Veterinary Microbiology, 33 (1992) 101-115 Elsevier Science Publishers B.V., Amsterdam
I01
Approaches to the identification of non-essential genes of African swine fever virus Christianne Mettraux a, Mathias Ackermann b, Jon-Duri Tratschin a and Ueli
Kihm a alnstitute for Viral Diseases, Basel, Switzerland blnstitute for Virology, Veterinary Medical Faculty, University of Zurich, Zurich. Switzerland (Accepted 30 June 1992 )
ABSTRACT Mettraux, C., Ackermann, M., Tratschin, J.°D. and Kihm, U., 1992. Approaches to the identification of non-essential genes of African swine fever virus. Vet. Microbiol.. 33:101 - 115. It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the fl-galactosidase (fl-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: ( 1 ) expression of B-gal; (2) detection offl-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene.
INTRODUCTION
Homologous recombination involving the appropriate flanking viral sequences and genetic markers has been used to insert foreign sequences into the genomes of large DNA viruses, e.g. herpes simplex virus (Roizman and Jenkins, 1985) and vaccinia virus (Mackett et al., 1982). These techniques have been especially useful for the introduction of mutations into genes which are not required for viral growth in tissue culture. African swine fever (ASF), a lethal infection of domestic pigs, is caused by Correspondence to: M. Ackermann, Institute for Virology, Veterinary Medical Faculty, University of Zurich, Winterthurerstr. 266a, CH 8057 Zurich, Switzerland.
0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
102
C. METTRAUX
a large double-stranded DNA virus, termed ASF virus (ASFV). For poorly understood reasons, no vaccines could be developed for the control and prevention of ASF. There are, however, indications that certain viral gene products may confer protection from death due to ASF (Kihm et al., 1987 ). Our work is based on the hypothesis that these viral gene products are non-essential for viral replication. For this reason, the aim of our study was, to prepare a system for the generation of recombinant ASFV in order to identify genes non-essential for ASFV replication. A cosmid library representing the genome of ASFV strain France 64 was established and characterized. In order to inactivate viral genes by insertion, the fl-galactosidase (fl-gal) gene was introduced either randomly (Jenkins et al., 1985 ) or at specific locations of selected subcloned viral DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted ASFV strain. The following methods were established for the selection of recombinant progeny virus: ( l ) expression offl-gal, (2) detection offl-gal specific DNA by plaque hybridization, and (3) absence of the functional product of the inactivated gene. MATERIALS AND METHODS
Viral strains and cell cultures ASFV strain LIS60C originated from strain Lissabon 1960 (Mebus, 1987) and was adapted to replicate in CV-1 cells. The highly virulent ASFV strain France 64 has been described previously (Pool, 1986; Kihm et al., 1987). Vaccinia virus type 156, from American type cell collection (ATCC) was kindly provided by Dr. K. McCullough. CV- 1 cells and the BHK-2 l-derived TK negative cell line T K - t s l 3 were obtained from ATCC. The cells were propagated in Dulbecco's minimum essential medium (D'MEM) supplemented with fetal calf serum and buffered with 7.5% sodium bicarbonate. Bacterial strains and vectors Information concerning the bacterial strains and vectors used throughout this study is summarized in Table 1. DNA manipulations Standard techniques were used (Maniatis et al., 1982). The cloning strategies and the/tMu techniques are described briefly below. Genomic libraries and clones ofASFV. Genomic DNA of ASFV strain France 64 was extracted from purified virus particles and partially digested with Sau3A (Frischauf, 1987). ASFV DNA fragments ranging in size from 25 to 45 kbp were ligated to cos sequences of vector pHC79 and packaged into empty heads of Lambda phage before the appropriate E. coli cells were transduced
n.a. = not applicable.
m8820 Mucts
DL MuTRI6 p C M I 5 / I / B pCM25/6
pHC79
n.a.
m8820 "I'R; Rec A- ; double lysogenic for helper Mu as well as for a transposable/~Mu variant with an insertion in the BamHl site 116 bases upstream from the right-hand end. The insertion consisting of the fl-galactosidase gene lacking promoter sequences and the translation initiation signal. Rec A + ; helper Mu in lysogeny; immune to double lysogeny
pBR322-derived cosmid vector
Cosmid receptor cells
Containing vaccinia virus promoters p I 1 and p7.5 and the fl-galactosidase gene under the control of p 11
pSCII
n.a.
pBR322 derivative
pAl IP.Xba
ED 8767
pBR322 derivative
pATI53
XLI-Blue
Specifications
Vector
E. colistrain
Bacterial strains and vectors
TABLE I
Reference
Castilho et al., 1984
Propagation of Dr. F. Jenkins, ,uMu Bethcsda, MD, USA mutagenized ASFV DNA fragments
Hohn and Collins, 1980
Boehringer, Mannheim, Germany
Jenkins, unpublished data, 1990
Murray et al., 1977
Chakrabarti et al., 1985
Dr. B. Hohn, Basel, Switzerland
Dr. B. Moss, Bethcsda, MD, USA
St ratagene, Heidelberg, Twigg and Germany; Amersham Sherrat, 1980 International, UK Laughlin et al., 1983
Supplied by
Mutagenesis of Dr. F. Jenkins, selected ASFV Bethesda, MD, USA DNA clones by random insertion
Construction of the cosmid library Cosmid cloning
Subcloning from cosmid library Subcloning and mutagenesis of ASFV TK gene Mutagenesis of ASFV TK gene
Purpose
104
C. METTRAUX
VARIABLE .
.
.
.
.
CONSERVED
VARIABLE
.
A
......
ii, ii
1
2
3
4
6
13
7
8
i ii
, i i:i
9
12
5
10
cosCM54
C
(
...........
----
..........
13 14
15
11 II
16
)
cosCM69 pca33
tq
cosCM26
{ cosCM15 cosCM25 p C M 1 5 / 2 / B
.;
,,
:,,~
DCM25/8
D
" ~----
z
10
20
0
" ~
30
-
T
40
T
50
60
" ~
70
" " Z
80
---"
~,---
"
I
" ' ~ f ~
T
- T ~
l
-7
9 0 100 110 120 130 140 150 160 170
Fig. 1. Map positions of cloned ASFV DNAs with schematic representation ofA. ASFV genome. B. Plasmid cloned D N A library of ASFV strain BA71V. Each number above the bars represents a specific plasmid clone. C. Cosmid (open bars) and plasmid (stippled bars) cloned fragments representing the genome of ASFV strain France 64. D. Ruler: numbers represent kilobasepairs
(kbp).
in order to establish the cosmid library (Collins and Hohn, 1978). For the construction of pCM 15/1/B, cosCM 15 was digested with BamHI and the fragments were subcloned into the BamHI site of pAT153, pCM25/6 represents an EcoRI subfragment ofcosCM25 cloned into the EcoRI site ofpAT153. pCM33 is a BglII-XhoI fragment of cosCM69 subcloned into pAl 1P.Xba. For the cloning ofpCM33/14, pCM33 was opened at the unique EcoRV site and a PstI/SmaI fragment of pSC11 was integrated. This fragment contains the lacZ gene under the control of vaccinia virus promoter pl I (p7.5 was oriented in the opposite direction). A plasmid library representing the genome of the ASFV VERO cell-cultureadapted strain BA71V was kindly supplied by Dr. Eladio Vinuela, Madrid, Spain (Almendral et al., 1984; Ley et al., 1984). The approximate map position of each cloned fragment is indicated in Fig. 1.
Mutagenesis of cloned ASFV DNA fragments by the #Mu procedure. Selected plasmids containing fragments of ASFV DNA were used to transform double lysogenic bacterial cells which contained a temperature-sensitive (ts) helper Mu phage as well as a ts/~Mu derivative consisting of the transposon elements, a gene specifying resistance to kanamycin, and a truncated fl-galacto-
NON-ESSENTIAL GENES OF AFRICAN SWINE FEVER VIRUS
H
A
i-L
,'
Kana R ,~-///~
B
D-l_ E
B
'3
•
random
'
105
|
LacZ
ii
!! ]
~
Fig. 2. Schematic representation of the/zMu construction and mutagenesis. A. Genomic map of /~Mu showing relevant restriction enzyme sites (H =HindIII; B =BamHI) and the kanamycin resistance gene (KanaR). B. fl-galactosidase gene (LacZ) with EcoRI sites (E) which was inserled into the BamHi site of/tMu. The inserted gene (lacking promoter sequences as well as translation initiation signal) was oriented in the direction of the arrow. C. Cloned fragment of ASFV DNA into which the pMu construction was randomly inserted. The box represents a viral gene. D. Map of randomly inserted/zMu within a viral gene. E. Fusion protein specified by recombinant virus, provided that pMu had been integrated in frame and in the correct orientation within the viral gene.
sidase (fl-gal) gene. The construction is shown in Fig. 2. Upon induction, the transposition was initiated and the/~Mu sequences were introduced into the plasmid sequences before being packaged into the phage heads specified by the helper Mu phage. The phages containing the mutagenized ASFV DNA were used to infect non-permissive bacterial cells which were resistant to lysogeny as well as to the lytic phage cycle. The cells were selected for double resistance to ampicillin and to kanamycin. Thus, only cells which had been infected with phages carrying the intact ampicillin gene of the original plasmid, the kanamycin resistance specified by the transposed/tMu with ASFV DNA sequences as well as two homologous fragments of Mu DNA (in order to allow recombination to a new plasmid ) were able to replicate. Transient expression assays. Transient expression of the fl-galactosidase was tested as described by Panicali et al. (1986). Briefly, CV- 1 cells were seeded into 24-well tissue culture plates. The next day, the cells were infected with ASFV 1 h before transfection with 3 #g of CaPO4-precipitated DNA per well. After 2 h incubation at 37 °C the cells were overlayed with fresh medium containing 1% agarose and 200/tg X-gal/ml. Finally, the monolayers were incubated at 37 °C for 1-3 days to allow blue color to develop.
106
c. METTRAUX
Production, selection and identification of recombinant ASFV. For the production of ASFV recombinants, the appropriate cells were seeded into six well dishes (diam. 3.5 cm). The next day, the cells were infected with ASF virus prior to transfection with the mutagenized and linearized plasmids. The medium was replaced 4 h later. The infected cells were harvested after 24 h incubation at 37°C. Finally, the virus was released by repeated freezing and thawing and transferred to fresh cells. For the selection of T K - recombinant virus progeny, T K - t s l 3 cells were used and the medium was supplemented with 50/tg/ml BUdR. Candidate recombinants which were expected to express the fl-galactosidase gene were propagated under 1% agarose overlay. After 2 days of incubation, a second agarose overlay containing X-gal at a final concentration of 300/~g/ml was added and the monolayer was screened for the development of blue plaques. The plaque hybridization for the detection of parent- and recombinant progeny-virus in infected cell monolayers was done as described by Villarreal and Berg (1977). Briefly, CV- 1 cells or T K - t s 13 cells were infected and propagated under agarose. At various times after infection, the agarose layer was gently removed and a dry plaque lift membrane (Nylon N +, Amerham) was laid carefully on top of the moist cell monolayer. After a few minutes, the membrane was moistened and carefully peeled off the plate. The adhering DNA was denatured by exposing the filter to 0.5 N NaOH. The membrane was neutralized, dried and fixed before hybridization with the appropriate probes and exposure to Hyperfilm X-ray. Positive signals were localized on the agarose layer and agarose pieces containing the candidate viruses were cut out and stored at - 7 0 °C before further characterization. RESULTS
Construction and characterization of a cosmid library of ASFV strain France 64 A cosmid library representing the genome of ASFV strain France 64 was established. Clones which hybridized to genomic ASF viral DNA were further
Fig. 3. Mapping of cosmid cloned ASFV DNAs to the previously characterized plasmid cloned genomic library of ASFV strain BA71V. A. Each of the plasmid clones of strain BA71V was digested with the appropriate restriction enzyme which excised the ASFV-specific DNA, the fragments were electrophoretically separated on 0.8% agarose gels and stained with EtBr. The numbers at the top of the figure correspond to the numbers ofthe plasmid clones in Fig. l B. In lane 17, Lambda HindllI digested DNA was applied as a molecular weight marker. B. through F. Autoradiograms of blots obtained following transfer of the restricted and separated plasmid clones (Fig. 3A) to nylon membranes and hybridization with cosmid clones cosCM54 (B), cosCM69 (C), cosCM26 (D), cosCMl5 (E), cosCM25 (F). The ASFV-specific bands are indicated by an arrowhead.
NON-ESSEN"rlAL GENES OF AFRICAN SWINE FEVER VIRUS
2 1
4 3
IIIIIII A
B
C
D
E
F
6 5
8 7
10 9
12 11
14 13
IIIIIIIIIII
107
16 15
17
108
c. MET'rRAUX
characterized as follows: each plasmid of a previously characterized library representing nearly the entire genome of strain BA7 IV of ASFV was digested with the appropriate enzyme which excised the ASFV DNA fragment. The complete panel of the restricted plasmids was electrophoresed in an agarose gel and transferred to a nylon membrane. In order to map the cosmids to the ASFV genome, the DNA of each cosmid was radioactively labelled and hybridized to the blotted panel of the restricted plasmid clones. The results obtained for five individual cosmids are shown in Fig. 3. Because the whole cosmid was used as a probe, in each lane one band representing the plasmid vector lighted up. The appearance of a second band indicated that the cosmid used as a probe contained ASFV-specific sequences corresponding to the ASFV DNA sequence present in the particular plasmid clones. Fig. 1 shows the map positions of the BA71V plasmid library as well as selected cosmid clones which cover more than 90% of the ASFV genome. Selected cosmids were further characterized by restriction enzyme mapping (EcoRI, BamHI, SalI; unpubl. data).
Mutagenesis of subcloned ASFV DNAfragments by the p.Mu procedure Plasmids pCM 15/1/B (subcloned from cosCM 15 ) and pCM25/6 (subcloned from cosCM25 ) were mutagenized by the/tMu procedure (see Material and Methods) in order to insert at random a cassette containing a truncated ~gal gene within the/~Mu sequences (Fig. 2). A total of 28 mutagenized clones ( 15 derived from p C M I S / 1 / B and 13 derived from pCM25/6) were digested with either BamHI (pCM 15/1/B ) or EcoRI (pCM25/6) in order to test if random integration of the Mu cassette had occurred. These restriction enzymes cut the original plasmids into two fragments, whereas integration of the/~Mu cassette into either the vector or the cloned ASFV DNA added two restriction enzyme sites to the respective fragments. For this reason the fragment which had integrated the/tMu sequences disappeared and three new fragments appeared instead. Upon hybridization with plasmid pAT 153, either the integral vector band appeared on the autoradiogram, indicating that pMu had been inserted into ASFV sequences or the original vector band disappeared and two new fragments lighted up, indicating that/tMu had integrated into the vector DNA. The third new band did not hybridize in either case because it consisted exclusively of DNA of the/~Mu cassette. The variation of insertion within the ASFV sequences could be judged from the variability of the pattern of the EtBr-stained agarose gel. The results are shown in Fig. 4. Interestingly, it seemed that random insertion had occurred in plasmid pCM15/1/B, whereas plasmid pCM25/6 was apparently resistant to these randomized insertions. Transposition of/tMu into this ASFV DNA fragment could be observed only rarely.
109
NON-ESSENTIALGENESOFAFRICANSWINEFEVERVIRUS 2
4
1
6
3
8
5
10
7
9
12 11
14 13
16 15
17
liilllllllUlllliU A
B
C
•
d
..:.j.
".
.i
.,
::~". ~
"
"
",~
~:li:: Z: l" !:~.~:i~'.~i! l ~" ~ •
.
~
:::" ~:~? :~:~}:~i!:::i:': :-,T~I:~::.... • .'
:..
:
.
Illlllllllllllll 2 1
4 3
6 .=i
8 7
10 9
12 11
14 13
15
Fig. 4. Photographs of EtBr-stained agarose gels (A and C) and autoradiograms (B and D) showing the results of/zMu mutagenesis of pCM25/6 (A, B) and pCM 15/1/B (C, D). These pAT153 cloned ASFV DNA fragments were subjected to #Mu mutagenesis. Fifteen (A, B) and ! 3 (C, D), respectively, of the mutagenized clones were randomly selected and grown individually. The extracted plasmid DNA was digested with EcoRI (clones originating from pCM25/ 6) and BamHI (clones originating from pCMI 5/1/B), respectively, before electrophoresis in 0.8% agarose gels, transfer to nylon membranes and hybridization with radioactively labelled pAT I53. Lane 1: molecular weight marker (Lambda HindlIl). Lane 2: original plasmid, digested with either EcoRl (A and B) or BamHl (C and D). Lanes 3 through 17 (A and B) and Lanes 3 through 15 (C and D): individual preparations of mutagenized clones. The arrows on the left of the figure point to the integral vector band.
1 lO
C. METTRAUX
A
cosCM69
B
pCM33
C
p7.5-011-galactosidase D
.
'
I
I
I pCM33/14
Fig. 5. Schematic representation of cloning and mutagenesis of the viral TK gene. A and B. Cloning o f a BgllI-Xhol (Bg2, X) fragment excised from cosmid cosCM69 (A) into plasmid pA 1 l p.xba. The new construct was designated pCM33 (B). C. A cassette containing the vaccinia virus promoters p7.5 and pl 1 and the fl-galactosidase gene (orientations are indicated by arrows) was inserted into the unique EcoRV site (EV) of pCM33. D. The new construct was designated pCM33/14. The EcoRV sites were lost during the cloning procedure.
Identification, cloning, and mutagenesis of the ASFV TK gene A synthetic oligonucleotide, synthesized according to the published sequence of the ASFV TK gene (Blasco et al., 1990; Hernandez and Tabares, 1991 ), was used to demonstrate by hybridization that cosCM69 carried the ASFV thymidine kinase (TK) gene. Fig. 5 and Fig. 6 show that the TK gene was localized on an approximately 700 bp XhoI/BglII fragment of cosCM69. This fragment was therefore subcloned and the new plasmid, designated pCM33, was opened at the unique EcoRV site and a p-gal gene under the control of the vaccinia virus promoter pl I was inserted into the body of the TK gene (Fig. 5 ). The new plasmid was termed pCM33/14.
Transient expression of fl-galactosidase in African swine fever virus infected cells In order to test if the above-mentioned fl-gal gene would be expressed in ASFV-infected cells under the control of the vaccinia virus promoter p l 1, the original pSCI 1 and the newly constructed p C M 3 3 / 1 4 were transfected separately into CV-I cells which had been infected with ASFV. The blue color developing in these monolayers (Fig. 7 ) indicated that the vaccinia virus promoter was recognized by the ASFV enzymes. Similar reactions were observed in vaccinia-virus-infected cells (not shown).
Construction and selection of recombinant ASF viruses In order to delete genes non-essential for ASFV replication, the above-mentioned cloned and mutagenized ASFV DNA fragments were transfected either
NON-ESSENTIAL
1
2
GENES
3
OF AFRICAN
4
5
SWINE
FEVER
6
7
VIRUS
111
8 .o
._.~.~'~..u.~,i~, i.- • ~,~_
:
• .....:
~. ,"i ..~'~'~-::,~.
• • "
i
:••i?.
:~" ~
Fig. 6. Photographs of an EtBr-stained 1.2% agarose gel (lanes 1-4) and of the corresponding autoradiogram (lanes 5-8 ). DNA ofcosCM69 was digested with Bglll and Xhol before electrophoresis (lane 2 ) or was electrophoresed undigested (lane 3). BarnHI-digested genomic DNA of ASFV strain France 64 (lane 4) and a molecular weight marker (Lambda HindIll; lane I ) were used as controls. After electrophoretic separation, the DNA was transferred to a nylon membrane and hybridized with a radiolabelled TK-specific oligonucleotide. Lanes 5-8 of the autoradiogram correspond to lanes 1-4 of the gel. The arrows point to the bands which hybridizcd with the oligonucleotide probc.
1
2
A B Fig. 7. Transient expression offl-galactosidase in ASFV-infected cells. A photograph of ASFVinfected (A l, A2, B2) and mock infected (B l) cells is shown. Following infection or mock infection, the cells were transfected either with pCM33/14 (Al and B l ) or with pSCl 1 (A2) or mock transfected (B2). Thereafter, they were overlayed with a medium containing agarose and X-gal. Blue (dark) color was observed exclusively in cells which expressed ~galactosidase.
112
c. METTRAUX
into T K - t s 13 cells or else into CV- 1 cells which had been infected with ASFV. The following three methods were used to select recombinant progeny viruses.
Selection for TK- viralprogeny virus. T K - t s l 3 cells were infected with ASFV (TK ÷, cell culture adapted) before transfection with pCM33/14. Recombinants having the TK gene inactivated by the insertion of the fl-gal gene were expected to be generated. The growth and replication of such recombinant T K - viral progeny was selectively favoured by addition of BUdR to the cell culture medium. T K - virus isolates are currently being characterized.
Selection for phenotypically blue plaque recombinant ASF virus. In parallel, separate cell cultures were transfected with/zMu-mutagenized plasmids and with p C M 3 3 / 1 4 following the infection with ASFV. Progeny virus was harvested and various dilutions were used to infect fresh monolayers of CV-I cells. The cells were overlayed with a m e d i u m containing X-gal and agarose. Although the cells were observed for various time periods post infection, no recombinant virus expressing fl-gal could be identified up to date (data not shown ).
Identification of ASFV-infected cells by plaque hybridization. Additional samples of the above-mentioned infected and transfected cell cultures were used to infect fresh cell cultures which were kept under an agarose overlay without X-gal. Plaque imprints were taken 6 days post infection. They were hybridized with a fl-gal-specific DNA probe. Although the method appeared to work properly as indicated by hybridization assays where ASFV DNA was used as a probe, no recombinant viruses could be selected with this method (data not shown). DISCUSSION
Although no vaccines for the prevention of African swine fever (ASF) exist, previous work from our laboratory indicated that at least death due to ASF can be prevented by the administration of adjuvanted, inactivated vaccines. As reviewed by Kihm et al. (1987), the protective effect could be attributed to structural viral proteins. As long as the virus was propagated in animals a n d / o r in macrophage cultures, the protective components were maintained. They were lost, however, upon adaption of the virus to replication in conventional cell cultures. We therefore suggested that viral gene products which are not required for replication are essential for this protecting effect. The purpose of our study was therefore to set up a system which allows the identification of genes non-essential for ASFV replication. In vitro genetic manipulation followed by homologous recombination of the altered DNA fragments with the genomes of the wild type viruses is a very efficient way to
NON-ESSENTIAL GENES OF AFRICAN SWINE FEVER VIRUS
| 13
study the functions of viral genes and their products (Mackett et al., 1982; Roizman and Jenkins, 1985 ). The successful application of these techniques (reviewed in Ackermann, 1988) for the identification of such non-essential genes of large DNA viruses is not possible, however, without previously having established the following: ( 1 ) molecular cloning and characterization of the parent viruses; (2) conditions favouring recombination; and (3) strategies for the selection of recombinant progeny virus. For these reasons, it was our first goal to clone and characterize the genomic DNA of the virus which specified the functions to be studied, i.e. which conferred protection from death due to ASFV infection. Accordingly, a genomic cosmid library of wild type (wt) ASFV strain France 64 was established and characterized by restriction enzyme analysis and by comparing the newly established genomic library to a previously characterized library (Almendral et al., 1984; Ley et al., 1984) by hybridization studies. In a second series of experiments, selected DNA fragments were mutagenized in vitro. Therefore we deleted the function of specific viral genes by the insertion of foreign DNA carrying the marker gene fl-gal. These ASFV DNA fragments could be used for in vivo recombination into the genome of non-mutated ASFV and subsequent selection of recombinant progeny virus. According to Ackermann ( 1988 ), there are three alternative ways for the selection of recombinants: ( 1 ) Application of selective pressure to favour the growth of recombinants. This approach was the most successful in our hands so far. We were able to select several isolates of apparently T K - ASFV; (2) Alternatively, recombinants may be selected based on the expression of a phenotypic marker. We attempted, without success so far, to select viral recombinants expressing the fl-galactosidase gene. In a first approach, a fl-galactosidase gene lacking both promoter sequences and translation initiation signal, was introduced into selected DNA fragments of ASFV strain France 64. Since the foreign gene was supposed to be inserted at random into the cloned DNA fragments (Casadaban, 1975; Castilho et al., 1984), the expression of /~-galactosidase was expected to be regulated by a natural viral promoter if the construction had been inserted in frame with a gene non-essential for viral replication. A second approach involved the use of a vaccinia virus promoter fused to the fl-galactosidase gene. This construction was inserted into the ASF viral thymidine kinase (TK) gene. One advantage of this alternative was the possibility to select for T K - virus before testing for the expression offl-galactosidase. Furthermore, we could demonstrate by transient expression assays that the vaccinia virus promoter p l 1 was functional in ASFV-infected cells. This finding was in agreement with other reports concerning the reciprocal use of vaccinia virus and ASFV promoters by vaccinia and ASF viruses, respectively (Talavera and Beloso, 1987; Bostock, 1990; Hammond and Dixon, 1991 ). (3) Finally, it is possible to select recombinants by screening the viral
114
C. METTRAUX
progeny for the presence of the inserted foreign DNA. For this reason, we established the technique of plaque hybridization. Radiolabelled D N A probes were used for the detection of foreign D N A sequences among ASFV D N A in plaque imprints of cell monolayers infected with candidate recombinant viruses. Apparently, either the recombination efficiency or the number of plaques tested was too low to detect any recombinants by this method. CONCLUSIONS Essential preconditions for the construction of recombinant ASF viruses were established. In the course of the mutagenesis of cloned ASFV D N A fragments, several interesting features o f ASFV were observed. ( 1 ) While the transposase specified by phage Mu was able to integrate/tMu sequences at random into certain ASFV D N A fragments ( p C M 1 5 / I / B ) , other fragments appeared to be resistant to random integration ( p C M 2 5 / 6 ) . (2) The vaccinia virus promoter p I 1 was used by enzymes specified by ASFV in transient expression assays. If this promoter is also recognized when integrated in the ASF viral genome remains unclear. (3) T K - virus isolates could be selected. ACKNOWLEDGEMENTS We thank Eladio Vinuela for the plasmid library representing the genome of ASFV strain BA71 V, Frank Jenkins for supplying t h e / t M u system and for the construction o f D L M u T R 16, Barbara H o h n for ED8767, and Bernard Moss for pSC 11. The photographic work o f Anita Hug is greatly appreciated. This work was supported by the Swiss National Science Foundation grant No. 31-8832.86.
REFERENCES Ackermann, M., 1988. The construction, selection, and application of recombinant herpes viruses. J. Vet. Med. B, 35: 379-396. Almendral. J.M., Blasco, R., Ley, V., Beloso, A., Talavera, A. and Vinuela, E., 1984. Restriction site map of African swine fever virus DNA. Virology, 133: 258-270. Blasco, R., Lopez-Otin, C., Munoz, M., Bockamp, E.-O., Simon-Mateo, C. and Vinuela, E., 1990. Sequence and evolutionary relationships of African swine fever virus thymidine kinase. Virology, 178: 301-304. Bostock, C,J., 1990. Viruses as vectors. Vet. MicrobioI., 23:55-7 I. Casadaban, M., 1975. Fusion of the Escherichia coil lac genes to the ara promoter: a general technique using bacteriophage Mu-1 insertions. Proc. Natl. Acad. Sci. USA, 72: 4399-4403. Castilho, B.A., Olfson, P. and Casadaban, M.J., 1984. Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons. J. Bacteriol., 158: 488-495. Chakrabarti, S., Brechling, K. and Moss, B., 1985. Vaccinia virus expression vector: co-expres-
NON-ESSENTIAL GENES OF AFRICAN SWINE FEVER VIRUS
115
sion of fl-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol., 5: 3403-3409. Collins, J. and Hohn, B., 1978. Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads. Proc. Natl. Acad. Sci. USA, 75: 4242-4246. Frischauf, A.-M., 1987. Digestion of DNA: size fractionation. Methods Enzymol., 152: 183199. Hammond, J.M. and Dixon, L.K., 1991. Vaccinia virus-mediated expression of African swine fever virus genes. Virology, 181 : 778-782. Hernandez, A.M.M. and Tabares, E., 199 I. Expression and characterization of the thymidine kinase gene of ASF virus. J. Virol., 65: 1046-1052. Hohn, B. and Collins, J., 1980. A small cosmid for efficient cloning of large DNA fragments. Gene, I l: 291-298. Jenkins, F.J., Casadaban, M.J. and Roizman, B., 1985. Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome. Proc. Natl. Acad. Sci. USA, 82: 4773-4777. Kihm, U., Ackermann, M., Miiller, H.K. and Pool, R., 1987. Approaches to vaccination. In: Y. Becket (Editor), Develop. in Vet. Virol. "African swine fever". Martinus Nijhoff, Boston, MA, pp. 127-144. Laughlin, C.A., Tratschin, J.-D., Coon, H. and Carter, B.J., 1983. Cloning of infectious adenoassociated virus genomes in bacterial plasmids. Gene, 23: 65-73. Ley, V., Almendral, J.M., Carbonero, P., Beloso, A., Vinuela, E. and Talavera, A., 1984. Molecular cloning of African swine fever Virus DNA. Virology, 133: 249-257. Mackett, M., Smith, G.L. and Moss, B., 1982. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA, 79:7415-7419. Maniatis, T., Fritsch, E.F, and Sambrook, J. (Editors), 1982. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mebus, C.A., 1987. Pathobiology and pathogenesis. In: Y. Becker (Editor), Develop. in Vet. Virol. "'African swine fever". Martinus Nijhoff, Boston, MA, pp. 21-3 I. Murray, M.E., Brammar, W.J. and Murray, K., 1977. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet., 150: 53. Panicali, D.L., Grzelecki, A. and Huang, C., 1986. Vaccinia virus vectors utilizing the fl-galactosidase assay for rapid selection of recombinant viruses and mcasurement of gene expression. Gene, 47: 193-199. Pool, R., 1986. Immunisierungsversuche mit Afrikanischem Schweinepestvirus-antigen. Thesis, Veterinary Medical Faculty, University of Zfirich. Roizman, B. and Jenkins, F.J., 1985. Genetic engineering of novel genomes of large DNA viruses. Science, 229:1208-1214. Talavera, A. and Beloso, A., 1987. RNA polymerase-promoler recognition in African swine fever and vaccinia viruses, 18P. In: Proc. International Congress "African swine fever and pig immunology", Salamanca, Spain. Twigg, A.J. and Sherrat, D., 1980. Transcomplementable copy-number mutants of plasmid ColEl. Nature, 283:216-218. Villarreal, L.P. and Berg, P., 1977. Hybridization in situ of SV 40 plaques: detection of recombinants SV 40 virus carrying specific sequences of nonviral DNA. Science, 196: 183-185.
I01
Approaches to the identification of non-essential genes of African swine fever virus Christianne Mettraux a, Mathias Ackermann b, Jon-Duri Tratschin a and Ueli
Kihm a alnstitute for Viral Diseases, Basel, Switzerland blnstitute for Virology, Veterinary Medical Faculty, University of Zurich, Zurich. Switzerland (Accepted 30 June 1992 )
ABSTRACT Mettraux, C., Ackermann, M., Tratschin, J.°D. and Kihm, U., 1992. Approaches to the identification of non-essential genes of African swine fever virus. Vet. Microbiol.. 33:101 - 115. It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the fl-galactosidase (fl-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: ( 1 ) expression of B-gal; (2) detection offl-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene.
INTRODUCTION
Homologous recombination involving the appropriate flanking viral sequences and genetic markers has been used to insert foreign sequences into the genomes of large DNA viruses, e.g. herpes simplex virus (Roizman and Jenkins, 1985) and vaccinia virus (Mackett et al., 1982). These techniques have been especially useful for the introduction of mutations into genes which are not required for viral growth in tissue culture. African swine fever (ASF), a lethal infection of domestic pigs, is caused by Correspondence to: M. Ackermann, Institute for Virology, Veterinary Medical Faculty, University of Zurich, Winterthurerstr. 266a, CH 8057 Zurich, Switzerland.
0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
102
C. METTRAUX
a large double-stranded DNA virus, termed ASF virus (ASFV). For poorly understood reasons, no vaccines could be developed for the control and prevention of ASF. There are, however, indications that certain viral gene products may confer protection from death due to ASF (Kihm et al., 1987 ). Our work is based on the hypothesis that these viral gene products are non-essential for viral replication. For this reason, the aim of our study was, to prepare a system for the generation of recombinant ASFV in order to identify genes non-essential for ASFV replication. A cosmid library representing the genome of ASFV strain France 64 was established and characterized. In order to inactivate viral genes by insertion, the fl-galactosidase (fl-gal) gene was introduced either randomly (Jenkins et al., 1985 ) or at specific locations of selected subcloned viral DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted ASFV strain. The following methods were established for the selection of recombinant progeny virus: ( l ) expression offl-gal, (2) detection offl-gal specific DNA by plaque hybridization, and (3) absence of the functional product of the inactivated gene. MATERIALS AND METHODS
Viral strains and cell cultures ASFV strain LIS60C originated from strain Lissabon 1960 (Mebus, 1987) and was adapted to replicate in CV-1 cells. The highly virulent ASFV strain France 64 has been described previously (Pool, 1986; Kihm et al., 1987). Vaccinia virus type 156, from American type cell collection (ATCC) was kindly provided by Dr. K. McCullough. CV- 1 cells and the BHK-2 l-derived TK negative cell line T K - t s l 3 were obtained from ATCC. The cells were propagated in Dulbecco's minimum essential medium (D'MEM) supplemented with fetal calf serum and buffered with 7.5% sodium bicarbonate. Bacterial strains and vectors Information concerning the bacterial strains and vectors used throughout this study is summarized in Table 1. DNA manipulations Standard techniques were used (Maniatis et al., 1982). The cloning strategies and the/tMu techniques are described briefly below. Genomic libraries and clones ofASFV. Genomic DNA of ASFV strain France 64 was extracted from purified virus particles and partially digested with Sau3A (Frischauf, 1987). ASFV DNA fragments ranging in size from 25 to 45 kbp were ligated to cos sequences of vector pHC79 and packaged into empty heads of Lambda phage before the appropriate E. coli cells were transduced
n.a. = not applicable.
m8820 Mucts
DL MuTRI6 p C M I 5 / I / B pCM25/6
pHC79
n.a.
m8820 "I'R; Rec A- ; double lysogenic for helper Mu as well as for a transposable/~Mu variant with an insertion in the BamHl site 116 bases upstream from the right-hand end. The insertion consisting of the fl-galactosidase gene lacking promoter sequences and the translation initiation signal. Rec A + ; helper Mu in lysogeny; immune to double lysogeny
pBR322-derived cosmid vector
Cosmid receptor cells
Containing vaccinia virus promoters p I 1 and p7.5 and the fl-galactosidase gene under the control of p 11
pSCII
n.a.
pBR322 derivative
pAl IP.Xba
ED 8767
pBR322 derivative
pATI53
XLI-Blue
Specifications
Vector
E. colistrain
Bacterial strains and vectors
TABLE I
Reference
Castilho et al., 1984
Propagation of Dr. F. Jenkins, ,uMu Bethcsda, MD, USA mutagenized ASFV DNA fragments
Hohn and Collins, 1980
Boehringer, Mannheim, Germany
Jenkins, unpublished data, 1990
Murray et al., 1977
Chakrabarti et al., 1985
Dr. B. Hohn, Basel, Switzerland
Dr. B. Moss, Bethcsda, MD, USA
St ratagene, Heidelberg, Twigg and Germany; Amersham Sherrat, 1980 International, UK Laughlin et al., 1983
Supplied by
Mutagenesis of Dr. F. Jenkins, selected ASFV Bethesda, MD, USA DNA clones by random insertion
Construction of the cosmid library Cosmid cloning
Subcloning from cosmid library Subcloning and mutagenesis of ASFV TK gene Mutagenesis of ASFV TK gene
Purpose
104
C. METTRAUX
VARIABLE .
.
.
.
.
CONSERVED
VARIABLE
.
A
......
ii, ii
1
2
3
4
6
13
7
8
i ii
, i i:i
9
12
5
10
cosCM54
C
(
...........
----
..........
13 14
15
11 II
16
)
cosCM69 pca33
tq
cosCM26
{ cosCM15 cosCM25 p C M 1 5 / 2 / B
.;
,,
:,,~
DCM25/8
D
" ~----
z
10
20
0
" ~
30
-
T
40
T
50
60
" ~
70
" " Z
80
---"
~,---
"
I
" ' ~ f ~
T
- T ~
l
-7
9 0 100 110 120 130 140 150 160 170
Fig. 1. Map positions of cloned ASFV DNAs with schematic representation ofA. ASFV genome. B. Plasmid cloned D N A library of ASFV strain BA71V. Each number above the bars represents a specific plasmid clone. C. Cosmid (open bars) and plasmid (stippled bars) cloned fragments representing the genome of ASFV strain France 64. D. Ruler: numbers represent kilobasepairs
(kbp).
in order to establish the cosmid library (Collins and Hohn, 1978). For the construction of pCM 15/1/B, cosCM 15 was digested with BamHI and the fragments were subcloned into the BamHI site of pAT153, pCM25/6 represents an EcoRI subfragment ofcosCM25 cloned into the EcoRI site ofpAT153. pCM33 is a BglII-XhoI fragment of cosCM69 subcloned into pAl 1P.Xba. For the cloning ofpCM33/14, pCM33 was opened at the unique EcoRV site and a PstI/SmaI fragment of pSC11 was integrated. This fragment contains the lacZ gene under the control of vaccinia virus promoter pl I (p7.5 was oriented in the opposite direction). A plasmid library representing the genome of the ASFV VERO cell-cultureadapted strain BA71V was kindly supplied by Dr. Eladio Vinuela, Madrid, Spain (Almendral et al., 1984; Ley et al., 1984). The approximate map position of each cloned fragment is indicated in Fig. 1.
Mutagenesis of cloned ASFV DNA fragments by the #Mu procedure. Selected plasmids containing fragments of ASFV DNA were used to transform double lysogenic bacterial cells which contained a temperature-sensitive (ts) helper Mu phage as well as a ts/~Mu derivative consisting of the transposon elements, a gene specifying resistance to kanamycin, and a truncated fl-galacto-
NON-ESSENTIAL GENES OF AFRICAN SWINE FEVER VIRUS
H
A
i-L
,'
Kana R ,~-///~
B
D-l_ E
B
'3
•
random
'
105
|
LacZ
ii
!! ]
~
Fig. 2. Schematic representation of the/zMu construction and mutagenesis. A. Genomic map of /~Mu showing relevant restriction enzyme sites (H =HindIII; B =BamHI) and the kanamycin resistance gene (KanaR). B. fl-galactosidase gene (LacZ) with EcoRI sites (E) which was inserled into the BamHi site of/tMu. The inserted gene (lacking promoter sequences as well as translation initiation signal) was oriented in the direction of the arrow. C. Cloned fragment of ASFV DNA into which the pMu construction was randomly inserted. The box represents a viral gene. D. Map of randomly inserted/zMu within a viral gene. E. Fusion protein specified by recombinant virus, provided that pMu had been integrated in frame and in the correct orientation within the viral gene.
sidase (fl-gal) gene. The construction is shown in Fig. 2. Upon induction, the transposition was initiated and the/~Mu sequences were introduced into the plasmid sequences before being packaged into the phage heads specified by the helper Mu phage. The phages containing the mutagenized ASFV DNA were used to infect non-permissive bacterial cells which were resistant to lysogeny as well as to the lytic phage cycle. The cells were selected for double resistance to ampicillin and to kanamycin. Thus, only cells which had been infected with phages carrying the intact ampicillin gene of the original plasmid, the kanamycin resistance specified by the transposed/tMu with ASFV DNA sequences as well as two homologous fragments of Mu DNA (in order to allow recombination to a new plasmid ) were able to replicate. Transient expression assays. Transient expression of the fl-galactosidase was tested as described by Panicali et al. (1986). Briefly, CV- 1 cells were seeded into 24-well tissue culture plates. The next day, the cells were infected with ASFV 1 h before transfection with 3 #g of CaPO4-precipitated DNA per well. After 2 h incubation at 37 °C the cells were overlayed with fresh medium containing 1% agarose and 200/tg X-gal/ml. Finally, the monolayers were incubated at 37 °C for 1-3 days to allow blue color to develop.
106
c. METTRAUX
Production, selection and identification of recombinant ASFV. For the production of ASFV recombinants, the appropriate cells were seeded into six well dishes (diam. 3.5 cm). The next day, the cells were infected with ASF virus prior to transfection with the mutagenized and linearized plasmids. The medium was replaced 4 h later. The infected cells were harvested after 24 h incubation at 37°C. Finally, the virus was released by repeated freezing and thawing and transferred to fresh cells. For the selection of T K - recombinant virus progeny, T K - t s l 3 cells were used and the medium was supplemented with 50/tg/ml BUdR. Candidate recombinants which were expected to express the fl-galactosidase gene were propagated under 1% agarose overlay. After 2 days of incubation, a second agarose overlay containing X-gal at a final concentration of 300/~g/ml was added and the monolayer was screened for the development of blue plaques. The plaque hybridization for the detection of parent- and recombinant progeny-virus in infected cell monolayers was done as described by Villarreal and Berg (1977). Briefly, CV- 1 cells or T K - t s 13 cells were infected and propagated under agarose. At various times after infection, the agarose layer was gently removed and a dry plaque lift membrane (Nylon N +, Amerham) was laid carefully on top of the moist cell monolayer. After a few minutes, the membrane was moistened and carefully peeled off the plate. The adhering DNA was denatured by exposing the filter to 0.5 N NaOH. The membrane was neutralized, dried and fixed before hybridization with the appropriate probes and exposure to Hyperfilm X-ray. Positive signals were localized on the agarose layer and agarose pieces containing the candidate viruses were cut out and stored at - 7 0 °C before further characterization. RESULTS
Construction and characterization of a cosmid library of ASFV strain France 64 A cosmid library representing the genome of ASFV strain France 64 was established. Clones which hybridized to genomic ASF viral DNA were further
Fig. 3. Mapping of cosmid cloned ASFV DNAs to the previously characterized plasmid cloned genomic library of ASFV strain BA71V. A. Each of the plasmid clones of strain BA71V was digested with the appropriate restriction enzyme which excised the ASFV-specific DNA, the fragments were electrophoretically separated on 0.8% agarose gels and stained with EtBr. The numbers at the top of the figure correspond to the numbers ofthe plasmid clones in Fig. l B. In lane 17, Lambda HindllI digested DNA was applied as a molecular weight marker. B. through F. Autoradiograms of blots obtained following transfer of the restricted and separated plasmid clones (Fig. 3A) to nylon membranes and hybridization with cosmid clones cosCM54 (B), cosCM69 (C), cosCM26 (D), cosCMl5 (E), cosCM25 (F). The ASFV-specific bands are indicated by an arrowhead.
NON-ESSEN"rlAL GENES OF AFRICAN SWINE FEVER VIRUS
2 1
4 3
IIIIIII A
B
C
D
E
F
6 5
8 7
10 9
12 11
14 13
IIIIIIIIIII
107
16 15
17
108
c. MET'rRAUX
characterized as follows: each plasmid of a previously characterized library representing nearly the entire genome of strain BA7 IV of ASFV was digested with the appropriate enzyme which excised the ASFV DNA fragment. The complete panel of the restricted plasmids was electrophoresed in an agarose gel and transferred to a nylon membrane. In order to map the cosmids to the ASFV genome, the DNA of each cosmid was radioactively labelled and hybridized to the blotted panel of the restricted plasmid clones. The results obtained for five individual cosmids are shown in Fig. 3. Because the whole cosmid was used as a probe, in each lane one band representing the plasmid vector lighted up. The appearance of a second band indicated that the cosmid used as a probe contained ASFV-specific sequences corresponding to the ASFV DNA sequence present in the particular plasmid clones. Fig. 1 shows the map positions of the BA71V plasmid library as well as selected cosmid clones which cover more than 90% of the ASFV genome. Selected cosmids were further characterized by restriction enzyme mapping (EcoRI, BamHI, SalI; unpubl. data).
Mutagenesis of subcloned ASFV DNAfragments by the p.Mu procedure Plasmids pCM 15/1/B (subcloned from cosCM 15 ) and pCM25/6 (subcloned from cosCM25 ) were mutagenized by the/tMu procedure (see Material and Methods) in order to insert at random a cassette containing a truncated ~gal gene within the/~Mu sequences (Fig. 2). A total of 28 mutagenized clones ( 15 derived from p C M I S / 1 / B and 13 derived from pCM25/6) were digested with either BamHI (pCM 15/1/B ) or EcoRI (pCM25/6) in order to test if random integration of the Mu cassette had occurred. These restriction enzymes cut the original plasmids into two fragments, whereas integration of the/~Mu cassette into either the vector or the cloned ASFV DNA added two restriction enzyme sites to the respective fragments. For this reason the fragment which had integrated the/tMu sequences disappeared and three new fragments appeared instead. Upon hybridization with plasmid pAT 153, either the integral vector band appeared on the autoradiogram, indicating that pMu had been inserted into ASFV sequences or the original vector band disappeared and two new fragments lighted up, indicating that/tMu had integrated into the vector DNA. The third new band did not hybridize in either case because it consisted exclusively of DNA of the/~Mu cassette. The variation of insertion within the ASFV sequences could be judged from the variability of the pattern of the EtBr-stained agarose gel. The results are shown in Fig. 4. Interestingly, it seemed that random insertion had occurred in plasmid pCM15/1/B, whereas plasmid pCM25/6 was apparently resistant to these randomized insertions. Transposition of/tMu into this ASFV DNA fragment could be observed only rarely.
109
NON-ESSENTIALGENESOFAFRICANSWINEFEVERVIRUS 2
4
1
6
3
8
5
10
7
9
12 11
14 13
16 15
17
liilllllllUlllliU A
B
C
•
d
..:.j.
".
.i
.,
::~". ~
"
"
",~
~:li:: Z: l" !:~.~:i~'.~i! l ~" ~ •
.
~
:::" ~:~? :~:~}:~i!:::i:': :-,T~I:~::.... • .'
:..
:
.
Illlllllllllllll 2 1
4 3
6 .=i
8 7
10 9
12 11
14 13
15
Fig. 4. Photographs of EtBr-stained agarose gels (A and C) and autoradiograms (B and D) showing the results of/zMu mutagenesis of pCM25/6 (A, B) and pCM 15/1/B (C, D). These pAT153 cloned ASFV DNA fragments were subjected to #Mu mutagenesis. Fifteen (A, B) and ! 3 (C, D), respectively, of the mutagenized clones were randomly selected and grown individually. The extracted plasmid DNA was digested with EcoRI (clones originating from pCM25/ 6) and BamHI (clones originating from pCMI 5/1/B), respectively, before electrophoresis in 0.8% agarose gels, transfer to nylon membranes and hybridization with radioactively labelled pAT I53. Lane 1: molecular weight marker (Lambda HindlIl). Lane 2: original plasmid, digested with either EcoRl (A and B) or BamHl (C and D). Lanes 3 through 17 (A and B) and Lanes 3 through 15 (C and D): individual preparations of mutagenized clones. The arrows on the left of the figure point to the integral vector band.
1 lO
C. METTRAUX
A
cosCM69
B
pCM33
C
p7.5-011-galactosidase D
.
'
I
I
I pCM33/14
Fig. 5. Schematic representation of cloning and mutagenesis of the viral TK gene. A and B. Cloning o f a BgllI-Xhol (Bg2, X) fragment excised from cosmid cosCM69 (A) into plasmid pA 1 l p.xba. The new construct was designated pCM33 (B). C. A cassette containing the vaccinia virus promoters p7.5 and pl 1 and the fl-galactosidase gene (orientations are indicated by arrows) was inserted into the unique EcoRV site (EV) of pCM33. D. The new construct was designated pCM33/14. The EcoRV sites were lost during the cloning procedure.
Identification, cloning, and mutagenesis of the ASFV TK gene A synthetic oligonucleotide, synthesized according to the published sequence of the ASFV TK gene (Blasco et al., 1990; Hernandez and Tabares, 1991 ), was used to demonstrate by hybridization that cosCM69 carried the ASFV thymidine kinase (TK) gene. Fig. 5 and Fig. 6 show that the TK gene was localized on an approximately 700 bp XhoI/BglII fragment of cosCM69. This fragment was therefore subcloned and the new plasmid, designated pCM33, was opened at the unique EcoRV site and a p-gal gene under the control of the vaccinia virus promoter pl I was inserted into the body of the TK gene (Fig. 5 ). The new plasmid was termed pCM33/14.
Transient expression of fl-galactosidase in African swine fever virus infected cells In order to test if the above-mentioned fl-gal gene would be expressed in ASFV-infected cells under the control of the vaccinia virus promoter p l 1, the original pSCI 1 and the newly constructed p C M 3 3 / 1 4 were transfected separately into CV-I cells which had been infected with ASFV. The blue color developing in these monolayers (Fig. 7 ) indicated that the vaccinia virus promoter was recognized by the ASFV enzymes. Similar reactions were observed in vaccinia-virus-infected cells (not shown).
Construction and selection of recombinant ASF viruses In order to delete genes non-essential for ASFV replication, the above-mentioned cloned and mutagenized ASFV DNA fragments were transfected either
NON-ESSENTIAL
1
2
GENES
3
OF AFRICAN
4
5
SWINE
FEVER
6
7
VIRUS
111
8 .o
._.~.~'~..u.~,i~, i.- • ~,~_
:
• .....:
~. ,"i ..~'~'~-::,~.
• • "
i
:••i?.
:~" ~
Fig. 6. Photographs of an EtBr-stained 1.2% agarose gel (lanes 1-4) and of the corresponding autoradiogram (lanes 5-8 ). DNA ofcosCM69 was digested with Bglll and Xhol before electrophoresis (lane 2 ) or was electrophoresed undigested (lane 3). BarnHI-digested genomic DNA of ASFV strain France 64 (lane 4) and a molecular weight marker (Lambda HindIll; lane I ) were used as controls. After electrophoretic separation, the DNA was transferred to a nylon membrane and hybridized with a radiolabelled TK-specific oligonucleotide. Lanes 5-8 of the autoradiogram correspond to lanes 1-4 of the gel. The arrows point to the bands which hybridizcd with the oligonucleotide probc.
1
2
A B Fig. 7. Transient expression offl-galactosidase in ASFV-infected cells. A photograph of ASFVinfected (A l, A2, B2) and mock infected (B l) cells is shown. Following infection or mock infection, the cells were transfected either with pCM33/14 (Al and B l ) or with pSCl 1 (A2) or mock transfected (B2). Thereafter, they were overlayed with a medium containing agarose and X-gal. Blue (dark) color was observed exclusively in cells which expressed ~galactosidase.
112
c. METTRAUX
into T K - t s 13 cells or else into CV- 1 cells which had been infected with ASFV. The following three methods were used to select recombinant progeny viruses.
Selection for TK- viralprogeny virus. T K - t s l 3 cells were infected with ASFV (TK ÷, cell culture adapted) before transfection with pCM33/14. Recombinants having the TK gene inactivated by the insertion of the fl-gal gene were expected to be generated. The growth and replication of such recombinant T K - viral progeny was selectively favoured by addition of BUdR to the cell culture medium. T K - virus isolates are currently being characterized.
Selection for phenotypically blue plaque recombinant ASF virus. In parallel, separate cell cultures were transfected with/zMu-mutagenized plasmids and with p C M 3 3 / 1 4 following the infection with ASFV. Progeny virus was harvested and various dilutions were used to infect fresh monolayers of CV-I cells. The cells were overlayed with a m e d i u m containing X-gal and agarose. Although the cells were observed for various time periods post infection, no recombinant virus expressing fl-gal could be identified up to date (data not shown ).
Identification of ASFV-infected cells by plaque hybridization. Additional samples of the above-mentioned infected and transfected cell cultures were used to infect fresh cell cultures which were kept under an agarose overlay without X-gal. Plaque imprints were taken 6 days post infection. They were hybridized with a fl-gal-specific DNA probe. Although the method appeared to work properly as indicated by hybridization assays where ASFV DNA was used as a probe, no recombinant viruses could be selected with this method (data not shown). DISCUSSION
Although no vaccines for the prevention of African swine fever (ASF) exist, previous work from our laboratory indicated that at least death due to ASF can be prevented by the administration of adjuvanted, inactivated vaccines. As reviewed by Kihm et al. (1987), the protective effect could be attributed to structural viral proteins. As long as the virus was propagated in animals a n d / o r in macrophage cultures, the protective components were maintained. They were lost, however, upon adaption of the virus to replication in conventional cell cultures. We therefore suggested that viral gene products which are not required for replication are essential for this protecting effect. The purpose of our study was therefore to set up a system which allows the identification of genes non-essential for ASFV replication. In vitro genetic manipulation followed by homologous recombination of the altered DNA fragments with the genomes of the wild type viruses is a very efficient way to
NON-ESSENTIAL GENES OF AFRICAN SWINE FEVER VIRUS
| 13
study the functions of viral genes and their products (Mackett et al., 1982; Roizman and Jenkins, 1985 ). The successful application of these techniques (reviewed in Ackermann, 1988) for the identification of such non-essential genes of large DNA viruses is not possible, however, without previously having established the following: ( 1 ) molecular cloning and characterization of the parent viruses; (2) conditions favouring recombination; and (3) strategies for the selection of recombinant progeny virus. For these reasons, it was our first goal to clone and characterize the genomic DNA of the virus which specified the functions to be studied, i.e. which conferred protection from death due to ASFV infection. Accordingly, a genomic cosmid library of wild type (wt) ASFV strain France 64 was established and characterized by restriction enzyme analysis and by comparing the newly established genomic library to a previously characterized library (Almendral et al., 1984; Ley et al., 1984) by hybridization studies. In a second series of experiments, selected DNA fragments were mutagenized in vitro. Therefore we deleted the function of specific viral genes by the insertion of foreign DNA carrying the marker gene fl-gal. These ASFV DNA fragments could be used for in vivo recombination into the genome of non-mutated ASFV and subsequent selection of recombinant progeny virus. According to Ackermann ( 1988 ), there are three alternative ways for the selection of recombinants: ( 1 ) Application of selective pressure to favour the growth of recombinants. This approach was the most successful in our hands so far. We were able to select several isolates of apparently T K - ASFV; (2) Alternatively, recombinants may be selected based on the expression of a phenotypic marker. We attempted, without success so far, to select viral recombinants expressing the fl-galactosidase gene. In a first approach, a fl-galactosidase gene lacking both promoter sequences and translation initiation signal, was introduced into selected DNA fragments of ASFV strain France 64. Since the foreign gene was supposed to be inserted at random into the cloned DNA fragments (Casadaban, 1975; Castilho et al., 1984), the expression of /~-galactosidase was expected to be regulated by a natural viral promoter if the construction had been inserted in frame with a gene non-essential for viral replication. A second approach involved the use of a vaccinia virus promoter fused to the fl-galactosidase gene. This construction was inserted into the ASF viral thymidine kinase (TK) gene. One advantage of this alternative was the possibility to select for T K - virus before testing for the expression offl-galactosidase. Furthermore, we could demonstrate by transient expression assays that the vaccinia virus promoter p l 1 was functional in ASFV-infected cells. This finding was in agreement with other reports concerning the reciprocal use of vaccinia virus and ASFV promoters by vaccinia and ASF viruses, respectively (Talavera and Beloso, 1987; Bostock, 1990; Hammond and Dixon, 1991 ). (3) Finally, it is possible to select recombinants by screening the viral
114
C. METTRAUX
progeny for the presence of the inserted foreign DNA. For this reason, we established the technique of plaque hybridization. Radiolabelled D N A probes were used for the detection of foreign D N A sequences among ASFV D N A in plaque imprints of cell monolayers infected with candidate recombinant viruses. Apparently, either the recombination efficiency or the number of plaques tested was too low to detect any recombinants by this method. CONCLUSIONS Essential preconditions for the construction of recombinant ASF viruses were established. In the course of the mutagenesis of cloned ASFV D N A fragments, several interesting features o f ASFV were observed. ( 1 ) While the transposase specified by phage Mu was able to integrate/tMu sequences at random into certain ASFV D N A fragments ( p C M 1 5 / I / B ) , other fragments appeared to be resistant to random integration ( p C M 2 5 / 6 ) . (2) The vaccinia virus promoter p I 1 was used by enzymes specified by ASFV in transient expression assays. If this promoter is also recognized when integrated in the ASF viral genome remains unclear. (3) T K - virus isolates could be selected. ACKNOWLEDGEMENTS We thank Eladio Vinuela for the plasmid library representing the genome of ASFV strain BA71 V, Frank Jenkins for supplying t h e / t M u system and for the construction o f D L M u T R 16, Barbara H o h n for ED8767, and Bernard Moss for pSC 11. The photographic work o f Anita Hug is greatly appreciated. This work was supported by the Swiss National Science Foundation grant No. 31-8832.86.
REFERENCES Ackermann, M., 1988. The construction, selection, and application of recombinant herpes viruses. J. Vet. Med. B, 35: 379-396. Almendral. J.M., Blasco, R., Ley, V., Beloso, A., Talavera, A. and Vinuela, E., 1984. Restriction site map of African swine fever virus DNA. Virology, 133: 258-270. Blasco, R., Lopez-Otin, C., Munoz, M., Bockamp, E.-O., Simon-Mateo, C. and Vinuela, E., 1990. Sequence and evolutionary relationships of African swine fever virus thymidine kinase. Virology, 178: 301-304. Bostock, C,J., 1990. Viruses as vectors. Vet. MicrobioI., 23:55-7 I. Casadaban, M., 1975. Fusion of the Escherichia coil lac genes to the ara promoter: a general technique using bacteriophage Mu-1 insertions. Proc. Natl. Acad. Sci. USA, 72: 4399-4403. Castilho, B.A., Olfson, P. and Casadaban, M.J., 1984. Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons. J. Bacteriol., 158: 488-495. Chakrabarti, S., Brechling, K. and Moss, B., 1985. Vaccinia virus expression vector: co-expres-
NON-ESSENTIAL GENES OF AFRICAN SWINE FEVER VIRUS
115
sion of fl-galactosidase provides visual screening of recombinant virus plaques. Mol. Cell. Biol., 5: 3403-3409. Collins, J. and Hohn, B., 1978. Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads. Proc. Natl. Acad. Sci. USA, 75: 4242-4246. Frischauf, A.-M., 1987. Digestion of DNA: size fractionation. Methods Enzymol., 152: 183199. Hammond, J.M. and Dixon, L.K., 1991. Vaccinia virus-mediated expression of African swine fever virus genes. Virology, 181 : 778-782. Hernandez, A.M.M. and Tabares, E., 199 I. Expression and characterization of the thymidine kinase gene of ASF virus. J. Virol., 65: 1046-1052. Hohn, B. and Collins, J., 1980. A small cosmid for efficient cloning of large DNA fragments. Gene, I l: 291-298. Jenkins, F.J., Casadaban, M.J. and Roizman, B., 1985. Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome. Proc. Natl. Acad. Sci. USA, 82: 4773-4777. Kihm, U., Ackermann, M., Miiller, H.K. and Pool, R., 1987. Approaches to vaccination. In: Y. Becket (Editor), Develop. in Vet. Virol. "African swine fever". Martinus Nijhoff, Boston, MA, pp. 127-144. Laughlin, C.A., Tratschin, J.-D., Coon, H. and Carter, B.J., 1983. Cloning of infectious adenoassociated virus genomes in bacterial plasmids. Gene, 23: 65-73. Ley, V., Almendral, J.M., Carbonero, P., Beloso, A., Vinuela, E. and Talavera, A., 1984. Molecular cloning of African swine fever Virus DNA. Virology, 133: 249-257. Mackett, M., Smith, G.L. and Moss, B., 1982. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA, 79:7415-7419. Maniatis, T., Fritsch, E.F, and Sambrook, J. (Editors), 1982. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mebus, C.A., 1987. Pathobiology and pathogenesis. In: Y. Becker (Editor), Develop. in Vet. Virol. "'African swine fever". Martinus Nijhoff, Boston, MA, pp. 21-3 I. Murray, M.E., Brammar, W.J. and Murray, K., 1977. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet., 150: 53. Panicali, D.L., Grzelecki, A. and Huang, C., 1986. Vaccinia virus vectors utilizing the fl-galactosidase assay for rapid selection of recombinant viruses and mcasurement of gene expression. Gene, 47: 193-199. Pool, R., 1986. Immunisierungsversuche mit Afrikanischem Schweinepestvirus-antigen. Thesis, Veterinary Medical Faculty, University of Zfirich. Roizman, B. and Jenkins, F.J., 1985. Genetic engineering of novel genomes of large DNA viruses. Science, 229:1208-1214. Talavera, A. and Beloso, A., 1987. RNA polymerase-promoler recognition in African swine fever and vaccinia viruses, 18P. In: Proc. International Congress "African swine fever and pig immunology", Salamanca, Spain. Twigg, A.J. and Sherrat, D., 1980. Transcomplementable copy-number mutants of plasmid ColEl. Nature, 283:216-218. Villarreal, L.P. and Berg, P., 1977. Hybridization in situ of SV 40 plaques: detection of recombinants SV 40 virus carrying specific sequences of nonviral DNA. Science, 196: 183-185.
