A dominant selectable marker for the construction of recombinant poxviruses

123

Gene, 65 (1988) 123-128

Elsevier GEN 02356

A dominant

selectable

(Recombinant

DNA;

transferase

marker vaccinia

for the construction virus;

eukaryotic

of recombinant

cloning

poxviruses

E. coli

vector;

xanthine-guanine

phosphoribosyl

gene; plasmid)

David B. Boyle and Barbara E.H. Coupar Commonwealth

Scientific and Industrial Research

Organisation, Australian Animal Health Laboratory,

Geelong, Victoria, 3220

(Australia)

Received 20 October 1987 Revised and accepted 23 November 1987 Received by publisher 26 January 1988

SUMMARY Mycophenolic

acid has been shown to be a potent inhibitor of vaccinia virus growth. By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome. When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes. Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.

A variety of techniques have been developed for the construction of recombinant poxviruses based on VV. In general, they rely on homologous recombination between VV sequences flanking the foreign gene of interest and the virus genome during simul-

taneous infection of cells with virus and transfection with recombinant plasmid. From the progeny virus, recombinant viruses representing less than 0.1 y0 or 0.0 1% of the population have to be selected and plaque-purified (Mackett and Smith, 1986). This is achieved by (i) plaque hybridization with an appropriate radiolabeled probe; (ii) selection for Tk-

Correspondence to: Dr. D.B. Boyle, CSIRO,

HSV, Herpes simplex virus; IMP, inosine

INTRODUCTION

Health

Laboratory,

(Australia)

P.O.

Bag

24,

Australian

Geelong,

Victoria,

Animal 3220

Tel. (052)265222.

1000 bp; moi, multiplicity MXHAT,

1 or 2.5 @g MPA/ml,

hypoxanthine/ml, Abbreviations: Ecogpt, GMP,

bp, base pair(s);

E. coli xanthine-guanine guanosine

monophosphate;

0378-I 119/88/$03.50 0 1988 Elsevier

BUdR,

5-bromodeoxyuridine;

phosphoribosyl

transferase;

gpt, gene coding for Ecogpt;

Science Pubbshers

B.V. (Biomedical

TK, thymidine ts, temperature

monophosphate;

kb,

MPA, mycophenohc

acid;

25Opg

0.2 pg aminopterine/ml,

xanthine/ml,

14 pg

4 pg thymidine/ml;

kinase; tk, gene coding for TK; Tk, tk phenotype; sensitive;

VV; XMP, xanthosine

Division)

ofinfection;

VV, vaccinia

monophosphate.

virus; VV-WR, wild-type

124

recombinants serted

when the foreign

gene has been in-

into the tk gene of the virus;

expression

of a marker gene product

(iii) the co-

such as /l-galac-

1981)

MPA

tosidase, or (iv) selection for a dominant marker such as HSV tk or neomycin resistance concurrently

inhibition

inserted into the virus genome. The proportion

xanthine

recombinant

progeny virus can be increased

of the by alter-

is used

at 25 ,ug/ml.

In

addition,

aminopterine is required to block de novo synthesis of purines from precursors to allow expression of the MXHAT,

by MPA. Under the selective conditions cells expressing as a precursor

of

thegpt gene are able to use

for XMP and thus synthesize

GMP. Normal mammalian

cells convert xanthine

to

native transfection protocols involving the use of ts mutants (Kieny et al., 1984) or single-stranded

XMP very poorly and thus are unable to grow under these selective conditions (for the original principle

recombinant

of HAT

DNA molecules

Many of these approaches type selection adaptable

in tk

(Wilson

cell lines

to other poxviruses

et al., 1986).

rely on the Tk phenoand are thus not

for which suitable tk

cell lines are unavailable. Although Tk- recombinants are selectable using BUdR, the mutagenic effect of BUdR and the resulting background of Tk mutants does not allow selection at the recombination step or enrichment at subsequent passage. The neomycin-resistance gene can be used as a dominant selectable marker; however, high concentrations of G418 antibiotic are required with 48 h pretreatment of cells for the selection to operate (Franke et al., 1985). Here we report that the gpt gene of E. cd’ can be used as a dominant selectable marker for insertion into VV. The antibiotic, MPA, which provides the basis for selection using the gpt gene is a very potent inhibitor of poxvirus growth. Since the gpt gene has been used as a dominant selectable marker in a variety of mammalian cells, this obviates the need for tk cell lines for the construction of recombinant poxviruses (Mulligan and Berg, 1980). When coupled in tandem with another gene of interest, recombinant VVs can be positively selected carrying both genes. Potentially the gpt gene provides a dominant selectable marker for insertion into any non-essential region of the VV genome and equally important into such regions of other host-specific poxviruses.

EXPERIMENTAL

AND

DISCUSSION

(a) Inhibition of vaccinia virus plaque formation by mycophenolic acid To inhibit mammalian cell growth and to select for cells carrying the gpt gene (Mulligan and Berg, 1980;

selection

see Szybalski

et al., 1961, and

Szybalska and Szybalski, 1962). Preliminary experiments established confluent

monolayers

that

of CV-1 cells used for the VV

plaque assay survived for more than seven days in MXHAT with up to 25 pg MPA/ml. When MXHAT was tested against vaccinia virus on preformed monolayers of CV- 1 cells (Jensen et al., 1964) doses of 1 to 25 pg MPA/ml completely inhibited plaque formation and even at 0.1 pg MPA/ml there was significant reduction in plaque size (Fig. 1). Inhibition of mammalian cell growth by MPA is reversible by guanine. Addition of guanine, 25 pg/ml, to plaque assays in the presence of MXHAT reversed the inhibition of plaque formation and plaque sizes approached normal (Fig. 1). These results suggested that MPA was inhibiting VV growth by acting as an inhibitor of IMP dehydrogenase preventing the formation of XMP and therefore GMP in a manner similar to its action on mammalian cells (Mulligan and Berg, 1981). MPA alone failed to inhibit VV plaque formation on CV- 1 cells. HAT (Szybalska and Szybalski, 1962) was required to inhibit de novo synthesis of IMP to allow expression of the MPA inhibition. HAT alone had no effect on VV plaquing on CV-1 cells. The cell monolayers did not require pretreatment with MXHAT for inhibition of virus plaquing. These observations suggest that insertion of the gpt gene into VV under the control of a VV promoter should lead to VV recombinants being able to grow in the presence of MXHAT. (b) Insertion of the gpt gene into vaccinia virus The gpt gene and attached SV40 polyadenylation signals from pSV2A-gpt (Mulligan and Berg, 1980) was subcloned as a HindIII-BumHI fragment into pUC9. The translation start codon of the gpt gene is located 200 bp from the Hind111 site. A unique BgfII

125

Virus

dilution MPA

-6

1

2

25

3

25

4

Fig. 1. Inhibition

of VV plaque

Plaque formation

by the wild type, VV-WR, was completely

inhibition

ofplaque

VV-Ecogpt, plaques

formation

was constructed

in the presence

formation

by mycophenolic

by 25 pg MPA/ml in MXHAT

acid. Ten-fold inhibited

dilutions

was reversed

by the addition

with the &PCgene within the tk gene under the control

of MXHAT

containing

1 ng MPA/ml;

of virus were plated

by 1 or 25 pg MPA/ml

row 4. Rows

l-4

contained

ofguanine

on monolayers in MXHAT;

at 25 pg/ml; row 3. The recombinant,

of the P-7.5 promoter. are specified

of CV-I cells.

rows 1 and 2. The

This virus was able to form

on the left margin.

126

pSV2A-gpt

puc9

Hindlll/BamHI

Hindlll/BamHI

Ligate

Bglll/BamHI

BamHl

fragment

CIAP

Ligate

/

Fig. 2. Construction

of plasmids

SV40 polyadenylation

\

for the insertion

signals from pSVZA-gpt

of the E. (Mulligan

To position the gpt gene as close to the VV promoter, Recombinant

plasmids

to the VV promoter H, tiindII1;

pGpt07/14

and pGpt07/15

E, EcoRI;

MCS, multiple

(Ecogpt)

P-7.5, a BglII-BamHI have the

and flanked by the tk gene sequence.

B, BarnHI;

coligpf

gene into the tk (TK) gene of VV. The gpt gene and attached

and Berg, 1980) were subcloned gpt

gene in the correct

These plasmids

cloning

fragment

site; CIAP,

as a HindIIl-BarnHI

was subcloned

and incorrect

orientations,

respectively,

into pUC9. et al., 1987). with respect

gene into the tk gene of VV, VV-WR.

were used to insert the

gpt

calf intestinal

phosphatase.

alkaline

fragment

into pBCB07 (Andrew

127

site is located

120 bp closer to the translation

codon (Pratt and Subramani,

start

1983). To position

gpt gene as close to the VV promoter

as possible,

BglII-BumHI

into

fragment

was cloned

(Andrew et al., 1987; Fig. 2). Recombinant pGpt07/14 restriction

and pGpt07/15 enzyme

a

pBCB07 plasmids

were demonstrated

digest analyses

the

by

to have the gpt

gene in the correct and incorrect orientation with respect to the VV promoter and flanked by the tk gene sequences. Only the plasmid pGpt07/14 having the gpt gene in the correct orientation with respect to the VV promoter

was able to generate

recombinant

viruses

which could grow in MXHAT. In a typical experiment 0.1 to 0.2% of the output virus from the recombination step where selection was not applied at this step were recombinants. A single passage in CV-1 cells at low moi (0.0 1) from the original recombination step under MXHAT selection resulted in 10 to 30% of the output virus being recombinants. When MXHAT selection was applied at the recombination step, the proportion of recombinants in the output increased dramatically. At a moi of 1 the proportion of recombinants increased from 0.1% to 0.5%. When low moi’s (0.01 and 0.001) were used and the incubation in MXHAT extended to five to seven days for the first step, recombinants represented 10 to 40% of the output compared with the normal 0.1 to 0.2% (Fig. 3; the total output of virus was reduced by lOO- to lOOO-fold). The ability to positively select at the recombination and subsequent steps as well as the absence of spontaneous background mutants greatly facilitated the construction and selection of recombinants. A recombinant VV has been constructed using pGpt07/ 14 and the insertion of the gpt gene into the tk region confirmed by restriction enzyme analysis and Southern hybridization analysis. This recombinant, VV-Ecogpt, is able to plaque under the MXHAT selection on CV-1 monolayers whilst plaquing of the wild-type virus, VV-WR, is completely inhibited (Fig. 1). Large fragments of fowlpox virus genome, up to 20 kb, have been inserted into VV using the pGpt07/14 vector as a selectable insertion vehicle (C.T. Prideaux and D.B.B., unpublished). Additional genes of interest could be inserted and expressed in VV by inserting another promoter plus gene into pGpt07 then using the gpt gene to select for recombinants.

./‘.‘\ 0.1’

i

.4 0.1

0.001

0.01

moi Fig. 3. The effect of selection

and moi at the recombination

on the “/, of the output virus as recombinants gene. Monolayer

cultures

cultures

were transfected

plasmid

DNA (2 pg of pGpt07/14

essential

and Smith,

step the

gpl

of CV-I cells ( lo6 cells/25 cm’ flask)

were infected with VV-WR at various

(Mackett

expressing

moi’s. Two hours later the

with calcium

phosphate-precipitated

and 20 pg of calf thymus DNA)

1986). Eight hours later Eagle’s minimum

medium plus 5% foetal calf serum was added with (m)

or without

(0)

harvested.

To determine

MXHAT

nants expressing

selection.

the gpt gene following

applied at the recombination or absence

of MXHAT

on CV-1 monolayers

selection,

the output

in the presence

treatments

virus was assayed

(recombinants

or absence

expressing

(total virus output)

From these titres the “6 of the output

nants was calculated scale against

the different

step, namely moi and the presence

the gpt gene only able to plaque) of MXHAT.

Six days later the cells were

the titre of the output virus as recombi-

for each treatment

as recombi-

and plotted

the moi used in the recombination

on a log,,,

step.

(c) Conclusions The use of the gpt gene as a dominant selectable marker for poxvirus recombinant construction has a number of advantages over other selectable markers. It does not require tk ~ cell lines and will operate in most if not all eukaryotic cell types. The ability to maintain confluent monolayers for extended periods and to plaque poxviruses should make it the marker of choice for the construction of recombinants based on slow-growing host-specific poxviruses. Furthermore, the ability to select at both the recombination and subsequent steps should allow the construction of recombinants where the frequencies of recombi-

128

nation

may be low as is the case with the insertion

of very large fragments

of foreign DNA. By coupling

the gpt gene in tandem with a gene of interest we have successfully construct

used this dominant recombinant

virus (D.B.B.

selectable

viruses

and B.E.H.C.,

based

marker to

on fowlpox

unpublished).

Franke,

C.A.,

Rice,

resistance

selection

and isolation

Strauss,

J.H.

as a dominant of vaccinia

and

Hruby,

selectable

D.E.:

marker

for

virus recombinants.

Mol.

Cell. Biol. 5 (1985) 1918-1924. Jensen,

F.C., Girardi,

Infection

R.V. and Koprowski,

and simian

tissue

cultures

H.:

with Rous

virus. Proc. Natl. Acad. Sci. USA. 52 (1964) 53-59.

M.P.,

Lathe,

Schmitt,

R., Drillien,

D., Wiktor,

Expression vaccinia Mackett,

A.J., Gilden,

of human

sarcoma Kieny,

ACKNOWLEDGEMENTS

CM.,

Neomycin

R., Spehner,

T., Koprowski,

of rabies virus glycoprotein

virus. Nature

D., Skory,

H. and Lecocq,

S.,

J.-P.:

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312 (1984) 163-166.

M. and Smith, G.L.: Vaccinia

virus expression

vectors.

J. Gen. Virol. 67 (1986) 2067-2082. Mulligan,

This research was carried out whilst D.B.B. and B.E.H.C. were on secondment from CSIRO, Australian Animal Health Laboratory to the John Curtin School of Medical Research, The Australian National University, Canberra. The authors wish to acknowledge the technical and Mrs. V. Corrigan.

assistance

of Mr. C. Rolls

R.C. and Berg, P.: Expression

mammalian Mulligan,

cells. Science

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Berg,

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of a bacterial

gene in

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for animal

cali gene coding

phosphoribosyltransferase.

cells that

for xanthine-guanine

Proc. Nat]. Acad.

Sci. USA 78

(198 1) 2072-2076. Pratt,

D. and

Escherichia

Subramani,

S.: Nucleotide

coli xanthine-guanine

sequence

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gcnc. Nucl. Acids Res. 11 (1983) 8817-8823. Szybalska,

E.H. and Szybalski,

IV. DNA-mediated

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