. However, HpalI cannot
cleave at sites modified by methylation, whereas MspI cleaves at modified or unmodified sites.
For comparison our plaque-purified isolate (6R) of A c M N P V was digested under the same
conditions as those for L d M N P V D N A (Fig. 2). Since the resulting restriction patterns
appeared similar, as judged by agarose gel electrophoresis, it was concluded that extensive
methylation did not occur at the nucleotide sequence recognized by these isoschizomers.
However, limited methylation may have occurred and simply not been detected by agarose gel
electrophoresis.
Localization of HRs
Since HRs would complicate the interpretation of hybridization data, their location was
determined by a Southern cross blot hybridization of L d M N P V D N A digested with BgllI (Fig.
3). Analysis of the Southern cross blot hybridization revealed a diagonal line of dark spots with a
number of spots occurring off the diagonal. The diagonal line represents the hybridization
response of each restriction fragment to itself, whereas spots off the diagonal represent cross
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2306
J. T. M c C L I N T O C K
2
3
4
5
A N D E. M. D O U G H E R T Y
6
--23.1
--9.1
--6-6
--4.4
-2-3
-2-0
Fig. 2. Digestion of AcMNPV (lanes 2 and 3)
and LdMNPV (lanes 4 and 5) DNA with
methylation-sensitive restriction enzymes.
Viral DNA was digested with HpalI (lanes 2
and 4) and MspI (lanes 3 and 5), electrophoresed in a 1.0~ agarose gel, stained, and
photographed under u.v. illumination. The
numbers on the left and right represent the
relative size (kb) of lambda phage D N A
fragments digested with EcoRI (lane 1) or
HindlII (lane 6).
hybridization between additional restriction fragments of different sizes. The BgllI cross blot
revealed spots that hybridized not only between themselves to form the diagonal line but to
fragments BgllI-B, -C/D, -J, -K, -Q and -R which formed symmetrical spots off the diagonal
line. In addition, several asymmetrical hybridization spots appeared off the diagonal. For
example, BgllI-C/D, -J and -K cross-hybridized to BgllI-I, and BgllI-B cross-hybridized to
BgllI-I and -M. Since no other coordinates of hybridization were observed these results suggest
that one sequence of L d M N P V D N A is repeated in at least four regions of the genome.
Construction of the LdMNP V restriction map
A BamHI restriction map of L d M N P V was constructed by comparing overlapping BamHI
fragments between cosmid clones containing partial digests of viral D N A (Table 2). The
positions of the BgllI, EcoRI and HindlII restriction sites were subsequently determined by
Southern cross blot hybridization and aligned to the BamHI restriction map. The Southern cross
blots were constructed using a 3zP-labelled BgllI digest of L d M N P V D N A . Using these data,
BgllI and EcoRI restriction maps were constructed. Likewise, a cross blot hybridization of
L d M N P V D N A digested with BgllI, radiolabelled, and probed against HindlII-digested
L d M N P V D N A resulted in the construction of the HindlII restriction map.
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LdMNPV restriction mapping
2307
Table 1. Molecular size of restriction fragments of LdMNP V 5-7D DNA
Molecular size (kb)
Fragment
BgllI
EcoRI
HindlII
BamHI
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
18.80
12.61
11.80
11.20
10-00
9.65
8.62
8-35
7-20
6.30
5.50
5-00
4.61
3-79
3.61
3.42
3.11
1.58
1.20
1-02
22.00
16.75
15.30
14.40
13.00
10.85
9.60
9-30
8.75
4.53
3.35
2.32
1.65
1-42
22.50
21.50
18.00
16.50
15.50
12.76
10.61
8-50
6.95
3.62
2.12
1.86
1.38
27.00
25.75
22.50
13.50
12.76
6-45
3.50
1-83
1.40
Total
146.44
133.22
141.79
114-69
Table 2. BamHI fragment order of the LdMNPV genome and in pHC79 cosmid clones
Isolate
(pHCLD)
BamHlFragment
Insert size (kb)
33
8, 34
27, 32
2
20, 42, 26
6, 12
16
29
17,21
39
35
43
B
BC
BCH
BCHG
CH
CHG
GE
GED
DF
FI
IA
IAB
25.75
48-25
50.08
53.38
24-23
27.83
16-26
29.76
19.95
7.85
28-40
54-15
Since we detected several HRs in the BgllI-BgllI cross blots we used this information in
conjunction with several cross blot hybridizations to map precisely the HRs (Fig. 4). Using
EcoRI-digested L d M N P V D N A to probe Southern blots containing HindlII-digested
L d M N P V D N A , we successfully mapped this region (data not shown). The four HRs were
designated hr~ to hr 4 and occur between the following BgllI fragments: hrl, BgllI-H to BgllI-T;
hr2, BglII-N to BgllI-G; hr3, BgllI-F to BgllI-L; hr4 BgllI-L to BgllI-S.
The restriction enzyme map of L d M N P V D N A for BamHI, BgllI, EcoRI and HindliI is
presented in Fig. 4. The positions of the BgllI, EcoRI and HindlII fragments were further
confirmed by double digestion of selected cosmid clones with BamHI and the appropriate
enzyme.
Identification and localization of the polyhedrin gene
Using clones containing the HindlII-V fragment of A c M N P V to probe Southern blots of
recombinant cosmid clones and restricted L d M N P V D N A , the position of the L d M N P V
polyhedrin gene was mapped. U n d e r the stringency of conditions used, the HindlII-V fragment
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2308
J. T. M c C L I N T O C K
A N D E. M. D O U G H E R T Y
BglII
B
EG
(C
BgllI
A
I
I
\\OF.
\'~1
I
I
II
O
I
J K LM N P ?
R
S
,T
R-
S-
T-
Fig. 3. Southern cross blot hybridization of unlabelled BgllI-digested L d M N P V D N A probed with 32p
end-labelled BgllI-digested L d M N P V D N A . The ~2p-labelied BgliI digest was oriented top to bottom,
with the higher Mr fragments on the top. The non-radioactive digest was oriented with the higher M r
fragments from left to right.
displayed, in most instances, single fragment hybridizations (Fig. 5 a). To determine the position
of the polyhedrin gene-containing fragments the same immobilized digests were subsequently
hybridized to 3:p-labelled total LdMNPV D N A (Fig. 5b). Thus the restriction fragments were
observed and identified. The strongest hybridization responses were observed with LdMNPV
BamHI-B/C EcoRI-C, HindlII-B, BgllI-B and -C, CIaI-A and PstI-A fragments. Weak
hybridization responses were observed with LdMNPV EcoRI-A, ClaI-N and PstI-C fragments.
Based on these findings the restriction map was oriented with the polyhedrin gene of LdMNPV
as the zero point. Because of the low number of restriction sites in LdMNPV for ClaI and PstI
the hybridization data were included for purposes of cloning into cosmid or ptasmid vectors.
DISCUSSION
To study the molecular organization of the LdMNPV genome, we characterized the D N A
from a plaque isolate of LdMNPV using several restriction endonucleases. This information
was used to analyse the cleavage patterns for recognition and identification, to determine the Mr
of the viral DNA, to investigate methylation patterns along the viral DNA, and to determine the
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L d M N P V restriction mapping
hr]
Bglll
L B
EcoRI
C
I
•
I
H
i
E
I
I
hr2
hr3
BI J i I
A
BamHl
D
H
I
i
JiLt
A
i
L
F
C
i
B
ILG
ru
C
i
20
i
30
i
HG
la
,
I
10
NM
G
I
A
B
F !K!
I_,,
I
C
i
I
]
MI
C
K
H
'
Genome (%) t
0
Kbp
i] i
hr4
TR
I IOIQ!N! D , G iPi E i F ,K L J i'lS
M
HindlII
2309
I
40
i
J
50
A
E
D
t
D
i
I
60
E
,i
i
i
70
F I
A
,,
I
80
J
I
t
I
I
I
I
I
I
I
10
20
30
40
50
60
70
80
90
100
I
B
i
I
90
I
I
100
I
I
110 120 130
Fig. 4. A physical map of LdMNPV DNA for the restriction enzymes BgllI, EcoRI, HindlII and
BamHI. The circular DNA is presented in a linear form. Orientation of the map was based on the
location of the polyhedrin gene as determined from hybridization experiments using AcMNPV
HindlII-V clones to probe Southern blots of restricted LdMNPV DNA.
appropriate enzymes for cosmid cloning and restriction enzyme mapping. Restriction enzymes
BamHI, BgllI, EcoRI and HindlII generated distinguishable fragments giving an approximate
molecular size for L d M N P V D N A of 88.5 x 106 M r or 134.04 kbp. Inconsistencies reported
earlier for molecular sizes of L d M N P V D N A were ascribed to the heterogeneity of uncloned
isolates (McCarthy et al., 1979; Stiles et al., 1983).
Methylation of D N A at specific sequences serves to inactivate gene expression. K n e b e l et al.
(1985) demonstrated that the in vitro methylation of the A c M N P V 1OK promoter sequence at the
5 ' - C C G G - 3 ' site led to inactivation of gene expression. However, extensive methylation in
A c M N P V following replication in permissive (Tjia et al., 1979) or semi-permissive cell-virus
systems (McClintock et al., 1986a) has not been detected. W e have shown using methylationsensitive isoschizomers and agarose gel electrophoresis, that the D N A of L d M N P V does not
appear to be extensively methylated, at least not at the nucleotide sequence recognized by HpalI
and MspI. However, we have not ruled out the possibility of limited methylation. Additional
enzymes which recognize different sequences, bases (methylated adenine), or the position of the
methylated residue in a sequence m a y provide further evidence on the role of methylation or lack
of it in baculoviruses.
A restriction m a p of L d M N P V was constructed by examining overlapping B a m H I fragments
between cosmid clones. The positions of the BgllI, EcoRI and HindlII restriction sites were
determined by the Southern cross blot hybridization procedure. To determine whether H R s
occurred in the L d M N P V genome Southern cross blots of L d M N P V D N A digested with BgllI
were performed. W e have shown that the genome of L d M N P V contains at least four H R s
interspersed throughout its length and that these regions are contained in the following
restriction fragments: BgllI-J, -K, -L, -Q and -R.
The four H R s of L d M N P V appear in some respects to be similar to those described for
A c M N P V (Cochran & Faulkner, 1983) and the M N P V of Choristoneurafumiferana ( C f M N P V )
(Kuzio & Faulkner, 1984). In the first instance, the H R s of A c M N P V were shown to consist of
sequences rich in EcoRI sites. Using Southern cross blots ofEcoRI-digested L d M N P V D N A we
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2310
J . T . McCLINTOCK AND E. M. DOUGHERTY
(b)
2
3
4
5
6
(a)
2
1
3
:
.....
4
5
A ~ B
6
7
~'A
~BB
~
A
?
N
Fig. 5. Identification and localization of the polyhedrin gene. (a) Southern blots of LdMNPV DNA
digested with BamHl (lanes 2), EcoRI (lanes 3), HindlII (lanes 4), BgllI (lanes 5), ClaI (lanes 6) and PstI
(lanes 7) were probed with pBR325 clones containing the HindlII-V fragment of AcMNPV. Lane 1
contains pBR325 linearized with EcoRI. (b) Following autoradiography and without prior melting of
the hybridized AcMNPV clones containing the polyhedrin sequence, the same blot was probed with
total nick-translated LdMNPV DNA. Lanes as in (a),
demonstrated sequence homology along the diagonal line. However, we also observed a unique
hybridization response in a region of the blot which indicates fragments of a size equivalent to
those reported for the HRs of A c M N P V (data not shown). We also observed that two
hybridization signals which were off the diagonal disappeared upon EcoRI digestion. Such a
hybridization response suggests that not all of the HRs observed in the L d M N P V genome
contain EcoRI sites. The lack of such sites in a given HR(s) is similar to that reported for
C f M N P V , where none were observed. We also observed cross-hybridization between BgllI-B
and several BgllI fragments of L d M N P V . This response may suggest that an additional H R
exists upstream from the polyhedrin gene in the L d M N P V genome. This would be consistent
with reports for A c M N P V and C f M N P V .
The origin of HRs has been of interest and their conserved nature suggests that they are
important in the replication and expression of the N P V genome. Based on Southern cross blots
the HRs do not appear to share any homology to host L. dispar D N A . For example, when blots of
HindlII-digested 652Y cellular D N A were probed with radiolabelled EcoRI-digested L d M N P V
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LdMNPV restriction mapping
2311
D N A we were unable to detect any hybridization response even under conditions of low
stringency.
NPVs have been reported to share certain regions of sequence homology as demonstrated
with the polyhedrin gene (Jewell & Miller, 1980; Smith & Summers, 1982). We investigated the
degree of homology between the HRs of LdMNPV and AcMNPV. Using cross blot
hybridizations of PstI-digested AcMNPV D N A to probe Southern blots of LdMNPV D N A
digested with BglII, EcoRI and HindIII, we were unable to detect any shared homology between
the HRs of these distantly related baculoviruses.
The cosmid library and the physical map of the LdMNPV genome described in this study will
facilitate further investigations on the identity and location of specific genes. The HRs detected
in baculoviruses have been implicated in viral transcription and/or as origins of D N A
replication (Cochran & Faulkner, 1983), It will be of interest to determine whether the HRs of
LdMNPV serve as origins of replication or function as enhancers of specific genes (Guarino et
al., 1986).
The technical assistance of A. Davis is gratefully acknowledged. We wish to thank Dr M. A. Cochran for his
useful suggestions on this project. Most of this work was first presented at the Seventh International Congress of
Virology, August 1987, Edmonton, Canada.
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