179,276-286

VIROLOGY

(1990)

Vaccinia Virus Host Range Genes MARION

E. PERKUS, SCOT-J-J. GOEBEL, STEPHEN W. DAVIS, GERARD P. JOHNSON, KEITH LIMBACH, ELIZABETH K. NORTON, AND ENZO PAOLElTI’

Virogenetics

Corporation,

465 Jordan Received

Road, May

Rensselaer 15, 1990;

Technology accepted

Park,

Troy,

New

York

12 180-8349

July 9, 1990

A gene encoding an 18-kDa polypeptide (ORF C7L) located in the vaccinia virus Hindlll C fragment was shown to be functionally equivalent to previously described host range gene (ORF Kl L) spanning the HindIll K/M fragment junction. Either C7L or Kl L host range gene is necessary and sufficient by itself to allow replication of vaccinia virus on human cells. Deletion of both C7L and Kl L genes from the wild-type vaccinia genome is required to derive a virus deficient for replication on human cells. Further, an ORF encoding a 77-kDa polypeptide derived from cowpox (CP77kDa) and previously shown to allow vaccinia to overcome the restriction for replication on Chinese hamster ovary cells could substitute for the vaccinia host range genes C7L and Kl L in permitting replication of the virus on human cells. Additionally, the three unique host range genes C7L, Kl L, and CP77kDa were functionally equivalent for vaccinia replication on pig kidney cells, but not on rabbit kidney cells. o 1990 Academic PWSS. IIIC.

The rabbitpox mutants of Gemmell and Fenner (1960) that failed to replicate on pig kidney cells (McClain, 1965; McClain and Greenland, 1965) provided the first description of poxvirus host range mutants. These mutants provided additional markers to further poxvirus genetics and were interpreted to be defective in a control function required for virus replication (Fenner and Sambrook, 1966). Genomic analysis of rabbitpox host range mutants (Lake and Cooper, 1980; Moyer and Rothe, 1980) demonstrated extensive terminal deletions up to 30 kbp of DNA. Host restriction of vaccinia virus have also been described (Drillien et a/., 1981, 1978; Hruby et a/., 1980; Mayr et al., 1975; Tagaya et a/., 1961). The host range mutant of Drillien et a/. (1981) was defective in replication on human cells and contained an 18-kbp deletion toward the left terminus of the genome. Marker transfer studies initially mapped the genetic function responsible for host range to a 5.2-kbp EcoRI fragment (Gillard et a/., 1985) and subsequently to an 855-bp open reading frame overlapping the vaccinia /-/indIll M/ K junction (Gillard et a/., 1986). This host range gene is transcribed leftward from /-/indIll K into HindIll M and is described as the KlL gene following recommended nomenclature (Rose1 et a/., 1986). We have recently reported the construction of a cloning and expression system that utilizes the Kl L host range gene as a selectable marker for the introduction of foreign genes into a vaccinia (WR) virus mutant deleted of 21 kb near the left terminus (Perkus et

’ To whom 0042-6822190 Copyright All rights

reprint

requests

should

$3.00

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

al., 1989). When we constructed a virus specifically deleted of the Kl L host range gene, we were surprised to find that growth on human cells was unimpaired. These results suggested the existence in vaccinia virus of a second host range gene. In this paper we report the identification of a second unique host range gene, C7L, mapping in the vaccinia HindIll C fragment (Goebel eta/., 1990) that is functionally equivalent to Kl L. Furthermore, we demonstrate that a unique cowpox gene encoding a 77-kDa protein (CP77kDa) that overcomes the restriction of vaccinia virus for replication on Chinese hamster ovary cells (CHO) (Spehner et al., 1988) is functionally equivalent to the vaccinia C7L and Kl L host range genes in supporting growth on human cells. Additionally, we report that any of the unique Kl L, C7L, or CP77kDa human host range genes is sufficient to overcome restriction of vaccinia virus replication on pig kidney cells and that either the Kl L or the CP77kDa gene, but not the C7L gene, allows vaccinia virus replication on rabbit kidney cells. MATERIALS

AND

METHODS

Cells and viruses The origins and cultivation of the WR (Panicali et a/., 1981) and Copenhagen (Guo et a/., 1989) strains of vaccinia virus have been previously described. The Brighton Red strain of cowpox (ATCC VR-302) was propagated on VERO cells (monkey kidney, ATCC CCL 81). Other cell lines used were MRC-5 (human embryonal lung, ATCC CCL 171) HeLa (human cervix,

be addressed. 276

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VIRUS

epithelioid carcinoma, ATCC CCL Z), WISH (human amnion, ATCC CCL 25), Detroit (human foreskin, ATCC CCL 54) LLC-PKl (pig kidney, ATCC CCL 101) RK-13 (rabbit kidney, ATCC CCL37), and CHO (Chinese hamster ovary ATCC CCL 61). All cells except CHO were cultivated at 37” in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (GIBCO Laboratories Life Technologies, Inc., Grand Island, NY), penicillin (100 IU/ml), and streptomycin (100 mg/mI) with additional supplements of nonessential amino acids (Eagle, 1959) for HeLa and Detroit and sodium pyruvate (1 mM) plus 0.19/o lactalbumin hydrolysate for Detroit cells. CHO cells were grown in Alpha MEM with 10% FBS. For virus cultivation the medium was changed to Eagle’s MEM (or Alpha MEM for CHO cells) supplemented with 5-10% newborn calf serum (Flow Laboratories, McLean, VI).

DNA cloning and oligonucleotide

synthesis

Plasmids were constructed, screened, and grown by standard procedures (Maniatis et al,, 1982; Perkus et a/., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from Bethesda Research Laboratories (Gaithersburg, MD), New England Biolabs, Inc. (Beverly, MA), and Boehringer-Mannheim Biochemicals (Indianapolis, IN). Klenow fragment of Escherichia co/i polymerase was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Ba131 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs, Inc. (Beverly, MA). T4 polymerase was obtained from Bethesda Research Laboratories (Gaithersburg, MD). The reagents were used as specified by the suppliers. Plasmid PBS-SK was obtained from Stratagene (La Jolla, CA). Plasmid pSV2gpt (ATCC 37 145) was obtained from the American Type Culture Collection (Rockville, MD). Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et a/., 1977) using Sequenase (Tabor and Richardson, 1987) as previously described (Guo et a/., 1989). DNA amplification for cloning by polymerase chain reaction (PCR) (Saiki eta/., 1988) was performed using custom synthesized oligonucleotide primers and GeneAmp DNA amplification reagent kit (Perkin-Elmer Corp., Norwalk, CT) in an automated Perkin-Elmer Cetus DNA thermal cycler. Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by Ba/31 exonuclease and mutagenesis using synthetic oligonucleotides (Mandecki, 1986).

HOST

RANGE

GENES

Construction

277

of recombinant

viruses

Generation of recombinant virus by in viva recombination and in situ hybridization on nitrocellulose filters were as described (Panicali and Paoletti, 1982). Screening for P-galactosidase activity and host range selection were as previously described (Perkus et al., 1989). Restriction analysis, agarose gel electrophoresis and Southern blot hybridization (Southern, 1975) were as previously described (Perkus et al., 1986; Piccini et al., 1987).

RESULTS Deletion of the host range gene, Kl L, from vaccinia virus Gillard et al. (1986) identified a single open reading frame coding for a polypeptide of 32.5 kDa which when recombined into the genome of a vaccinia virus host range deletion mutant restored host range function. This gene is transcribed leftward from HindIll K into HindIll M and is described herein as the Kl L gene following the recommended nomenclature (Rose1 et a/., 1986). The vaccinia virus host range mutant employed by Gillard et a/. (1986) to identify the Kl L host range function contained an 18-kbp deletion near the left end of the genome (Drillien et a/., 1981). To more precisely appreciate the contribution of the Kl L host range gene to viral host range, cytopathology, and virulence, we decided to delete specifically the Kl L gene from wild-type vaccinia. The Kpnl fragment D which completely contains the Kl L gene was cloned into the Kpnl site of pUC18, resulting in plasmid pSD435 (Fig. 1). Additional subcloning generated plasmid pSD453 wherein the Kl L gene and its promoter were deleted (Fig. 1). P-Galactosidase coding sequences, under the control of the late 11 -kDa promoter (Bertholet et al., 1985) were inserted into the Bglll site of pSD453, generating pSD453BG (Fig. 1). Plasmid pSD453BG was used as donor plasmid for in viva recombination with vaccinia virus vP410, a TK- mutant of the Copenhagen strain of vaccinia virus (Guo et a/., 1989). Recombinant progeny were identified as blue plaques in the presence of X-gal, the plaque was cloned on VERO cells, and the recombinant virus was designated vP548. As expected, vP548 lacked Kl L sequences since no hybridization signal was obtained with a 32P-labeled Kl L internal probe (data not shown). Surprisingly, however, the ability of vP548 to replicate on human MRC-5 cells was not abrogated. To eliminate the possibility that the P-galactosidase gene was somehow responsible, the 1 1 -kDa promoter/ggalac-

278

PERKUS ET AL. C

Hindll

N

M

K KdM

KP~’

{Ligate pSD452 IHpol(portial) )Bglll linkers, lSglll ILigate

ligate

”l-p3

HPQl

EamHl 6 galactosidose ,: KIL (rabbit, human, porcine) > C7L (human, porcine). Clearly, there exist other poxvirus host range genes still to be discovered. One of the major criteria used in the classification of the poxvirus family is the natural host range of the viruses. Hence, as examples, avipoxviruses are restricted to replication in avian species and suipox to swine. This suggests a complex family of host range regulatory functions responsible for these host restrictions. The extensive information currently availablecomplete DNA sequence of the vaccinia genome; cloned and defined unique host range genes Kl L, C7L, and CP77kDa; defined host range deletion mutants of vaccinia as well as naturally host-restricted poxviruses; and defined cell substrates-provides unique opportunities to experimentally detail the regulatory functions of the poxvirus host range genes initially reported by Fenner. We have recently reported (Taylor et al., 1988a, 198813) the use of fowlpox recombinants as useful vaccine vectors for both avian and nonavian species. In nonavian species, avipoxviruses are restricted for replication. Nevertheless, a recombinant fowlpox virus ex-

VACCINIA

VIRUS

pressing the rabies glycoprotein effectively immunized and protected the nonavian species mice, cats, and dogs from rabies challenge. The manipulation of host range deleted vaccinia viruses as recombinant vectors for vaccine applications provides another vaccination strategy. Indeed, we have already demonstrated that host~restr~cted vaccinia re~ombinants can be used to successfully vaccinate animals (Tartaglia eta/., unpublished data). This approach provides useful attenuation of the vaccinia vectors. ACKNOWLEDGMENTS This study was supported in part by U.S. Army Support Contract DAMDl7-85-C-5232. We thank J. Tartaglia and 1. Taylor for helpful drscussions. We also thank Karen Dombrowski for typing the manuscript.

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ET AL. TABOR, S., and RICHARDSON, C. C. (1987). DNA with a modified bacteriophage T7 polymerase.

Sci. USA 84,4767-477

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Vaccinia virus host range genes.

A gene encoding an 18-kDa polypeptide (ORF C7L) located in the vaccinia virus HindIII C fragment was shown to be functionally equivalent to previously...
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