Virus Reseurch, 24 (1992) 187-196 0 1992 Elsevier Science Publishers

VIRUS

187 B.V. All rights

reserved

0168-1702/92/$05.00

00785

Mapping of the RD phenotype of the Nancy strain of coxsackievirus B3 A. Michael Lindberg ‘, Richard L. Crowell b, Roland Zell ‘, Reinhard Kandolf ’ and Ulf Pettersson a a Department of Medical Genetics, Uppsala CJniL,ersity,Biomedical Center, Uppsala, Sweden, ’ Department of Microbiology and Immunology, Hahnemann Uninicersity School of Medicine, Philadelphia, PA 19102-1192, USA and ’ Department of Virus Research, Max Planck Institute for Biochemistry, Martinsried, Germany (Received

6 December

1991; revision

received

and accepted

18 February

1992)

Summary

The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the Pl region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the Pl region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the Pl region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The Pl region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the Pl region. Furthermore, a chimera which only contained the 5’ part of the Pl region derived from CB3NRD and the remaining Pl sequence from CB3N was able to

Correspondence tot A.M. Lindberg. Present College of Kalmar, Box 905, S-391 29 Kalmar,

address: Sweden.

Department

of Natural

Sciences,

University

AbbreL,iations: CB, coxsackievirus group B; CB3. coxsackievirus group B type 3; CB3N, coxsackievirus B3 strain Nancy; CB3NRD, RD variant of coxsackievirus B3 strain Nancy; PV, poliovirus; HRV, human rhinovirus; nt, nucleotide; aa, amino acid; nc, noncoding.

replicate in RD cells, suggesting that determinant for the RD phenotype. Host-range mutant; Virus sequence; Chimera virus

capsid

the VP2 polypeptide

protein

VP2;

Host

cell

contains

at least one

receptor;

Nucleotide

Introduction Coxsackievirus group B (CB) with its six serotypes (CBl-6) is a picornavirus belonging to the genus Enterovirus. They are small, icosahedral, positive-stranded RNA viruses that cause enteric infections in humans (reviewed in Melnick, 1990). The RNA genome is about 7400 nucleotides (nt) in length and has a poly(A) tail at its 3’ end. The first 750 nt comprise the 5’ noncoding region (5’nc) and contain signals that are important for viral replication and initiation of translation (Melnick, 1990; Rueckert, 1990). The major portion of the CB genome encodes a long open translational reading frame which terminates about 100 nt before the poly(A) tail. The 3’ noncoding region (3’nc) contains signals that are important for replication of the viral genome (Rueckert, 1990). Several serotypes and variants of CB have been sequenced completely or partially (Iizuka et al., 1987; Jenkins et al.. 1987: Klump et al., 1990; Lindberg et al., 1987; Tracy et al., 1985). Comparisons at the nucleotide and amino acid sequence levels have revealed a close relationship between different serotypes of CB and also to other enteroviruses such as coxsackievirus group A, echovirus, poliovirus (PV) and even to the human rhinovirus type 14 (HRV14). An interesting host range variant of coxsackievirus group B type 3 strain Nancy (CB3NRD) was isolated by Reagan et al. (1984). This variant has acquired the capacity to replicate in the human rhabdomyosarcoma cell line, RD, as well as in Buffalo green monkey kidney (BGM) cells and HeLa cells while the parental strain Nancy of CB3 (CB3N) is able to replicate only in the two latter cell types. CB3NRD also, unlike the parental strain, is able to agglutinate human erythrocytes (Reagan et at., 1984). Saturation experiments have demonstrated that the CB3NRD variant most likely has acquired the ability to utilize a second cellular receptor (Reagan et al., 1984; Crowell et al.. 1986; Hsu et al., 1990). In the present communication we report the complete nucleotide sequence of the region of the genome of CB3NRD which encodes all the capsid proteins, and show that mutations which are located in the gene for capsid polypeptide VP2 appear to be of importance for the RD phenotype of CB3NRD.

Materials Propagation

and Methods and cloning of CBSNRLI

The characterization of CB3NRD is described elsewhere (Crowell et al., 1986; of virus, monolayer cultures Reagan et al., 1984; Hsu et al., 1990). For propagation

189

CB3N genome

550

VP2

,VP4,

I

I

I I

VP3

I I

3500

VP1

I

PCR products

(pCB3RD-31)

(pCB3RD-19)

cBo+

CB-2

(pCB3RD-10) m-1

CBS-2 proCB3+

cB-4

CB-3

(pCB3RD-44)

cB-ll

(CB3RD-53) CBCO

CB-12

Fig. 1. A schematic picture of the Pl region in the CB3 genome. PCR products which were cloned are indicated in parentheses. The oligonucleotides used in each PCR amplification (Table 1) are also indicated.

of RD cells were infected with CB3NRD. After incubation at 37°C for 9 h the cells were harvested and washed in phosphate buffered saline and freeze-thawed three times. The lysates were cleared by centrifugation and supernatants containing CB3NRD virions were purified as descibed by Iizuka et al. (1987). Single stranded cDNA was prepared using oligo(dT) as a primer. Regions of the cDNAs were amplified using polymerase chain replication (PCR) (Saiki et al., 1988). Approximately 10% of the PCR products were analyzed by gel electrophoresis. The remaining parts of the PCR products were purified by low-melting agarose gel electrophoresis and the PCR fragments were excised and mixed with an equal volume of TE buffer and incubated at 65°C for 5 min. The mixtures were then phenol extracted twice. After ethanol precipitation and washing, the ends of the PCR fragments were converted to blunt ends by a fill-in reaction using the Klenow enzyme. The PCR product obtained using the oligonucleotides CB-4 (contains a Sac1 restriction cleavage site) and CB-3 (contains a NcoI restriction cleavage site) was then cleaved with Sac1 and NcoI and ligated into a SacI/NcoI cleaved pGem-5Zf( + I plasmid vector thus obtaining the clone pCB3RD-19 (Fig. 1). The remaining clones were obtained by ligating the PCR products into SmaI cleaved pGem3 plasmid vector (Fig. 1). All constructs were then transformed into E. coli strain DH5a. Clones thus obtained cover the entire Pl region of CB3NRD. DNA obtained by several clones for each construct were pooled and compared by dideoxy sequence analysis @anger et al., 1977) to the one selected clone indicated in Fig. 1. No sequence heterogeneity was observed. Primers used in the PCR and other oligonucleotides derived from the cDNA sequence of CB3N (Lindberg et al., 1987) were used in the dideoxy sequencing reactions.

The nucleotide sequences vereux et al., 19841.

were analyzed using the ‘UWGCG’ software (De-

AR infectious full-length cDNA clone, pCB3-M2, was used for all constructions. pCB3-M2 was derived from the full-length cDNA clone pCB3-Ml (Kandolf and Hofschneider, 1985, 19891. Compared to pCB3-Ml pCB3-M2 has, in addition, an SV40 promoter/enhancer element inserted 5’ of the cDNA. The PI region of pCB3-M2 was removed by cleavage at the unique XmaI and PflMI cleavage sites. Two clones, designated pCB3NRD19 (5’ part of Pll and pCB3NRD-10 (3’ part of Pl), were chosen as the sources of fragments from CB3NRD (Fig. I). For the construction of the CB3-M2/PlRD clone the XmaI/NcoI fragment of pCB3NRD-19 was inserted into the pCB3-M2 clone together with the NcoI/PfMI fragment of pCB3NRD-10. In the CB3-M2/PlN virus the XmaI/P~MI fragment of the pCB366 clone (Lindberg et al., 1987) replaced the corresponding fragment in pCB3-M2. For construction of CB3M2/P15’RD3’N, the XmaI/Z?g~II fragment in CB3-M2/PlN was replaced by the corresponding fragment from clone pCB3NRD-19. The structures of all chimeric viruses were verified by nucleotide sequencing. T~ansfectjo~ of HeLa ceils The recombinants were transfected into HeLa cells by the calcium phosphate method (Graham and van der Eb, 19731. Briefly, HeLa cell monolayers were grown in Dulbecco minimal essential medium using 2.5% fetal calf serum and, 2.5% newborn calf serum. 10 pg of the appropriate pIasmids were added to the cells in a calcium phosphate precipitate. The cells were then incubated at 37°C overnight, treated with 15% glycerol, and incubated further until a cytopathic effect (CPE) was observed. The supernatant was then transferred to fresh HeLa or BGM cells, which were incubated until CPE was observed. Supernatants from these cultures were then harvested and assayed for growth on BGM and RD ceils by visualization of CPE or using the pIaque assay described by Reagan et al. (1984). CPE was scored after 1-4 days of cultivation. Viruses having an RD phenotype yielded plaques with similar titers on BGM and RD cells in contrast to viruses with an RD-negative phenotype which exhibited at least a lo”-fold lower plaquing efficiency on RD cells as compared to BGM cells (Reagan et al., 1984).

Results Sequence analysis of the PI region of the RD rjariant of CB3 strain Nancy Since the RD phenotype of CB3N is known to involve receptor interactions (Hsu et al., 19901 we assumed that the critical mutations are located in the Pl

191

TABLE

1

Oligonucleotides derived the PCR reactions

from the cDNA

Description

Nucleotide

CBI CB2 CB3 CB4 CBS-2 CBll CB12 proCB3 + CBCO CBO +

5'-ACTGCCCCTGATTGTTGCC-3'

sequence

of CB3 strain

Nancy (Lindherg

et al., 1987) used in

Strand

sequence

origin

_ +

5'-TGTGGTTCGGCCATGGCTACT-3'

_

5'-CCAGTAGCCATGGCCGAACCACA-3' 5'-AAAATGGGAGCTCAAGTATCAACGCAAAAGACTGGGGCACA-3'

+

5'-CTACATGTGGCAACGTCTGGTTGGGTCGGTTGGTCCTCTGCTGT-3'

~

5'-CATTGGTCAGGCAGCATAAAGCTTA-3'

+

5'-CAGAGAAGTCATTGCATGCTGA-3'

_

5'-AGCTAGAATTCTTTACC-3'

+

5'-GTTGTGCACTGACA-3'

_

5'-ATTCCTATACTGGCTGCTTA-3'

+

region of the CB3 genome since this region encodes all the capsid polypeptides. The nucleotide sequence of the Pl region of the CB3NRD genome was therefore determined from cloned cDNA copies, obtained by PCR amplification using the primers depicted in Table 1. Twelve sequence differences were revealed when the Pl regions of CB3N and CB3NRD were compared (Table 2) and together they resulted in six amino acid substitutions, two of which were located in each of VPl, VP2, and VP3 (Table 2 and Fig. 2). To determine which of these were of importance for the RD phenotype, chimeric viruses were constructed using an infectious cDNA clone of CB3.

TABLE

2

A comparison between and amino acid level Gene

Position

VP2

1270 1271 1399 1400 2273 2287 2377 2436 2.518 2567 2688 2879

VP3

VP1

The positions

the Pl regions

(nt)

are numbered

of CB3N (Lindberg

et al., 1987) and CB3NRD

CB3N

according

at the nucleotide

CB3NRD

nt

aa

nt

aa change

A T C G T G T G A G G T

D D T T V C V E N T E Y

T A G C C A C C C A A C

V V s s _

to the cDNA

sequence

of CB3N (Lindberg

Y _ Q T _ K _ et al., 1987)

192

VP4

VP2

I pCB3-MZ/Pl

Tl e “1

c

E h ”

I

I

I I

I

I

I

v

s;

Y ”

Q

h ”

I I

T

K

h

e

I I I

I

I

I

I

I

I

I

I

I

’

v

1 h

sl

c

E

I

hl v

n ”

P. ”

I

‘tt

Bgl II

RD phenotype

I

I

I

t’

N h

I

I

h

Xmal

I

I

I

RD

pCB3#2/P15’RD3’N

E h ”

”

VP1

I

I

l pCB3-M2/Pl

D

N

VP3

I I

N n

E

I

f-.

I

I

Nco I

t PflM

l

I

Fig. 2. A schematic drawing which shows the amino acid sequence differences that were observed when the PI regions from CB3N (pCB3-M2/PlN) (Lindberg et al., 1987), CB3NRD (pCB3-M2/PlRD) and the chimera between the two viruses, pCB3-M2/P15’RD3’N, were compared. The structure of a different chimeric virus is also shown and the ability of the different constructs to replicate in RD cells is shown to the right.

Construction of chimeras between CB3N and CB3NRD Kandolf and Hofschneider (1985) have cloned a full-length reverse-transcribed cDNA copy of CB3. This infectious cDNA was recloned behind the SV40 early promoter to obtain the construct pCB3-M2 which yields infectious CB3 with an at least 20-fold higher efficiency than the original clone. The virus from which the infectious cDNA was prepared is a variant of CB3N which has been propagated in human cardiac cells. Like the original CB3N it has an RD-negative phenotype. The complete nucleotide sequence of the infectious cDNA that is present in the construct pCB3-M2 was recently determined (Klump et al., 1990) and 17 nucleotide sequence differences were observed in the Pl region when it was compared with the sequence of CB3 strain Nancy. It was therefore necessary to first construct clone pCB3-M2/PlN in which the Pl region of clone pCB3-M2 was replaced by the Pl region of CB3N, to reconstruct a virus with capsid proteins identical with the parent of CB3NRD. To determine if the RD phenotype indeed was determined by the Pl region, the clone pCB3-M2/PlRD was constructed in which the Pl region of the RD variant was inserted into pCB3-M2. Both chimeric constructs yielded infectious virus with a high efficiency and the different virus preparations were assayed for their ability to grow on BGM and RD cells. The results showed that progeny from clone pCB3-M2/PlN, as expected, failed to grow on RD cells whereas progeny obtained from clone pCB3-M2/PlRD had an RD-positive phenotype. The results thus confirmed that the mutations in CB3NRD, which are of critical importance for the RD phenotype, are located in the Pl region of the genome.

193

Consequently, it is likely that one or more of the six amino acid substitutions, which were observed when the amino acid sequences of the capsid proteins of CB3N and CB3NRD were compared (Fig. 2), would determine the RD phenotype. To locate the critical mutations responsible for the RD phenotype more precisely the XmaI/BglII fragment of clone pCB3-M2/PlN, which covers the VP2 gene and only included two of the six observed amino acid differences, was replaced by a corresponding fragment from CB3NRD. The resulting clone, designated pCB3M2/P15’RD3’N, thus contained the VP2 gene derived from CB3NRD and the entire VP1 gene together with almost the entire VP3 gene derived from CB3N (Fig. 2). The growth properties of the resulting progeny were studied and the results showed that the chimeric virus produced CPE at the same rate on both BGM and RD cells demonstrating that it had an RD-positive phenotype. CB3N did not produce CPE on RD cells. From these results we suggest that the mutations that are critical for the RD phenotype are located in the VP2 gene. Table 2 and Fig. 2 show that the XmaI/BgfII 1153-bp fragment of CB3NRD includes four nucleotide substitutions which together cause two amino acid replacements. One of these is a ser/thr substitution that is located in a critical position of the VP2 gene. From a recently constructed model of the tertiary structure of CB3N (Liljas et al., manuscript in preparation) it can be predicted that the altered residue is located in a region of the CB3N virion which corresponds to antigenic site IIB in the PV type 1 (PVl) strain Mahoney capsid (Hogle et al., 1985). It is thus located in a position that is likely to be exposed on the surface of the CB3N virion. The second observed amino acid sequence difference in the XmaI/BglII fragment results in a ‘val/asp substitution. According to our tertiary structure model this alteration is unexposed on the surface of the virion.

Discussion The RD variant of CB3N is a host range mutant of CB3 which has acquired the ability to replicate in the human rhabdomyosarcoma cell line RD and RD variants of different CBS have been isolated by adaptation of different parental viruses to is growth in RD cells (Reagan et al., 1984). It appears that the RD mutation comparatively stable since the phenotype is maintained during several passages of the RD variants in HeLa or BGM cells (R.L. Crowell, unpublished observation). Results from previous studies (Hsu et al., 1990) indicate that the RD mutation allows the virus to interact with a second cellular receptor. The results obtained in the present study show that the RD mutation maps in the Pl region of the CB3 genome. Our results demonstrate, moreover, that the RD phenotype can be transferred to an RD-negative virus by the 1.1 kb long XmaI/BglII fragment. This fragment includes the entire VP2 gene and a small portion of the VP3 gene. Two amino acid substitutions were identified in the predicted amino acid sequence of VP2 from CB3NRD, a ser/thr and a val/asp

194

replacement. The position of the thr/ser mutation corresponds to the so called puff region of VP2 in PVl and HRV14 and the critical residue is located in a portion of VP2 which is equivalent to epitope IIB of PVI. It is noteworthy that two of the observed nucleotide substitutions in the VP2 gene of CB3NRD affect the same codon. This might explain why the RD phenotype remains stable after passaging of the virus for several generations in HeLa or BGM cells. Our results do not exclude the possibility that residues in the other capsid polypetides also play a role for the RD phenotype. The finding that the amino acid substitutions in VP2 are important for the RD phenotype of CB3N was unexpected. Studies of the structure of PVl and HRV14 have shown that there exists a canyon-like region in the capsid which is presumed to interact with the cellular receptor (Rossmann et al., 1985; Rossmann and Palmenberg, 1988; Rossmann, 1989). This canyon is also present in the tertiary structure model of CB3N (Liljas et al., manuscript in preparation). However, none of the amino acid substitutions in the VP2 gene of CB3NRD are exposed on the surface of the canyon. It is on the other hand clear that the affected region of VP2 constitutes an important antigenic site and it is noteworthy that Beatrice et al. (1980) have shown that polyclonal antisera obtained after immunization with purified VP2 from CB3N can neutralize virus. It thus appears that the immunogenic epitope IIB of VP2 is a major antigenic site in CB3. Further indications that epitope IIB is a major immunodominant epitope have been obtained by Auvinen et al. (submitted for publication). They have shown that peptides derived from epitope IIB of VP2 generate a strong immune response when injected into mice. They also show that Balb/c mice which were primed with synthetic peptides, strongly to a secondary challenge with covering epitope IIB in VP2, responded inactivated CB3 virus, while a primary challenge with peptides derived from epitope I only resulted in a weak secondary response. It is likely that other host range variant viruses have more than one binding site for specific attachment to cellular receptors, analogous to that described herein. For example, poliovirus type 2 (Lansing strain) may have acquired a second binding site to account for the change in its host range when adapted to mice (Armstrong, 1939). Furthermore, a chimera of PVl and PV2 that had six amino acids derived from PV2 (Lansing) in exchange with those of PVl (Mahoney) in the N-Ag I loop of VPl, conferred upon PVl the host range phenotype for mouse infection (Martin et al., 1988; Murray et al., 1988). Thus, in this case the second virion binding site also would be located on the surface of the virion within a neutralizing epitope (as for CB3NRD), whereas the primary virion binding site may be located within the canyon. Further studies are needed to determine the role of multiple receptor specificities for a given virus mutant, like CB3NRD, in the pathogenesis of infection (Landau et al., 1990).

Acknowledgements We thank Drs. H. Diderholm and Gun Frisk for interesting discussions and supply of RD cells. This study was supported by grants from the Swedish Medical

195

Research Council, the Marcus BorgstrGm Foundation and the Swedish Board for Technical Development (U.P.), by U.S. Public Health Service research Grant AI-03771 from the National Institute of Allergy and Infectious Disease (R.L.C.) and by Grant Ka 593/2-2 from the Deutsche Forschungsgemeinschaft (R.K.).

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