Journal of General Virology (1990), 71, 1265-1270.
1265
Printed in Great Britain
Physical map of Hz-1 baculovirus genome from standard and defective
interfering particles
Yu-Chan Chao,t Martha Hamblin and H. Alan Wood*
Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, New York 14853, U.S.A.
Restriction maps of the 228 kb genome of the Hz-1
standard baculovirus were constructed for XhoI,
HindIII, EcoRI, SstII and Sinai, using cosmid pVK102
and pBluescript vectors. The genome does not contain
a N o d restriction site. Three regions of the genome
were unclonable and were mapped by isolation of D N A
fragments, in vitro labelling and Southern hybridization procedures. Serial passage in tissue culture was
used to produce defective interfering (DI) particles.
The majority of DI particles in five virus isolates
contained genomic deletions ranging from 24 to 52 kb
in the 22 to 45 map unit region.
Introduction
Studies of Hz-1 viral DNA replication and virusinduced protein synthesis have been conducted following
inoculations with standard and standard plus DI
particles (Burand et al., 1983 a, b; Burand & Wood, 1986).
These studies documented translational events involved
in productive replication and the early events in
establishing persistent infections. However, the organization of the Hz-1 genome and regulation of gene
expression during persistent and productive infections
have not been investigated. As a first step towards a
more complete understanding of these processes, we
have generated a physical map of the Hz-1 genome.
Because deletions in the genome play a role in the
establishment of persistent infections, we have also
identified the areas of the genome that are deleted in DI
particles.
The Hz-1 virus is a non-occluded baculovirus (subgroup
C of Baculoviridae), and was originally isolated from the
persistently infected IMC-Hz-1 cell line established from
ovarian tissue of Heliothis zea (Granados et al., 1978;
Ralston et al., 1981). The Hz-1 virus can establish
persistent and productive infections in several lepidopteran cell lines (Wood & Burand, 1986). Burand et al.
(1983b) showed that defective virus particles are generated following serial passage in vitro and are required for
the establishment of persistent infections. The defective
virus particle samples always contained a small amount
of standard virus as a helper virus. The defective virus
particles were shown to interfere with infection or
replication of the standard particles (Burand & Wood,
1986).
The standard Hz-1 virus particles contain a superhelical, circular, double-stranded D N A genome of 228 to
245 kb (Huang et al., 1982; Burand et al., 1983b), which
is approximately twice the size reported for occluded
baculoviruses (Smith & Summers, 1978) and the nonoccluded Oryctes baculovirus (Crawford et al., 1985).
Based on restriction enzyme profiles and electron
microscopic contour measurements of D N A molecules,
the Hz-1 defective interfering (DI) particles were shown
to be heterogeneous in size (Huang et al., 1982; Burand &
Wood, 1986). DI particle DNAs were estimated to have
deletions ranging from 92 to 130 kb. Huang et al. (1982)
obtained two plaque-purified isolates which were
enriched in different types of defective particle DNA.
t Present address: Institute of Molecular Biology, Academia Sinica,
Nanking, Taipei, Taiwan, Republic of China.
0000-9223 © 1990 SGM
Methods
Cells and virus. The Trichoplusia ni (TN-368) and Spodoptera
frugiperda (IPLB-SF-21) cell lines were maintained at 26°C in a
modified TNMFH medium as described by Burand et al. (1983a).
Standard virus was plaque-purified from the Hz-1 Bl viral isolate
(Burand & Wood, 1986). Virus samples containing D1 particles were
obtained following five serial, undiluted passages of the standard virus
in cell cultures. Virus isolates enriched with particular types of DI
particles were obtained by plaque isolation procedures.
Purification of Hz-1 virus and viral DNA. TN-368 or IPLB-SF-21 cells
were infected with Hz-1 standard virus as described by Burand &
Wood (1986). The DNA from DI particle-containing samples was
32p-labelled during replication in TN-368 cells according to Summers
et aL (1980). The virus was purified from the cell culture supernatant at
48 h post-inoculation by the sucrose gradient centrifugation method of
Huang et al. (1982). The virus band was collected and recovered by
centrifugation at 25000 r.p.m, in a Beckman type 30 rotor for 30 min.
The virus pellet was resuspended at a concentration of I mg/ml in
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1266
Y.-C. Chao, M. Hamblin and H. A. Wood
10 mM-Tris-HC1pH 7.6, 1 mM-EDTAbuffer. The virus suspension was
then brought to a final concentration of 0.5~ sodium Sarkosyl, 20 mMEDTA, pH 8.0 and 100 ~tg/ml proteinase K. The solution was
incubated at 55 °C for 1 h, phenol-chloroform-extracted three times,
extracted twice with chloroform, and then dialysed against 10 mMTris-HCl, 1 mM-EDTA, pH 8.0 buffer.
+
X
+
H
H
E
H
X
Restriction enzyme analyses. Viral DNA was digested with restriction
enzymes using manufacturer's recommended buffers. Fragments were
separated by electrophoresis through 0.6 to 1% agarose gels in Trisacetate buffer (Maniatis et al., 1982). The fragments were labelled in
descending size from A to Z and then a to e. Fragment size and molar
ratio determinations were made by photometric scanning of photographic film negatives or autoradiograms with a Helena R&D
Densitometer (Helena Laboratories) and computer analysis with
Appligration software (Dynamic Solutions). High Mr and 1 kb ladder
standards (Bethesda Research Laboratories) were used in adjacent
lanes.
Cosmid and plasmid cloning. To construct cosmid libraries, viral
DNA was partially digested with HindlII or XhoI and subjected to
agarose gel electrophoresis. Fragments with sizes between 18 and 35 kb
were recovered from agarose with Gene Clean (BIO 101).The pVK102
cosmid (Knauf & Nester, 1982) DNA was digested with a corresponding restriction enzyme and was then intestinal alkaline phosphatasetreated (Leisy et al., 1984). Two ktg of phosphatase-treated cosmid
DNA was mixed with 0.2 gtgof size-selectedviral DNA in a 10 i.tlligase
reaction mixture containing 2 units ofT4 DNA ligase and incubated at
12 °C overnight. After in vitro packaging using Gigapack (Stratagene)
and transduction into Escherichiacoli HB 10l, tetracycline-resistant and
kanamycin-sensitive colonies were selected. Selected clones were
grown overnight at 37 °C in 5 ml LB medium containing 12.5 ~tg/ml
tetracycline. Cosmid DNA was isolated by the alkaline lysis procedure
as described by Maniatis et al. (1982).
Viral restriction fragments were cloned into pBluescript (Stratagene)
using standard methods (Maniatis et al., 1982). The plasmids were
propagated in E. coli DH5ct cells.
'X
Southern hybridization. Hz-1 viral DNA was digested with HindlI1,
XhoI and EcoRI restriction enzymes. The fragments were fractionated
by gel electrophoresisin a IB1 model HRH gel chamber at 60 V for 20 h
or 60 h. DNA fragments were then transferred unidirectionally
(Southern, 1975) or bidirectionally (Smith & Summers, 1980) to
GeneScreen filters (New England Nuclear) according to the manufacturer's instructions. Cloned or gel-purifiedviral DNA was either nicktranslated (Rigby et al., 1977) or random primer-labelled (Feinberg &
Vogelstein, 1983) using [ct-32p]dATP (New England Nuclear).
Results
Viral D N A f r a g m e n t s generated following digestions
with EcoRI, E c o R I plus HindIII, HindIII, XhoI plus
HindIII a n d XhoI are illustrated in Fig. 1. T h e f r a g m e n t
sizes are g i v e n in T a b l e 1. A total of 21, 31 a n d 31
g e n o m i c fragments were detected following digestion
with EcoRI, HindIII, a n d XhoI, respectively. T h e
fragments with sizes larger t h a n 10 k b could be
f r a c t i o n a t e d after 60 h electrophoresis (data n o t shown).
Hz-1 viral D N A samples partially digested with
HindIII or XhoI were used to construct a g e n o m i c library
in the p V K 1 0 2 cosmid vector. T a b l e 2 shows HindIII
cosmid clones which c o n t a i n e d the HindIII restriction
Fig. 1. Hz-1 viralDNA restriction fragments followingelectrophoresis
at 60 V for 16 h in 0.8~ agarose gel and staining with ethidium
bromide. The DNA samples were digested with EcoRl (E), EcoRl and
HindlII (E + H), HindlII (H), XhoI and HindllI (X + H) and XhoI (X)
restriction enzymes.
fragments from Q to K, b to c, W to V a n d H to G. T h e
derived HindIII l i b r a r y c o n t a i n e d four u n c o n n e c t e d
regions. The partial physical m a p derived from the XhoI
cosmid library included restriction f r a g m e n t s from S to
V, O to Q a n d W to U , leaving three u n c o n n e c t e d
regions. T h e three u n c o n n e c t e d regions in the XhoI
cosmid library were at the same locations as three of the
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1267
Physical maps of Hz-1 baculovirus genome
A
XhoI
S
&
B
V
.L
O U M o
F
E
P O
•
I T M
K
D X O
..
L
K
A
.
QN
Lc
E dK
,,,,~"
~,.''.
bY
d
.
H U
,,
T
I
,
,
R N TX
F aZYbO
,
,,,,,
B
M R N P
, . . . . . .
L ZSc
~
C
H W d
,
D
W
_
C
. q t . 0 4 ~ . O ~
A
D
J
dV
.
G
RPO
F
C
~
.
H
G
,"
G
.
S
U
_''_
I
.
B
EcoRI
• -
~
~o',
~
.o.~
s~tu
SmaI
0
10
'
2'o
30
'
40'
5o
% Genome
60'
70
'
80
'
9'o
100
Fig. 2. Physical m a p of standard Hz-1 virus genome for XhoI, HindIII, EcoRI, SstII and Sinai restriction fragments. The circular
genome has been linearized at the XhoI A / G junction. Fragments which were not successfully cloned into cosmids or plasmids are in the
XhoI G-A: HindIII I, Xho] D :HindIII A and XhoI H :HindIII D regions of the map. The distance in kb of each restriction site from this
point is indicated.
Table 1. Sizes (kb) of Hz-1 virus restriction enzyme
fragments
Fragment
HindI II
EcoRI
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
a
b
c
d
e
35.4
22.0
17.0
11.4
10.6
10.6
10.3
9.9
9.2
8.3
7.4
7.4
6.2
6-1
6.1
5-9
5-7
4-8
4.7
4-2
4.1
3.9
3.3
3-2
2.2
2-0
1.7
1.6
1-3
0.9
0-5
16.1
15.8
15.7
13.6
13.2
12.5
12.0
11.6
11.1
10.4
9.2
8.5
8-5
7.7
7.5
6.3
5-7
5.5
5-1
4.8
4.5
4.1
4-0
3.9
3-8
2.2
2.1
1-5
1-4
1.2
0-4
29.1
27.5
23.8
22-1
19-1
13-0
11-0
10.5
10-3
9.7
9.7
8.5
7.9
6.0
5.4
4.3
3-1
2.6
2.1
1.5
0.9
Total
227.9
229.9
228-1
A
B
C
D
E
F
G
H
I
Xhol
unconnected regions in the HindlII cosmid library. In an
unsuccessful effort to connect these three regions of the
genome, more than 2000 HindIII clones and 1000 XhoI
clones were screened. The 33 kb XhoI A fragment is the
only fragment which is beyond the size capacity for
cloning into the pVK 102 cosmid (Knauf & Nester, 1982;
Leisy et al., 1984). Attempts to clone the missing HindIII
D, I and A and XhoI H, G and D fragments into
pBluescript plasmid using recombinant-negative cells
were also unsuccessful.
In order to connect the regions mapped by overlapping
HindIII and XhoI clones, DNA fragments which
mapped at the border of the gaps were labelled and used
as probes for hybridization to Southern blots of total viral
DNA restricted with HindIII, XhoI and EcoRI. The
completed HindIII and XhoI physical maps were aligned
on the basis of data obtained from restriction digests of
the HindIII and XhoI cosmid clones and confirmed by
Southern hybridizations with restriction fragments. An
EcoRI physical map was constructed by digestion of
HindIII cosmid clones with EeoRI and HindIII. For
those fragments or regions missing in the cosmid clones,
the EcoRI map was completed by Southern hybridizations. Finally, gel-purified EcoRI G, E and C fragments
were used as probes to confirm the connections in the
three non-overlapping regions of the XhoI cosmid
library. Based on these results the Hz-1 genome is a
covalently closed, circular DNA as earlier described by
Huang et al. (1982).
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1268
Y.-C. Chao, M. Hamblin and H. A. Wood
Table 2. Genomic library of Hz-1 DNA cloned in cosmid pVKl02
(a) Hindlll fragments contained in four groups of overlapping clones isolated from HindllI libraries
Group 1
Group 2
HindllI
Isolate
no.
fragments
Isolate
no.
H0.4-8
H0.4-9
H3-15
H5-15
H8-21
H9-2l
H9-23
H15-23
H15-27
H21-31
H23-31
Q U M
Q U M a
U M a F
M a F
a F E
F E
F E P
E P
E P O
P O K
O K
H38-47
H40-50
H40-52
H45-54
H50-61
H52-61
H52-65
H52-66
H54-66
H54-68
H61-68
Group 4
Group 3
Isolate
no.
HindlII
fragments
b Y J R
J R N
J R N T
R N T X
T X B
X B
X B
X B
B
B
H74-85
H76-85
L
L
L
L
L
HindlII
fragments
W
C d V
C d V
HindlII
Isolate
no.
fragments
H85-96
H G
Z
Z
Z S
Z S c
(b) X h o l fragments contained in three groups of overlapping clones isolated from X h o l library
Group 1
Group 2
XhoI
Isolate
no.
fragments
X16-28
X18-29
S B
B V
Group 3
XhoI
Isolate
no.
fragments
X36-47
X38-47
X38-50
X42-50
X47-56
X50-61
X54-67
O L c E
L c E d
L c E d K
E d K
d K I T
I T F
T F a Z Y b Q
There was no recognition site in Hz-1 viral DNA for
the NotI restriction enzyme (data not shown). The viral
genome contained only a single recognition site for Sinai
and SstII (data not shown). Only the XhoI P, XbaI A and
BamHI D fragments were digested with SmaI. With
SstII, only the XhoI A, XbaI N and BamHI B fragments
were digested. The location of the SmaI and SstII sites
were determined by single and double digestion experiments with cloned fragments.
A linear representation of the circular Hz-1 genome
physical map with XhoI, HindIII, EcoRI, SstII and SmaI
sites is illustrated in Fig. 2. The only DNA fragments
which were not located in these maps are the HindIII e
and XhoI e fragments, which contain less than 600 bases.
The XhoI A fragment was selected as the beginning of
the map because the only RNA transcript detected from
Hz-1 persistently infected cell lines is transcribed from
this fragment (unpublished results).
The DNAs from plaque-purified isolates containing
DI particles were labelled in vivo with 3zp, purified and
XhoI
Isolate
no.
fragments
X71-79
X76-86
X81-94
X86-96
W J M
M R N P
N P C
C U
analysed following digestion with EcoRI. Because of the
presence of standard virus genomes in these samples,
deletions in DI particle DNA were assessed on the basis
of decreased or increased molar ratios and the appearance of additional fragments. Four types of major DI
particle populations are illustrated in Fig. 3.
A majority of the isolate no. 2 DI particles had
deletions which encompass the EcoRI A, C, H, N and U
fragments. The additional DNA fragment that comigrates with the EcoRI G fragment hybridizes with the
EcoRI A fragment (data not shown) and, therefore,
arises from a partial deletion in the EcoRI A fragment.
Isolate no. 4 contained a major DI particle population
with a deletion in the EcoRI C fragment resulting in the
appearance of a new 13 kb fragment. Isolate no. 6
illustrates a deletion in the EcoRI A, C, H and U
fragments. The new 15 kb fragment arises from a partial
deletion in the EcoRI A fragment. Isolate no. 7 contained
a major DI particle population with deletions which
encompass the EcoRI A, C, H and U fragments.
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Physical maps of Hz-1 baculovirus genome
4
2
,
H~
_ili
6
7
.
.
|
•
:v
•
JK~
u,_..
: 5 2 ::: ::
or
" i;'27a:2;
p~
='::
~r::r
i:
~: 57:
Qr
R~._
a~ :gi ;:'
SP-
: "
d
L;
:
::.:
Fig. 3. Autoradiogram of 32p-labelled Hz-1 D N A EcoRI restriction
fragments following electrophoresis at 60 V for 16 h in 0.8% agarose
gel. The D N A of the standard (S) Hz-1 virus and DI particlecontaining Hz-1 virus clones 2, 4, 6 and 7 were labelled in vivo. The
letters on the left identify fragments shown in Fig. 1. The dots indicate
submolar bands, and arrowheads indicate the positions of additional or
multimolar bands.
Discussion
Restriction site maps of the genome of Hz-1 virus have
been determined from XhoI, HindIII, EcoRI, SstII and
Sinai (Fig. 2). Missing from these maps are the locations
of the HindlII e and XhoI e fragments. The size of the
viral genome was calculated as 228 kb (Table 1 and Fig.
1269
2). The polyhedrin gene in the BamHI F fragment of the
Autographa californica nuclear polyhedrosis virus (NPV)
does not hybridize to either the Hz-1 (data not shown) or
non-occluded Oryctes virus D N A (Crawford et al., 1985).
Therefore the Hz-1 virus genomic map could not be
oriented according to the suggestion of Vlak & Smith
(1982).
Unlike the Oryctes (Crawford et al., 1985) and other
baculovirus genomes (Cochran et al., 1986), no reiterated
sequences were detected in this study. The Hz-1 virus
genome is approximately 100 kb larger than the nonoccluded Oryctes baculovirus genome (Crawford et al.,
1985) and D N A hybridization experiments have indicated that the Hz-1 and Oryctes viruses are unrelated
(J. P. Burand & H. A. Wood, unpublished data).
Using the plaque-isolation procedure of Huang et al.
(1982), we were also able to obtain DI particlecontaining virus isolates which had different restriction
profiles. Based on the alterations in D N A fragment sizes
and band intensities with several isolates containing DI
particles and the physical map of the standard viral
genome, each of our isolates contained a major DI
particle population with genomic deletions in the EcoRI
C fragment. The deletion may extend to the left to
include the EcoRI U, H and N fragment regions of the
genome and may extend to the right to include part or all
of the EcoRI A fragment. The deletions were limited to
between 22 and 45 map units (m.u.). Because it is not
possible to eliminate standard particles from DI particle
samples or to fractionate the DI particle populations, we
have been unable to characterize any minor DI particle
populations which contain smaller deletions or deletions
in additional areas of the genome.
The identified genomic deletions are consistent with
deletions in EcoRI A, C and H fragments previously
described for the Hz-1 B5 isolate (Burand & Wood,
1986). However, the B5 isolate DNA also had partial or
complete deletions of the EcoRI B, S, F, O, P, R, G and J
fragments which correspond to 62 to 89 m.u. Most of the
restriction fragment pattern alterations described for
Hz-1 isolates no. 179 and 181 (Huang et al., 1982)also
correspond to deletions in the 62 to 89 m.u. region of the
genome. However, deletions in the 22 to 45 m.u. region
are not apparent. Based on these data there appear to be
two regions of the Hz-1 genome in which deletions occur
at a high frequency. Through plaque isolation it is
possible to obtain isolates enriched with specific
deletions. Additional analysis of plaque-purified isolates
will be used to determine the deletion limits in these
regions and to determine whether deletions occur in
other regions of the genome.
Cusack & McCarthy (1989) reported the plaqueisolation of several defective particle-containing
Lymantria dispar multiple NPV populations. Each popu-
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1270
Y.-C. Chao, M. Hamblin and H. A. Wood
lation contained DI particle subpopulations with distinct
restriction enzyme patterns. Based on the physical map
of the L. dispar virus (Smith et al., 1988), the genomic
alterations appeared to be dispersed throughout the viral
genome.
Despite extensive screening, no cosmid or plasmid
clones were obtained which covered the 30-6 to 35.6, 68-5
to 71 or 95.6 to 0.4 m.u. areas. The 30-6 to 35-6 m.u.
unclonable region corresponds to the genomic deletion
area described with each of our enriched DI particle
populations and the B5 isolate (Wood & Burand, 1984).
The second deleted region in the B5 isolate (Wood &
Burand, 1984) and the deleted region in the 179 and 180
isolates (Huang et al., 1982) correspond to the unclonable 68-5 to 71 m.u. area of the genome. It is possible that
these areas of the genome contain unique sequences
which result in deletions during replication in both
bacterial and insect cells. Additional cloning vectors are
being evaluated in an effort to clone these areas so that
they can be sequenced.
The physical map of the viral genome now makes
possible the construction of an Hz-1 transcriptional map
during the course of productive infections. Based on
these studies, we hope to locate genes responsible for
turning off host protein synthesis and for cell lysis
(Burand et al., 1983a). Additionally, a comparison of
transcripts under productive and persistent infection will
be useful in locating regions of the genome involved in
the establishment and maintenance of persistent
infections.
This investigation was supported in part by U.S. Department of
Agriculture Grant No. 86-CRCR-I-2011.
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(Received 15 August 1989; Accepted 22 February 1990)
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