Restricted replication of ectromelia virus in cell culture correlates with mutations in virus-encoded host range gene

VIROLOGY

187,433-442

Restricted

(1992)

Replication

of Ectromelia Virus in Cell Culture Correlates in Virus-Encoded Host Range Gene’

WAN CHEN,* ROBERT DRILLIEN,t

with Mutations

DANIELE SPEHNER,t AND R. MARK L. BULLER*r2

*Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; and fUnit@ lnserm 74 et Laboratoire Commun ULP-SynthBlabo, lnstitut de Virologie de la Facultk de MBdecine de Strasbourg, 3 rue Koeberlg, 67000 Strasbourg, France Received October 28, 199 1; accepted

November

25, 199 1

Ectromelia virus (strain Moscow) was shown to replicate poorly or not at all in cell lines derived from the rabbit or hamster. The failure of ectromelia virus to replicate in cell lines derived from the hamster suggested that the virus lacked a functional CHO host range (hr) gene required for multiplication in these cells. A DNA fragment which hybridized to the CHO hr gene was cloned from the ectromelia virus genome and shown by sequence analysis to be deleted of 506 bp within the ectromelia virus CHO hr homologue. Two additional ectromelia viruses (Hampstead and Mill Hill strains) were also shown to lack an intact CHO hr gene. Insertion of the CHO gene from cowpox virus into the ectromelia virus genome extended the host range of ectromelia virus in tissue culture. These results demonstrate that an intact CHO hr gene is not required for maintenance of ectromelia virus in nature and provide a partial explanation for ectromelia virus’ narrow host range, as opposed to the broad host range of cowpox virus, which has a functional CHO hr gene. 0 1992 Academic PWSS, IIIC.

INTRODUCTION

between integral membrane proteins and cytoskeletal elements. The C7L gene of the Copenhagen strain of vaccinia virus is sufficient for replication of a Kl L minus vaccinia virus in human embryonic MRC-5 cells and pig LLCPKl cells (Perkus et al,, 1990). The deduced amino acid sequence of C7L has no homology with either the Kl L or the ankyrin proteins. The C7L open reading frame is conserved in tested vaccinia viruses (Kotwal and Moss 1988; Goebel et al., 1990). A host range (hr) gene from cowpox virus, hereafter referred to as CHO hr, maps to the X-101 J and I fragments approximately 27 kbp from the left terminus of the cowpox virus genome (Spehner et al., 1988). Upon transfer to the vaccinia virus genome, this gene enables vaccinia virus to overcome a replication block in CHO cells (Spehner et a/., 1988). The CHO hr gene encodes a protein which contains one ankyrin-like repeat but little other amino acid sequence homology with the Copenhagen Kl L or C7L translated gene products (Lux et a/., 1990). All tested strains of vaccinia virus fail to productively infect CHO cells (Drillien et a/., 1978; Hruby et al., 1980), which agrees well with the finding that the CHO hr gene is absent from the Copenhagen strain of vaccinia virus (Goebel et a/., 1990). The study of host range genes in tissue culture has not furthered our understanding of their contribution to cell, tissue, or species tropism in a natural infection, in part due to the lack of a validated animal model for vaccinia or cowpox viruses. In response to this con-

Orthopoxviruses are closely related as judged by restriction enzyme and DNA sequence analyses, crossprotection in animal studies, and cross-neutralization of infectivity in tissue culture; however, the host species tropisms of the individual viruses are quite distinct. Cowpox virus is isolated in nature from a large number of animal species, whereas the natural host of ectromelia virus is thought to be only the mouse. It is reasonable to assume that a basis of host species tropism lies in the efficiency of virus replication in the host cells which, in turn, is affected by a series of cell-specific virus genes, three of which (C7L, Kl L, CHO hr), have been identified. The Kl L gene of the Copenhagen strain of vaccinia virus encodes a 30-kDa protein which is sufficient to relieve the host restriction of a vaccinia virus deletion mutant on a number of human cells as well as rabbit RK-13 cells and pig LLC-PKl cells (Drillien eta/., 1981; Gillard et al., 1985, 1986; Perkus et al., 1990). Computer-assisted comparisons of the amino acid sequence of the Kl L gene with the protein gene bank revealed homology with an ankyrin repeat unit (Lux et al., 1990). Ankyrins are thought to control interactions

’ The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned Accession No. M83102. 2 To whom reprint requests should be addressed. 433

0042-6822/92 Copyright All rights

$3.00

@ 1992 by Academic Press. Inc. of reproduction in any form reserved.

CHEN ET AL.

434

straint, we have chosen to study these genes in ectromelia virus, since this virus infection of its natural host, the mouse, has long been accepted as a model for an acute viral disease (Fenner, 1948). In this study, we examine the ectromelia virus tissue culture host range and examine the basis of the block in ectromelia virus replication in CHO cells. METHODS AND MATERIALS Viruses A spleen homogenate from a BALB/c mouse sacrificed 6 days following infection with the Moscow strain of ectromelia virus was generously supplied by Dr. Pravin Bhatt (Section of Comparative Medicine and Division of Animal Care, Yale University School of Medicine, New Haven, CT). From this material and following three sequential plaque purifications, three virus isolates were obtained and used to prepare a passage 1 virus stock within BSC-1 cells. Groups of four male A/J mice were then infected by the foot-pad route. For each isolate, 100% mortality was achieved with approximately 3 PFU per mouse. Isolate Mos-3-Pl was chosen as the prototype virus for future studies, as at this multiplication of infection (m.o.i.) it gave a mean day of death of 9 days vs 10 days with isolates Mos-lPl and Mos-2-Pl . Mos-3-Pl was further passaged in L929 cells (as this cell line gives fourfold more ectromelia virus infectivity than BSC-1 cells), and the resultant virus stock, Mos-3-P2, was used in all further experiments (except the experiment described in Fig. 1). This virus was as virulent as Mos-3-Pl for foot-pad inoculations of male A/J mice (R. M. L. Buller, unpublished results). The ectromelia virus strains used in the experiments described in Fig. 1 were kindly provided by Dr. John .Williamson (St. Mary’s Hospital Medical School). The Brighton strain of cowpox and the Utrecht strain of rabbitpox were obtained from the ATCC.

nese hamster; ATCC CCL 14) Don (lung, Chinese hamster; ATCC CCL 16), and Dede (lung, Chinese hamster; ATCC CCL 39). All cell lines (except LLC-PKl , CHO, B14FAF28-G3, Don, and Dede) were grown in Eagle’s minimum essential medium (MEM). Dulbecco’s modified Eagle’s medium was used for the growth of B14FAF28-G3 and Don cells; Ham’s F-12 medium for CHO cells; and McCoy’s 5a medium for Dede cells. The growth of LLC-PKl cells was carried out in Medium 199. Unless noted all media contained 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin with the further addition of 12.5 pg/ml of BUdR to the media formulation for the HuTK143B cell line. (Whittaker Bioproducts, Inc., Walkersville, MD). Virus infection of cells Cultures containing 2 X 1O5cells were infected with viruses at a m.o.i. of l-5 PFU in 0.5 ml of MEM for 1 hr at 37”. The virus inocula were removed, the cultures were then washed twice with prewarmed MEM containing 1O/O FBS, and the cells were fed with the standard MEM cell culture medium. At the indicated time points, virus-infected cells were scraped into the culture supernatant, which was transferred to a 15-ml conical test tube and stored at -20”. Alternatively, virus-infected cells were extracted for total nucleic acid prior to Southern blot analysis, On occasion, this process was scaled up to produce large amounts of virus. Fifty cultures of L929 cells (20 X 10” cells per culture) were infected with 1 PFU/cell of Mos-3-P2 for 3-4 days. Cell-associated virus was purified as described by Joklik (1962). This virus was passage 3 (Mos-3-P3) from the original plaque isolate and was as virulent for A/J mice as passages 1 and 2, with 1.6 particles (estimated by electron microscopy with latex spheres as standards) sufficient for a LD,, (Miller, 1973).

Cells BSC-1 (kidney, African green monkey; ATCC CCL 26) or L929 (connective tissue, mouse; NCTC clone 929; ATCC CCL 1) cell cultures were used to propagate wild-type and recombinant viruses. HuTK- 143B (osteosarcoma, human ATCC CRL 8303) cells were used for isolation of TK- recombinant virus. The following cell lines were utilized for characterizing virus cell tropism: SIRC (cornea, rabbit; ATCC CCL 60), RK-13 (kidney, rabbit; ATCC CCL 37) LLC-PKl (kidney, pig; ATCC CCL lOl), MRC-5 (embryonic lung, human; ATCC CCL 171), HEp-2 (epidermoid carcinoma, human; ATCC CCL 23) CHO (ovary, Chinese hamster, ATCC CCL 61), Bl4FAF28-G3 (peritoneal cells, Chi-

Virus infectivity assays Virus was released from the cells by three freezethaw cycles and dispersed by two 30-set periods of sonication in a chilled-cup attachment to a Model W-385 sonicator (Heat Systems-Ultrasonics, Inc., Farmingdale, NY). Virus infectivity was estimated as described previously (Buller and Wallace, 1985). Briefly, virus plaques were visualized by the addition of 0.5 ml 0.3% crystal violet formalin solution to each well after incubation at 37” for 2 days for vaccinia and cowpox viruses, and after 4 days for ectromelia virus. During cloning of ectromelia virus, plaques were localized using a 1O/O low-melting-temperature agarose overlay

ECTROMELIA

VIRUS LACKS FUNCTIONAL

(GIBCO BRL, Inc., Gaithersburg, MD) in MEM and 5% FBS. After 4 days at 37”, a second agarose overlay supplemented with 0.01% w/v neutral red (GIBCO BRL, Inc.) was added for plaque visualization. Enzymes and chemicals Restriction enzymes were supplied by GIBCO BRL, Inc., New England Biolabs, Inc. (Beverly, MA), or Boehringer-Mannheim Biochemicals (Indianapolis, IN) and used as specified by the manufacturers; Random primer DNA labeling system, T, polynucleotide kinase, lipofectin and alkaline phosphatase from calf intestine were purchased from GIBCO BRL, Inc; DNA ligation and Klenow fill-in reagents were obtained from Stratagene (La Jolla, CA); and BUdR was purchased from Sigma Chemical Co. (St. Louis, MO). Cloning

procedures

In general, molecular cloning and analysis of DNA were essentially as described by Maniatis (1989). In particular, the ectromelia virus DNA was isolated from purified virions with SDS/proteinase K digestion followed by phenol extraction (Garon et al., 1978). The DNA migrated in pulse-field agarose gels as an intact band with a size of approximately 202 kbp as determined by coelectrophoresis with concatamers of hDNA (M. Merchlinsky, unpublished results). The DNA was partially digested with Sau3A, and fragments of approximately lo-20 kbp were ligated into the Xhol site of X FIX II vector (Stratagene) followed by packaging with Gigapack II XL extract (Stratagene). AX recombinant, L28, was identified by DNA hybridization with plV27 DNA containing the CHO hr gene of cowpox virus (R. Drillien, unpublished results). From L28, a 4.2kbp Sal1 fragment containing the ectromelia virus CHO hr homologue was subcloned into pBluescript II (Stratagene), and recombinant plasmid pL28-5 was isolated. This plasmid was then used for DNA sequence analysis of the ectromelia virus CHO hr homologue. The CHO gene was recombined into the ectromelia virus TK gene using a previously described strategy (Gillard et a/., 1985). Briefly, the cowpox virus strain Brighton CHO hr gene was fused in-frame to the last 29 codons of the Kl L gene from vaccinia virus (strain Copenhagen). This was done to permit (if needed) the precipitation of the CHO gene product by antisera against the terminal 29 codons of the Kl L gene (Gillard et a/., 1989). This fragment was recombined into the vaccinia virus strain WR TK gene downstream from a 7.5K promoter, and a plasmid designated plV41 was isolated and characterized (details to be published elsewhere). This plasmid was used to generate TK minus

CHO HR GENE

recombinant virus expressing low and Results).

435

the CHO gene (see be-

DNA sequencing Using the exonuclease III method (restriction enzyme sites /Votl and Sacl), a nested set of deletions was generated in the ectromelia virus DNA fragment in pL28-5. DNA sequence analysis was carried out by the dideoxy nucleotide method of Sanger (1980) using Sequenase version 2.0 (U.S. Biochemical, Cleveland, OH). Sequencing of the majority of the first DNA strand was from the plasmid universal primer site, while the remaining gaps in the sequence of the first strand and the complementary strand were sequenced using a series of synthetic oligonucleotides. The individual sequence data sets were overlapped and analyzed with Microgenie and Mac Vector software. Transfection recombinant

of plasmid and isolation viruses

of

A culture of 3 X lo6 CV-1 cells was infected with virus at a m.o.i. of approximately 0.1 PFU per cell followed by transfection of plasmid DNA by lipofectin (Felgner et al., 1987). After 48 hr at 37”, virus was released into the culture supernatant from cellular debris by three freeze-thaw cycles and sonication. Putative TK- recombinant viruses were plaque-purified three times on HuTK-143 cells in the presence of BUdR (12.5 pg/ml) and a passage 1 stock was prepared in BSC-1 cells. Viral DNAs were analyzed by Southern blot analysis to determine the virus pedigree. RESULTS The host range of ectromelia lines

virus in tissue culture

The replication of ectromelia, cowpox, and vaccinia viruses in 12 cell lines from six animal species are shown in Table 1. Ectromelia virus failed to grow in one rabbit cell line (SIRC) and two hamster cell lines (CCL 14 and CHO), and it replicated poorly in the remaining hamster and rabbit cell lines (CCL 39, CCL 16, RK-13). In RK-13 cells, virus replication was delayed, with virtually all of the virus infectivity detected arising between 12 and 24 hr p.i. CPV replicated in all cell lines tested, although with varying efficiencies. The Copenhagen strain of vaccinia virus most often gave the highest levels of infectivity in the tested cell lines, but failed to replicate in CHO cells and replicated poorly in a second hamster cell line, CCL 14, as well as mouse L929 cells. The failure of ectromelia virus to replicate efficiently in four hamster cell lines was consistent with the lack of a functional CHO hr gene. To test this hy-

436

CHEN ET AL. TABLE 1 MULTIPLICATIONOF ECTROMELIAVIRUS, COWPOXVIRUS, AND VACCINIAVIRUS IN DIFFERENTCELL LINES Cell type

Species

Tissue

Virus yield (X 1 O5 PFU/culture) Cell line

Monkey

Kidney

BSC-1

Mouse

Connective

L929

Pig

Kidney

LLC-PKl

Rabbit

Kidney

RK-13

Cornea

SIRC

EM

CPV

96.0 * 39.0 (4+Y 36.6 t 6.4 (3+) 14.2 + 8.6 (3+) 3.0 t 2.0

48.0 f 11.2 (3f) 9.2 + 3.0

Pf) Human

Embryonic

lung

MRC-5

Epithelial-like

HeLa

Epidermis

HEp-2

Lung

CCL 39

Peritoneal

cells

CCL 14

Lung

CCL 16

Ovary

CHO

0.19 + 0.02 (0) 16.6 f 5.0 (3+) 17.4 2~ 3.6 (3f) 59.2 + 6.0 (4+) 0.68 f 0.3 (I+) 0.27 + 0.13 (0) 0.87 !I 0.72 (1 +I 0.01 rt 0.02 (0)

24 hr p.i. vv 86.0 IL 42.0 (4+) 3.67 2 1.3

Pf)

(2+)

5.4 AI 3.4 (2+) 19.0 + 3.4 (3+) 37.2 + 4.6 (3+) 22.6 k 2.8 (3+) 9.0 f 2.2

68.0 + 17.4 (4f) 128 + 25.4 (4f) 56.6 2 20.8 (4+) 62.6 f 6.4 (4+) 66.0 zk 47.4 (4+) 88.6 f 48.0 (4+) 14.0 t 0.0 (3+) 0.112 f 0.05 (0) 72.6 f 32.0 (4+) 0.14 rt 0.16 (0)

P+) 68.0 + 5.6 (4+) 7.8 + 3.2

(2+) 39.0 + 40.8 (3+) 10.4 t 10.4

P+) 7.8 z!z3.4

@+I

a Each value represents a mean of three samples. See Materials and Methods for experimental detail. A replication indexwas given between 0 and 4+, with 0 for values at 24 hr less than or equal to the maximum values observed at 2 hr p.i., which was 0.4 X 1 O5 PFWcuRure; 1 t forvalues between 0.41 X 1 O5and 2.1 X 1 05; 2t forvalues between 2.2 X 1O5and 11 .O X 1 05; 3t forvalues between 1 1.1 X 1O5and 55. X 1 05; and 4t for values greater than 56 X 1 05.

pothesis, several strains of ectromelia virus, as well as other species of orthopoxviruses, were examined by Southern blot analysis for the presence of DNA sequences homologous to the CHO hr gene. Southern blot analysis of DNA from various orthopoxviruses for sequences homologous to the CPV CHO hr gene DNA from the Moscow, Hampstead, and Mill Hill strains of ectromelia virus, rabbit pox strain Utrecht, and cowpox virus strain Brighton were digested with Hpal restriction endonuclease and examined by Southern blot analysis for the presence of sequences homologous to the CHO hr gene. Prior studies with CPV have shown a 2.3-kbp Hpal fragment to contain the 2004-bp ORF of the CHO hr gene (Spehner et al,, 1988). DNA from the Utrecht strain of rabbit pox yielded a fragment of similar size to that observed with CPV DNA (Fig. 1, lane 1 and lane 5), while DNA from the Copenhagen strain of vaccinia virus showed no such hybridization (result not shown). This latter obser-

vation confirmed the study of Goebel el a/. (1990), who noted the absence of DNA sequences homologous to the CHO hr gene in the genome of the Copenhagen strain of vaccinia virus. In contrast to Hpal-digested CPV DNA, similar digests of DNAfrom the three strains of ectromelia virus revealed the presence of a single fragment of about 1.8 kbp that hybridized strongly with a probe containing the CHO hr gene (Fig. 1, lanes 2-4). The smaller 1.8-kbp Hpal fragments observed with the DNA from the ectromelia virus strains suggested that an intact CHO hr gene may not be present in ectromelia virus strains. The genetic basis of the block in ectromelia replication in CHO cells

virus

Since the failure of ectromelia virus to replicate in CHO cells could be due to an inactive CHO hr gene and/or the lack of another gene(s), an ectromelia virus recombinant was constructed to differentiate between these possibilities. The CHO hr gene was transfected into ectromelia virus-infected cells, an ectromelia virus

ECTROMELIA

VIRUS LACKS FUNCTIONAL

2.3 1.8

FIG. 1. Southern Blot DNA analysis of vaccinia and ectromelia virus strains for sequences homologous to the CPV CHO hr gene. Primary CEF cells were infected for 48 hr at 37” with cowpox virus strain Brighton (lane l), ectromelia virus strains Mill Hill (EVMH, lane 2) Moscow (EVM; a distinct isolate from the Mos-3-P2 isolate used in all other experiments, lane 3) and Hampstead (EVH, lane 4) or rabbitpox virus strain Utrecht (RPV, lane 5). Total nucleic acid was isolated from each culture and a portion was digested with Hpal restriction endonuclease priorto Southern analysis using a hybridization probe containing the entire CHO gene (plV27).

TK minus isolate R41 was plaque purified, and the genotype was examined by Southern analysis (Fig. 2). HindIll-digested ectromelia virus DNA probed with the CHO hr gene showed the presence of one band with a size of 20.9 kbp (lane 1). Similarly treated DNAfrom the R41 isolate showed the presence of this band as well as a 6.8-kbp fragment (lane 2) which was the correct size for the HindIll fragment containing the ectromelia virus TK gene (4.5 kbp) interrupted by the CHO hr gene (on a 2.3-kbp fragment). As expected, probing with the vaccinia virus TK gene (a replicate blot) detected only the 6.8-kbp fragment in R41 DNA (lane 4). Thus, the CHO hr gene had been recombined into the TK gene of the ectromelia virus isolate R41. Recombinant virus R41 was examined for its ability to replicate in various cell lines restrictive for parental virus. As shown in Table 2, the TK minus ectromelia virus recombinant R41 containing the CHO hr gene replicated in CHO and other cell lines derived from hamsters or rabbits to levels similar to those of CPV. This observation demonstrated that except for the CHO hr homologue, all genes required for replication in the nonpermissive cell lines are functional in ectromelia virus. To further study the ectromelia virus CHO hr homologue, it was molecularly cloned and sequenced. Molecular cloning of the ectomelia virus DNA sequences homologous to the CHO gene An ectromelia virus DNA fragment containing the CHO hr homologue was subcloned from a X library into

437

CHO HR GENE

a Bluescript II plasmid, and a recombinant was isolated and designated pL28-5 (Fig. 3A). The pedigree of pL28-5 was confirmed by Southern blot analysis of Hpal- and Hincll-digested pL28-5 and ectromelia virus DNAs (data not shown). As predicted from the results in Fig. 1, Hpal-digested DNAfrom the second isolate of the Moscow strain of ectromelia virus (Mos-3-P2) and pL28-5 gave a 1.8-kbp fragment, which hybridized to an oligonucleotide probe complementary to nucleotides 1492 to 1527 of the CHO hr gene. Also, Hincll digestion of either pL28-5 or the ectromelia virion DNA revealed the presence of an identical 1 .I-kb fragment which was 500 bp smaller than the corresponding fragment predicted by the DNA sequence analysis of the CHO hr gene. A restriction enzyme map of the ectromelia virus CHO homologue is presented in Fig. 3B. Nucleotide homologue

sequence

of the ectromelia

virus CHO hr

The ectromelia virus CHO hr DNA sequence (Fig. 4) showed strong DNA sequence homology with the CPV CHO hr gene, including the conservation of the early promoter critical domain (nucleotide 98, CAAAAATGATATTTA; underline; Davidson and Moss, 1989) the ATG initiation codon (nucleotide 175; underline), and the early transcriptional (TlTlTNT; nucleotide 1673; underline; Yuen and Moss, 1987) and translation (TAA; nucleotide 1666; underline) termination signal sequences (Spehner et al., 1988). Upstream of the CHO hr homologue, there is an early transcription termination signal (nucleotide 51; underline) and translation stop (nucleotide 37; TAA; underline) for an ORF that displays a very strong homology to a 5K ORF of vaccinia virus strain WR (Kotwal and Moss, 1988). Relative to the CPV CHO hr gene sequence, the ectromelia virus CHO hr homologue sequence had 12 deletions, 4 insertions, and 72 substitutions over the

FIG. 2. Genomrc structure of the TK minus ectromelia virus isolate. DNAs from cells infected with wild-type ectromelia virus (lanes 1 and 3) and ectromelia virus R41 (lanes 2 and 4) were digested with Hindill restriction endonuclease and analyzed by agarose gel electrophoresis followed by Southern analysis using a %labeled probe containing CHO hr genes (lanes 1 and 2) or the vaccinia virus HindIll J fragment (lanes 3 and 4).

438

CHEN ET AL. TABLE 2 MULTIPLICATIONOF ECTROMELIAVIRUS CHO HR RECOMBINANTIN VARIOUSTISSUECULTURECELL LINES Virus yield (X 1 O5 PFU/culture) Ectromelia BSC-1 RK-13 SIRC CCL 39 CCL 14 CCL 16 CHO

33 13.4 0.46 1.2 0.04 2.4 0.066

WT

k 7.6 rf: 2.2 t 0.1 + 0.9 t 0.03 + 0.5 k 0.09

a Three samples per value, and replication

Ectromelia

(3+) (3+) (1+) (1+) (0) (2t) (0)

31.4 51.4 32.3 9.4 11.4 45.2 12.8

8.0 23.2 6.4 3.0 3.2 22.4 2.54

R41

Cowpox virus

(3+) (3+) (3+) (2t) (3t) (3+) (3t)

21.4 44.6 98.6 8.2 20.8 14.2 7.6

f f t t + + 5

7.2 11.4 17.4 3.4 7.4 2.8 1.8

(3+) (3+) (4+) (2+) (3+) (3t) (2+)

indices per legend to Table 1.

ectromelia virus sequence (nucleotides 282 to 787 of the CPV CHO hr gene). It is of interest to note that this deletion in the ectromelia virus sequence is bounded in

course of the 1822-nucleotide sequence. By far, the major structural difference between the two DNA sequences was the absence of 506 nucleotides in the A C

+ + + t * + *

48 hr p.i.a

NM

11IKI F i

E

PO 1\1I I G ClJl H 1 D

(

A

1

B

vv

0

A

I~II

c u

recombinant

lJtKEM IOKbp

L28

I-

I

I Kbp

I/

Sal/

Hpa I

Him

/I

Hpa I

tipal

I

Sal I

I Kbp

FIG. 3. Molecular cloning of ectromelia virus DNA sequences homologous to the CHO hr gene. AX library of the ectromelia virus genome was screened with a CHO hr probe (plasmid plV27), and a X recombinant designated L28 was isolated. A 4.2.kbp DNAfragment generated by SalI restriction endonuclease digestion of L28 was shown to contain the entire DNA sequence homologous to the CHO hr gene, and this was subcloned into Bluescript II (viral sequences are stipuled; see Methods and Materials for details). The resulting plasmid recombinant, pL28-5, was used to fine map the ectromelia virus sequences (B). The vaccinia and ectromelia HindIll maps were from Earl and Moss (1990) and from Esposito and Knight (1985), respectively.

ECTROMELIA 1 1 61

VIRUS LACKS FUNCTIONAL

439

CHO HR GENE

GTTAACGGAGGTGTAAACTCTGG*GTTMTAAAATTTAAAcAcTAAATTATTTTTTATT* IIIIlIIIIIIIIIIII IIIIII I IIIIIIIIIIIIIIIIIIIII lIIIIlIII GTTAACGGAGGTGTAAAATCTGGMcTGGTAAAATTT~cAcTm**A TTTTTATTA 1195 TATACTRATTGAGTATATAACATTCAGCGATATCGATATCTCTCTAGTGG~TACATGTT IIllIIIIIIIIIIIlIIIIIIlIIIIIIIIIIIIlIIIIIIIIII

ATAATTGTACAAG

TTTTTG*TCTGGT*TAAATAcATTcAMAATG*TAATl"rAATG*c*

IIIIIIIIIIIII

IIIIIII

IIIIIIIIIIIIIIIlIIIIIIIIII

60

ATAATTGTACMGTTTTTTGACCTGGTATAAATACATTCAMAATGAT

120

TTAGTTGTGCGGGTGTATAGAGTTCACAGTAGCTCATTCACTTCTATTCAGT~TGT

IIIIIIIII

IIIIIIIIIIIIIIII

. .. .. . ..

llllllIIIII ATTTAATGACA . ..

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

TATACTAATTGAGTATATAACATTCAGCGATATCGATATCGATATCTCTCTATTGG~TGCATGTT

1255

GGAATATGGAGCTGTGGTAAATAAAGAGGCTATTCACGGAGGCTATTCACGGATACTTT~TATT~TAT

749

GGAATATGGAGCTGTTGTAT-GAGGCTATACACGGATACTTTAG-TGTT~TAT

IIIllIIIIIIIIII

IIIIIIIlIIlIIIIII

IIIIIlIIlIIII

119

TTAGTTGTGTGGGTGTATAGAGTTCATAGTAGCTCATAGTAGCTCATTCACTTCTATTCAGTC-~T

1315

TGATTCTTACACGATGAAATATCTACTAAAAAA

180

TTGATTATCTGG-TGAGGAGGTGGCTCTCGATGAACT

809

TGATTCTTACACGATGAAATATCTACTAAAAAA

1375

CGATGATGGAGAGATCCCGATTGGACACCTATGT-TCC~CTATGGACGTTATMTTT

868

CAATGATGGAGAGATCCCGATTGGACACTTATGTAAATCCTTT

III1

IIlIIIIIIIIIIIIIIIIIII

IIIIIII

179

TTGACTATCTGG-TGAGGAGGTGGTTCTCGATAAACT

240

GAGATCCTAATGATACCAGGAACCAATTCAAGAATAATGC

239

300

360

420

480

540

600

IIIIIIIIIIIlIIIIII

Ill

I

I IIIIllIIIIIIIIIIIIIIIIIIIIIIIIIIIlIIIIII

GGGATCCTAATGATACCAGGAACCAATTCAAGAATAATGCTC

780

Ill1

IIIIIII

lllllllrlllllllllllllllllllllllll~~~~~~~~~~~~~~~~~~~ GGAA

I IIIIIlIIlliIIlIIIIllIlIIII

GGGGAGATGCTGTCMTCCTCT

IlIIIIIIIIIIIIIIIIII

lIIIIIIIII

1435

CTACACTGATACATACAGACAGGGTTTTCGTGATATGTCTTATGCTTGCCC~TTCTTAG

928

CTACACTAATGCATACAGARAGGGTCTTCGCGACAGGTCGTATGATTGACCMTTCTTAG

IIIIIII

II IIlIIIII

IIIII

IIII

II I III

IIII

III

IIIIIIIIIII

1495

TACTATAAACTTTTGCCTACCTTATCTTAAAGACATTAACATTMCATGATTGAC-CGAGGAGA

988

TACTATAAACATTTGCCTACCTTATCTTAAAGACATTAACATT~CATGATTGAC-CGAGGAGA

1555

MCACTTCTTCACMGGCTGTTAGATATMT-C~TCTCTAGTGTCTTTACTGCTAGA

1048

AACACTTCTTCACAAGGCTGTTAGATATAATAAACAATC

IlIIIIIIII

IIIIIlIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlllII

TACACAAAAATTGGAGACAGCTTACTCCATTAGGGGMTACAC-TAGTAGACATGGTA

IIIIlIIIIIIIIIIlIIIIIIIIIIIIIIIIIIIIIII

III

IIIIIIIIIIIIII

AGTATCTTTACTGCTAGA

AAGTTAATAAGGATATAGCGATGGTTCTACTAGAAGCTACTG 1615

ATCCGGTTCAGATGTCAACATTAGATCAAATAACGGATATATACATGTATAGCCATTGC~T

1105

ATCCGGTTCAGATGTCAACATTAGATCAAATAACGGATATATACATGTATAGCCATTGCCAT

IIIIIIIIIIIIIIIIlIIIIIIIIIIIIIIIllIIIIIIlIIIIIIIIIlIIIIII

II

ACTTTAATATATTCACCTATATGAAATCCAAAAATGTAGATATTGACTTGAT-GGTAT 1675

CAACGAATCTAGAAACATTGAACTGCTGCTGCTATTATAGA

1165

CAACGAATCTAGAAACATTGAACTGCTG-TGCTATTATGTC

1735

TTGTGTGATTGATTCATTGAGAGARATATCTATCTMCATAGTAGATMTGCCTATGCTAT-

1223

TTGTGTGATTGATTCATTGAGTGARATATCTATCGTA

1795

ACAATGTATTAGATATGCCATGATTATAGATGACTGTATATCGTCT~GATTCCAGAGTC

1283

ACAATGTAT

1855

CATAAGTAAACACTATAATGATTATATAGATATTTGCAAT

1339

CATAAGTCAACGCTATAATGATTATATAGATCTCTGCAATGATG

1915

AAAAATAATAGTGGGAGGCACACTATGTTCTCATTAATA

1398

AAAAATAATGGTAGGAGGCACACTATGTTCTCATTAATA

1975

AATTATTCATCGGTATGCCAATAATCCAGRATTACGTGCGTGCGTATTATGAGTC-C-

1458

AATTATTTATCGATATGCCAT~TCCAGAATTACGTGAG

IIIIIIIIIIIIIIIlIlIIIIIIIlIIII

IIIIIlIIIlIII

IIIIIIIIIIIIII

AAACCTACATTAGA

TGGTAGAACATGGATTTGATTTTAGTGTTAAATGCGAAAA

ATTATGTAATGACAGATGATCCTGTTCCTGAAATTATTATTGATTTATTCATAG-TGGAT

CAC-TGACGATTATCAACCACGAAATTGCGGTACAGTACAGTATTACATCTGTATATCATCT

CTCATCTGTATTCAGAGTCGGATTCGAGATCATGTGTGAATGTC

III/lIIIIIIIIIIIIII/IIIIIIIIII

IIIIlIIIIIIIIIIIIIII

TATTCAGAGTCGGATTCGAGATCATGTGTG

281 840

IIIIII

ATGAGCACTGTAATAATGTTGAGGTTGTCAAACTACTACTACTAGACAGTGGTACTMTCCAT

660

720

IIIIII

689

CCCGGAAGTTGTTAAATGTC

TGATTMTCATGGMTCMCCCATCTTCTATAGATAAAAA

IlIIIIIIIIIIIIlIIIIIIIIIIIIIIl

IlIIIIIIIIIIIIIIIIIIIIIIIIIII

331

TGATTMTCATGGMTCMCCCATCTTCTAGAGATAAAAAT

900

ATTATATTAAGTCATCTCATATAGATATAGACATCGTTAAG

391

ATTATATTMGTCATCTCATATAGATATAGACATCGTTAATAG

960

ATAACACGGCTTATTC

451

ATMCACTGCTTATGCATATATATATAGACGATCTMCATGCTGCA~CGAGGMTCATG

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIII

IIIIII

IIIIIIIIIIIIIIIIll

ATATATAGACGATCTAACATGTTGTACTCGAGGAATTATG

I

IIIIlIIIIIIIIIIIIIIII

1016

GCTGATTATCTAAATAGTGATTATAGATACAATAAAGATG

511

GCCGATTATCTAAATAGTGATTATAGATACMT-GATGTAGATTTAGA~TGGTC-

II IIIIIIIIIII

III

II IIIIIIIIlIlIIIIIIIIIIIIIIIIIIIIIIIII!!IIIIIIIIIIIIIIIIIII

1076

TTGTTTTTGGAGMTGGAAAACCGCACGGMTAATGTGTA

GTATTGTACCACTATGGAG

571

TTGTTTTTGGAAAATGG-GCCGCACGGAATAATGTGTTAGGTATTGTACCACTATGGAG

IlIIIIIIIII

IIIIIIIIIIIIIIIIIII

1135

AAATGATAAGGAAACCATCTCTTTGATATTG-CMTGMCTCGGATGTCCTCCMCA

631

AAAGGATMGGMACCATCTCTTTGATATTGAAAACMTGAA

III

IlIIIIII

IIIIIlIIIIIIIIIIIII

IIIIIIIIIlIIIIlIIIIIIIlIIIIIIIIIIIIIII

IIIIIIIIIIIIIIII

CGGATGTCCTCCAACA

/IIIIIIIIIl/IIII/IIII IIlIIIIII III/III

IIIIIIIIIII

II IIIIIIIIIIIIIIIIIIIIIII

IIIIIlIIIIIIIIIIIIIIIIIII

IIIlIIIIIIIIIIIIIIIIl

TATGCCATGATTATAGATGACTGTACATCGTCTAAGATTCCAGAGTC

III

IIIllIIII

IIIIIIIlIIIIIIIIIII

I IIIIIIIIlIIIIIIII

IIIIII

II IlIIIIlIlIIIIIIIIIIIIIIIIIIIIlIIIII

IlIIIlI

IIII

IIIIIIIIIll

IlIIIIIIIIIIIIIIIIIIIIIII

IIIIIIIIIIIIIIlIIIIII

2035

T-TATATACGTGGAAGTATATGATATTATTTCCAAAT

1518

TAM

2095

MTTCATAAA

1575

AATTCAT-CCATCATAGMTCAGTTGATGATMTACCTACATTTCTMTCTTCCGTA

2152

TACCATCAAATACAAAATATTCGAGCAACAATAAGTATTTTTTATACCTTT-TTGAT

1635

TACCATC-TACAAAATATTCGAGCMCMT~GTATTTTTTATGCCTTT-TTGAT

2212

AAATAAATTTTTTCTAGTGATATTTTGGCMGATGAGMTCCTATTTCTCATCGCTTTCA

1695

AAATAAATTTTTTCTAGTGATATTTTGGCAAGATGATGAG~TCCTATTTCTCATCGCTTTCA

2272

TGTATGGGTGTGTTCACTC

1755

TGTATGGGTGTGTTCACTCATATGTTAACGCGCGGTTG-CC-TGTCC~TCTAGACA

1815

TTGTAACT

IIII

IIIIIIIIIIIII

IlllIIIIIIIIIIII

ATATACGTGGAAGCATATGATATTATTTCC

IIIIIllIII

I

A

IIII

/IIIIIlII/IIIIIII

GATGCAATAGTGAAGCATAATM

AACATAGAATCAGTTGATGACAATACCTACATTTCTAATTTGCCTTA

I IIIIIIIlIlIIIIIIII

IIIIIIIIIlIIIIIIII

IlIIIllIIIIIIIIlIIIIIIllIIIIIlIIIIIIIlllllIll

I II II IlIIIIIIlIIIII

IIIIIIllIIIIIIIIIIIIIIIIIlIIIIIIIIIIIIIIIIIIIlllIIIIIIIIlIII IIIIIIllIIIIIIIIIII

FIG. 4. Nucleotide sequence of the ectromelia virus CHO hr homologue. The nucleotide sequence of the ectromelia virus CHO hr homologue (bottom) compared to the CHO hr gene (top). Underlines: early promoter critical domain CAAAAATGATATTTA (consensus sequence; squares denote identity; Davidson and Moss, 1989); early transcription termination signal TTTTTNT; translation initiator, ATG and terminator code, TfA; overlines, pentamers.

the CPV sequence on one side by CTCTA (nucleotide 279; overline) and on the other by CTGTA (nucleotide 785; overline). The presence of this nearly perfect pentamer repeat surrounding the deletion may indicate that the sequences were lost as a result of homologous recombination or a slippage mechanism during DNA replication (Smith eta/., 1991). As a consequence

of this 506-bp deletion, the protein sequence (compared to CPV CHO gene) changes after amino acid 36 (Leu) and, after an additional 16 amino acids, encounters a stop condon. A further four ORFs (without regard for an initiation methionine) of greater than 30 amino acids were observed in the original ORF. Figure 5 compares the CPV CHO hr gene with ORFs

440

CHEN ET AL.

CPV

deletion

FIG. 5. Alignment of the CPV CHO gene with homologous sequences from vaccinia and ectromelia viruses. The CPV CHO hr gene was aligned with ORFs greater than 30 and 60 amino acids from ectromelia virus and vaccinia virus DNA sequences, respectively.

from ectromelia virus and vaccinia virus strain WR (nucleotide 37 11 to 1483; published by Kotwal and Moss, 1988) but without regard for an amino terminal methionine. Like the ectromelia virus DNA sequence, the vaccinia virus strain WR sequence was fragmented into smaller ORFs. DISCUSSION Cowpox virus has a very broad natural host range, and virus has been isolated from humans, rodents, cattle, and cats (Fenner eta/., 1989). Vaccinia virus has an equally broad experimental host range but, as a rule, is not maintained in nature (for exceptions, see Fenner et a/., 1989). Variola virus’ natural host was man, although certain nonhuman primates were susceptible to experimental infection. The natural host of ectromelia virus is thought to be only the mouse (Fenner, 1982; Buller and Palumbo, 1991) and there is considerable variation in the disease patterns observed among the various genera, species, and strains of mice (Schell, 1960; Buller et a/., 1986; Wallace et al., 1985; Brownstein, 1989). It is reasonable to assume that a basis for the very narrow ectromelia virus species tropism is the efficiency of virus replication in the host cells, which is affected probably by a series of cell-specific virus gene functions, three of which (C7L, Perkus et al., 1990; KIL, Drillien et a/., 1981; Gillard et a/., 1985, 1986; CHO hr gene, Spehner et al,, 1988) have been identified previously in other orthopoxviruses and recent data indicate that there maybe a fourth gene (Vero hr gene; F. Takahashi-Nishimaki et al., 1991). The mechanism of action for each of these genes has not been determined, but a study by Perkus and her colleagues (1990) has shown that, depending on the host cell, some of the genes (C7L, Kl L, and CHO hr) are functionally interchangeable. Thus, the block in

replication of a vaccinia virus mutant lacking Kl L and C7L can be overcome by Kl L, CHO hr, or C7L in a pig kidney cell line, LLC-PKl, or in a human embryonic fibroblast line, MRC-5. Likewise, Kl L and CHO hr are functionally equivalent in a rabbit kidney cell line, RK13. Although the CHO hr gene is required for multiplication on CHO cells, it is not necessary for orthopoxvirus replication in all cell lines derived from the Chinese hamster as the Copenhagen strain of vaccinia virus grew well in one lung cell line (CCL 16) and moderately well in a second lung line (CCL 39), but very poorly in the peritoneal line (CCL 14) and the ovary line (CHO). This confirms the early observation made by Drillien et al. (1978) and suggests that genetic elements other than the CHO hr are important for virus replication, at least in cell lines derived from the lung of the Chinese hamster. The relatively poor replication of cowpox virus compared to ectromelia virus in a mouse cell line L929 suggests that another host range gene (lacking in CPV) may be important for poxvirus replication in the mouse. Ectromelia virus replicated well in cell lines derived from monkey, mouse, pig, and human, but replicated poorly in rabbit cell lines (SIRC and RK13) and hamster cell lines (CCL 14, CCL 16, CCL 39, and CHO). From the work of Sphener et a/. (1988) and Perkus et al. (1990) the failure of ectromelia virus to replicate in RK13 and CHO cells suggested that the Kl Land CHO hr homologues, respectively, were not fully functional. The presence in ectromelia virus of an inactive CHO hr gene was indicated by three pieces of evidence. First, Southern analysis of Hpal-digested DNA from three strains of ectromelia virus detected a 1.8-kbp fragment instead of the 2.3-kbp fragment hybridizing with a CHO hr gene probe. This also argued that a deleted CHO hr gene was probably a property of all ectromelia strains. Second, DNA sequence analysis of molecularly cloned DNA from the Moscow strain of ectromelia virus revealed a 506-bp deletion as well as additional mutations in the ectromelia virus CHO homologue which would result in a premature termination of the expressed protein. Third, the recombination of the CHO hr gene into the ectromelia virus TK gene overcame the block for virus replication in cell lines derived from hamsters and rabbits. The nonfunctional CHO hr gene could explain, in part, the narrow host range of ectromelia virus in nature compared to CPV. Vaccinia virus also lacks a functional CHO hr gene and has not been able to establish itself in nature, although it has a broad experimental host range. The CHO hr gene is not the first gene described for orthopoxviruses that is functional in some species or strains and apparently not in others. The 38-kDa antiinflammatory gene (SERPIN-like) is present in CPV and

ECTROMELIA

VIRUS LACKS FUNCTIONAL

the WR strain of vaccinia virus, but not in the Copenhagen strain of vaccinia virus (Pickup et al,, 1984, 1986; Palumbo et al., 1989; Goebel, 1990). Homologues of the Copenhagen strain Kl L hr gene in variola major strain Harvey, variola minor strains Butler and Garcia, and monkeypox strain Denmark were found by DNA sequence analysis to have stop codons at position 67 of the putative 286 amino acid protein, which would possibly result in the production of a truncated, inactive version of the SO-kDa Kl L gene product (Cowley and Greenaway, 1990). An ORF for a protein with strong amino acid homology with superoxide dismutase is present in the Copenhagen and WR strains of vaccinia virus (Goebel et al., 1990; Blasco et al., 199 l), but appears to lack a domain critical for function (Smith et al., 1991). It may be that this gene is functional in other poxviruses. We speculate ttiat the ancestral poxvirus possessed a fully active repertoire of these types of genes and, during the evolution of the virus with its hosts, additional genetic information was gained and lost until a genetic equilibrium was attained that permitted virus replication, spread, and transmission within a susceptible host population without causing unduly high levels of mortality (Buller and Palumbo, 1991). This hypothesis may be especially pertinent to the poxvirus host range genes. ACKNOWLEDGMENTS We thank Mr. C. Duarte and Ms. B. R. Marshall for technical and editorial expertise, respectively; Dr. P. Earl for assistance in computer manipulation of DNA sequence information; Dr. M. Merchlinsky for pulse-field analysis of ectromelia virus DNA; and Drs. B. Moss, P. Earl, G. Karupiah, J. Baldick, and G. Palumbo for helpful discussions.

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viral protein that is related to plasma protein inhibitors of serine proteases. Proc. Natl. Acad. Sci. USA 83, 7698-7702. PICKUP, D. J., INK, 6. S., PARSONS, B. L., Hu, W., and JOKLIK, W. K. (1984). Spontaneous deletions and duplications of sequences in the genome of cowpoxvirus. Proc. Nat/. Acad. Sci. USA 81,68176821. SAMBROOK, J., FRITSCH, E. F., and MANIAIS, T. (1989). “Molecular Cloning,” 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. SANGER, F., COULSON, A. R., BARRELL, B. G., SMITH, A. J., and ROE, B. A. (1980). Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. 1. Mol. Ho/. 143, 161-178. SCHELL, K. (1960). Studies on the innate resistance of mice to infection with mousepox. II. Route of inoculation and resistance, and some observations on the inheritance of resistance. Aust, /. hp. Biol. Med. Sci. 38, 289-300.

SMITH, G. L., CHAN, Y. S., and HOWARD, S. T. (1991). Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat. J. Gen. Viral. 72, 1349-l 376. SPEHNER,D., GILLARD, S., DRILLIEN, R., and KIRN, A. (1988). A cowpox virus gene required for multiplication in Chinese hamster ovary cells. /. Viral. 62, 1297-l 304. TAKAHASHI-NISHIMAKI, F., FUNAHASHI, S.-I., MIKI, K., HASHIZUME, S., and SUGIMOTO, M. (1991). Regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins. virology 181, 158-l 64. WALLACE, G. D., BULLER, R. M. L., and MORSE, H. C., Ill (1985). Genetic determinants of resistance to ectromelia (mousepox) virusinduced mortality. J. Viral. 55, 890-891, YUEN, L., and Moss, B. (1987). Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes. Proc. Natl. Acad. Sci. USA 84, 6417-6421.