Intertypic recombination in poliovirus: Genetic and biochemical studies

VIROLOGY

124,121-132

Intertypic

(1933)

Recombination

in Poliovirus:

Genetic and Biochemical

Studies’

ELENA A. TOLSKAYA,* LYUDMILA A. ROMANOVA,* MARINA S. KOLESNIKOVA,* AND VADIM I. AGOL*s?’ *Institute

of Policnnyelitis and Viral Encephalitides, the USSR Academy of Medical Sciences, Moscow Region L&782, and TMosww State University, Moscow 117234, USSR Received June 2, 1982; accepted August

28, 1982

Poliovirus strains of type 1 and type 3 carrying genetically mapped ts mutations and differring in growth response to guanidine have been used to infect HeLa cells. With four heterotypic pairs of the mutants, recombinants with the crossover points between the loci coding for the antigenic properties, on the one hand, and for the sensitivity to guanidine, on the other, have been obtained. The recombinants have been indentified on the basis of their phenotypic properties and, in particular, of the pattern of inheritance of unselected markers. One recombinant has been characterized by fingerprinting virusspecific polypeptides. It has been found that the capsid proteins (VPZ, VP3, and VPl) of this recombinant originate from the type 3 parent, whereas the nonstructural polypeptides (X, 2, and 4) are inherited from the type 1 parent. Implications of the poliovirus intertypic recombination are discussed.

mately additive genetic maps (Cooper, 1977; Tolskaya and Kolesnikova, 1977; McCahon, et al., 1977; MacCahon, 1981). This fact was an indication that the viruses with altered phenotype were indeed of recombinational origin. What could not be done in such experiments, however, was unambiguous discrimination between the presumptive recombinants, on the one hand, and mutants or revertants, on the other. Such discrimination should involve the demonstration that different portions of the recombinant genome are actually inherited from the genomes of different parents. To demonstrate this, the parents should be as distantly related to each other as possible, and they should be compared with the presumptive recombinants with respect to the structure of their RNA or virus-specific proteins. With this considerations in mind, we began studies on intertypic recombination in poliovirus. The fact of such recombination was first established in genetic experiments, and we report here those experiments that were designed to discriminate as strongly as possible between the recombinants and mutants. Then, to obtain definitive evidence for our conclusion,

INTRODUCTION

Among RNA-containing viruses, picornaviruses appear to possess a unique ability to undergo intermolecular genetic recombination (Cooper, 1977). Although the phenomenon itself has been known for two decades (Hirst, 1962; Ledinko, 1963), until recently evidence for its existence was only provided by purely genetic experiments. Progeny clones obtained upon mixed infections were compared to their parents with respect to such phenotypic properties as, for example, growth sensitivities to elevated temperatures or inhibitors like guanidine. The distances between the mutant loci, as defined by the frequencies with which clones with an apparently recombinant phenotype emerged in crosses between appropriate mutants, could be arranged in linear and approxi1 Preliminary results have been reported in part at the 19th Scientific Meeting of the Institute of Poliomyelitis and Viral Encephalitides, Moscow, May 19-21, 1981, and at the USSR-FRG Symposium “Structure and Transcription of the Genome,” Yerevan, October 12-14, 1981. *To whom requests for reprints should be addressed. 121

0042~6922/83/010121-12!$03.00/0 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

122

TOLSKAYA

ET

AL.

Mahoney

I

Plaquiiq in the presence of quanidine. HCl, 60 pq/ml, and selection of a 91: clone

10 passages at 40° an,d cloning

-I-

l-+=+-l Plaquix!g presence 100 pg/ml, selection S7r clone

in of

the 57. and of a

I 1 passage in the presence of FU, 1 mu, and selection of a ts clone

passage in the presence of FU, 1 mu, and selection of a ,G clone

b

s129

Plan?inq in the pre&ce of guanidine. HCl, 60 pq/ml, and selection of a F clone c5 r129

FIG. 1. The scheme used for obtaining in HeLa cells and plaquing was performed otherwise.

mutants of poliovirus in rhesus monkey

a biochemical analysis of a recombinant and its parents was undertaken. In a previous paper (Romanova et al., 1980) we presented the first biochemical proof that the intermolecular recombination between RNA genomes does occur. Using a partial proteolysis mapping technique, we have found that the capsid proteins of a recombinant were inherited from a type 3 poliovirus, whereas a nonstructural protein, polypeptide 2, was indistinguishable from that of the type 1 parent. However, due to close similarity between the nonstructural polypeptides of type 1 and type 3 polioviruses, respectively (Romanova et al., 1981), the resolving power of the partial proteolysis technique was, in this case, not very high and, in particular, the origin of the proteins encoded in the central part of the recombinant genome could not be traced. Therefore, more sensitive two-dimensional fingerprinting analysis of the parental and recombinant virus proteins was utilized in the present study. The re-

type kidney

1. Passages were carried out cells at 37”, if not indicated

sults support our previous conclusion that the strain investigated is indeed an intertypic recombinant; they allow us also to localize, though relatively roughly, the crossover point. Meanwhile, a report describing biochemical studies of aphtovirus intratypic recombination has appeared (King et al., 1982). MATERIALS

AND

METHODS

I%xses. All of the type 1 poliovirus mutants used in this study have been derived from the Mahoney strain. The procedures utilized for their selection are presented in Fig. 1. Strain 452/62 3D, originally isolated from a polio case, was used as the progenitor of type 3 mutants. This strain was twice plaque-cloned at 40” and then passaged in HeLa cells at 37” in the presence of 1 mMfluorouraci1 (FU). A number of ts mutants, including ~557 used here, were isolated from this mutagenized stock.

POLIOVIRUS

RECOMBINATION

A guanidine-dependent mutant, d557, was isolated after plaquing ~557 in the presence of 60 pg/ml of guanidine-HCl. Viruses were titrated by the standard plaque assay in primary cultures of rhesus monkey kidney cells at 37” (Balayan et al., 1964). The plaque count for guanidine-sensitive (gs) and N-sensitive (S%) viruses was inhibited at least loo-fold when the agar overlay contained 60 pg/ml of guanidine-HCl or 57 (ethyl-2-methylthio-4methyl-5-pyrimidine carboxylate), respectively; the plaque count of ts mutants dropped at least lOOO-fold at 40” compared to 37”. A resistant phenotype was defined by the ability to form essentially the same number of plaques (less than twofold difference) at 37 and 40” for tr viruses, and in the absence or presence of 60 pg/ml of guanidine-HCl for gr viruses. Mutants with a guanidine-dependent (gd) phenotype produced at least 100 times less plaques in the absence of guanidine than in the presence of 100 kg/ml of guanidineHCl. Serodifferentiation of the strains was achieved by including type-specific hyperimmune antisera into the agar overlay during plaque assays; homologous antisera decreased the apparent titers of the virus more than 106-fold, whereas heterologous antisera exerted practically no inhibitory effect. TABLE PHENOTYPIC

PROPERTIES

1

OF POLIOVIRUS

STRAINS”

Growth sensitivity to Strain

Serotype

Mahoney MgrS’lr r153 r129

1 1 1 1

3D 8557

3 3 3

d557

Temperature 40” tr tr ts ts tr t.9 ts

“For definitions and designations properties, see text.

Guanidine gs w gr gr gs LJS &

S7 S7.9 S7r s7s s7s

S7S s7s s7s

of phenotypic

133

6’

3’ RNA IS

a

VW VP2 VP3 ----

lb

3b “PI

x

PROTEINS

2 4

I

FIG. 2. (a) A simplified cleavage map of the poliovirus polyprotein. (b) A genetic map of poliovirus type 1 (Tolskaya and Kolesnikova, 1977). The genetic distances between the appropriate loci (expressed as percentage of recombinants in the cross) are given under the map. The position of mutation ts202, which is the nearest to the 3’-end of the genome in our collection, is indicated (the ts202 mutant was kindly provided by Dr. P. Cooper). Symbol g denotes the locus controlling the virus growth response to guanidine. It is not known what portion of the poliovirus genome is encompassed between mutations tsl53 and ts202, and, therefore, the genetic and cleavage maps cannot be directly aligned.

The phenotypic properties of the viruses used in the recombination experiments are given in Table 1. The locations of the appropriate mutations on the genetic map of poliovirus type 1 are depicted in Fig. 2. The positions of the poliovirus type 3 mutations could not be determined accurately due to large variations in the apparent recombination frequencies; however, mutation ts557 is located to the right (towards the 3’ end) from the guanidine locus, at a distance of roughly 0.75 map units (unpublished). Crosses between viruses. Duplicate lday-old monolayer cultures of HeLa cells, 4 to 5 X lo5 cells, were infected by two parental viruses at an input multiplicity of ca. 20 PFU of each virus per cell. After 1 hr absorption at ambient temperature, the unadsorbed virus was thoroughly washed off with a balanced salt solution, and the maintenance medium that was suitable for the reproduction of both viruses was added. For a pair of gs and gr strains this medium consisted of basal Eagle’s medium, and for a pair of gs and gd strains

TOLSKAYA

124

it was Eagle’s medium containing 30 pg/ ml of guanidine-HCl. As shown previously (Tolskaya and Kolesnikova, 1977), either virus did not multiply at this concentration of the drug in single infections, but, due to reciprocal complementation, nearly optimal yields of both viruses were produced in mixed infections. The infected cultures were incubated at 36.5” for 6 hr, by which time a marked cytophatic effect developed. The cultures were frozen at -2O”, and stored at this temperature until the analysis. The progeny was titrated by a plaque assay in monkey kidney cells under several conditions, including those nonpermissive for one of the parents (to estimate the yield of the progeny of the other parent), or nonpermissive for the both parents (to estimate the yield of presumptive recombinants). This was achieved either by the addition of appropriate drugs, guanidine-HCl or S7 and/or type-specific antisera, or by conducting the plaque assay at 40”. The actual conditions are given under Results. The plaque counts were made after 2 days of incubation, if not stated otherwise (if incubation was prolonged, plaques produced by newly emerging mutants might appear). A standard pair of type 1 poliovirus mutants with a known recombination frequency was included in each experiment; experiments where the observed and expected values for the recombination frequency in the standard cross deviated significantly from each other were discarded. Isolation

of virus-spec@c

polypeptides.

Labeling of virus-specific polypeptides with [aS]methionine and preparation of cytoplasmic extracts from the virus-infected cells were performed as described by Romanova et al. (1980). To deplete the extracts from the bulk of capsid polypeptides, they were, after adjusting the KC1 concentration to 0.5M, centrifuged at 50,000 rpm for 4 hr in a Beckman SW65 rotor (Gorbalenya et al., 1981). The proteins were precipitated from the supernatant by 10% trichloroacetic acid, washed with acetone, and dried. Before electrophoresis, the dried preparations were dis-

ET

AL.

solved in the dissociation buffer, 2% sodium dodecyl sulfate (SDS), 50 mM TrisHCl, pH 6.8, 5% mercaptoethanol, 10% glycerol, 0.003% bromophenol blue, and were separated by disc electrophoresis in SDS-containing 15% polyacrylamide gel slabs using a Tris-glycine buffer system (Laemmli, 1970). The slabs were dried and autoradiographed. Capsid polypeptides were prepared by similar electrophoresis of purified (Medappa et al., 1971) virions. Fingerprinting tryptic peptides. The gel regions corresponding to individual labeled virus-specific polypeptides were excised from the slabs, washed with 50% methanol, and then with water. The gels were placed to 0.5 ml of 0.1 M NH4HC03, pH 8.0, and 25 ~1 of TPCK-treated trypsin (Serva), 7 mg/ml, was added. After incubation for 18 hr at 37”, a fresh portion of trypsin was added, and incubation was continued for 4 hr. The samples were diluted with water up to 3.0 ml, and subjected to two cycles of lyophilization. The hydrolyzate was dissolved in water and subjected to two-dimensional fingerprinting using cellulose plates (Merck), 10 X 10 cm. For electrophoresis, a solution containing 15% acetic acid and 5% formic acid, pH 1.9, was used. The samples were run at 500 V and at approximately 7’ for a time interval controlled by the movement of a dye, Orange G. For the seconddimension chromatography, a buffer containing butanol:pyridine:acetic acid:H20 (32.5:25:5:20) and 7% (w/v) PPO, which allowed direct fluorography of the dried plates (Gautsch et al., 1978), was used. RESULTS

Genetic Evidence for Intertypic nation in Poliovirus

Recombi-

The recombination between poliovirus belonging to serotypes 1 and 3 was investigated using four pairs of parents. In each pair, the parents differed from one another, in addition to antigenic properties,

POLIOVIRUS

RECOMBINATION

125

doubt was expressed as to whether intertypic recombination exists in picornavi-

r129.30

ruses at all (cf. King et al., 1982).

hQrs7rr5537 ::> ts557

gs te.153 rl53"P557

;:A

es

tz.557

FIG. 3. Schematic representation of the intertypic crosses investigated. Heavy lines denote parental genomes, and light lines symbolize genomes of the recombinants selected. The positions of the crossover points are indicated somewhat arbitrarily, but they should lie between the locus responsible for the antigenicity on the left and the guanidine locus on the right.

by at least two phenotypic properties. One of them, resistance to, or dependence on, low concentrations of guanidine, was utilized for the selection of the recombinants, while the other property served as a nonselective genetic marker that helped discriminate between recombinants and mutants. A schematic representation of the crosses is given in Fig. 3. The results obtained in each cross will be described separately and in some detail, inasmuch as TABLE PLAQUE

ASSAY OF THE PROGENY

r129 X SD. The data obtained upon titration of the progeny of this cross at three different sets of conditions are given in Table 2. It is seen that the proportion of plaques that developed under the agar overlay containing anti-polio 1 serum and 100 pg/ml of guanidine-HCl was 20-fold greater in the progeny of mixed infection than in the progeny obtained upon the infection with 3D alone. The viruses able to plaque under these conditions might, however, include not only recombinants but also mutants of the type 3 parent that acquired the ability to grow in the presence of guanidine. To discriminate between these possibilities, 10 plaques formed under the above conditions by the progeny of the mixed infection were picked up and the corresponding clones were investigated with respect to their antigenic properties as well as guanidine and temperature sensitivities. All 10 clones were identified as type 3 viruses; 9 of them exhibited a ts gr phenotype, as expected for the recombinants (cf. Fig. 3) and one clone exhibited a tr gd phenotype, which suggested that it was a mutant. Since upon titration of the progeny of single infection with 3D, only one plaque developed in this experiment in the presence of guanidine, the 2

OF THE CROSS r129

x 3D UNDER

DIFFERENT

CONDITIONS

Progeny virus assays (PFU/ml)

Infecting virus(es) r123 3D r129

X 3D

In the absence of guanidine A

In the presence of guanidineHCl, 100 pg/ml

In the presence of guanidine and antipolio 1 serum B

Frequency of presumptive recombinants + mutants”

5.4 x 10’ 19.2 x lo7 7.2 x 10’

8.8 x lo7 -

10.5 x lo2 1.0 x ld


4.8 X

10’

7.0 x 102

10-a

a Frequency of the poliovirus type 3 clones that are able to plaque in the presence of 100 gg/ml of guanidineHCl (B/A).

126

TOLSKAYA

ET

TABLE PLAQUE

ASSAY OF THE PROGENY

Mahoney d557 Mahoney

In the absence of guanidine (A)

X d557

virus

assays

-

<0.5 x lo2 <0.5 x lb 12.0 x lo3

6.2 X 10’ 5.8 x lo7

type 1 clones that for d557, and C/(A

are able to plaque in the presence + B) for the cross).

Infecting virus(es) MgrS7r s557 MgrS7r

x ~557

’ Frequency HCl (B/A).

In the absence of guanidine (4 9.0 x lo1 3.4 x lo7 4.9 x lo7 of the poliovirus

type 3 clones

virus

of 60 Fg/ml

assays

x ~557 UNDER DIFFERENT

of guanidine-

CONDITIONS

(PFU/ml)

In the presence of guanidineHCI, 60 pg/ml 12.5 X lo7 3.5 x 107 that

13 x 10-G
4

OF THE CROSS MgrS7

Progeny

Frequency of presumptive recombinants + mutants”

binant phenotype was ca. 10V5, whereas no (to.5 X 10m6) such clones were observed in single infections. Mahoney X d&57. The results of plaque assays under different conditions of the progeny of this cross are shown in Table 3. It is seen that the proportion of the type 1 virus producing plaques in the presence of guanidine was at least two orders of magnitude higher in the progeny of mixed infection than in the progeny of single infection with Mahoney. All of 12 clones isolated from the progeny of mixed infected cells were shown, in separate assays, to exhibit antigenic properties of the type 1 virus and a ts gd phenotype. This is just the phenotype expected for the recombi-

TABLE ASSAY OF THE PROGENY

CONDITIONS

(PFWml) In the presence of guanidine and antipolio 3 serum (Cl

phenotypic properties of five clones originating from the 3D plaques formed under similar conditions in independent experiments were analyzed. All five of the viruses were found to possess a tr gd phenotype. It has to be noted that upon the growth of 3D in the presence of guanidine, we were so far able to isolate only gd and never gr mutants. Whatever the reason for this peculiarity, it lends additional support to the conclusion that the clones of type 3 virus with a gr phenotype that were contained in the progeny of mixed infection were actually recombinants that inherited their gr as well as ts markers from the type 1 parent. Thus, in this cross the frequency of the clones with the recom-

PLAQUE

X d557 UNDER DIFFERENT

In the presence of guanidineHCl, 60 pg/ml (W

17.3 x lo7 <0.5 x loa 4.2 X lo7

a Frequency of the poliovirus HCl (C/A for Mahoney, C/B

3

OF THE CROSS MAHONEY Progeny

Infecting virus(es)

AL.

are able to plaque

In the presence of guanidine and antipolio 1 serum 09 <0.5 x 102 <0.5 x lb 4.5 x lo3 in the presence

Frequency of presumptive recombinants + mutants” <0.5 x 10-S <10-s 10-a

of 60 pg/ml

of guanidine-

POLIOVIRUS

nants (cf. Fig. 3). No plaques were produced in the presence of guanidine by the virus harvested from the cells singly infected with Mahoney in the experiment presented in Table 3. However, in the course of our studies a number of mutant plaques that formed in the presence of guanidine by Mahoney in separate experiments were picked up, and the phenotype of the corresponding clones was investigated: roughly 90% of these clones displayed a tr gd phenotype, the remainder of the clones were tr gr. Clones with a ts phenotype, if present, were rare enough and were not encountered. Collectively, the results described in this paragraph strongly suggest that intertypic recombinants were generated in the Mahoney X d557 cross. MgrS?‘r X ~557. The results obtained upon plaquing the progeny of this cross are presented in Table 4. Viruses with antigenic properties of type 3 and able to grow in the presence of guanidine were at least 100 times more abundant in the progeny of the mixed infection than in the progeny of the type 3 parent itself. Nine such clones isolated from the progeny of the mixed infection were investigated with respect to their phenotype. All nine were shown to possess antigenicity of type 3, and seven clones among them exhibited a

tr gr S7s phenotype,

exactly as expected for recombinants (cf. Fig. 3). Two clones were of a ts gd S7s phenotype, and should be considered mutants. No plaques developed in the presence of guanidine by the progeny of the ~557 single infection after 2 days of incubation, the standard time of plaque counting. However, after 4 days of incubation, several tiny plaques appeared. Five clones derived from these plaques were investigated, and all of them were found to exhibit a ts gd S7’s phenotype, as expected for mutants. It is worthwhile to note again that mutants of the type 3 virus that acquired ability to grow in the presence of guanidine were as yet invariably found to exhibit a gd phenotype. Therefore, a gr phenotype of some of the type 3 clones generated upon mixed infection is strong additional evidence for the recombinant origin of these clones. r153 X ~557. The selection conditions used for the detection of recombinants in the progeny of this cross differed substantially from those used in the previous cases. Now, clones that exhibit a tr phenotype (and hence assumed to inherit 5’- and 3’adjacent portions of the genome from the type 3 and type 1 parents, respectivelycf. Fig. 3) and that, in addition, were able to grow in the presence of guanidine (assumed to inherit the guanidine locus from

TABLE

5

PLAQUEASSAYOFTHEPROGENYOFTHECROSS~~~~ Progeny

127

RECOMBINATION

virus

assays

x 9557 UNDERDIFFERENTCONDITIONS (PFU/ml) At 40”

At 37’

Infecting virus(es) r153 s557 t-153

X ~557

‘Frequency (B/A).

In the absence of guanidine (4 10.6 X lo7 9.5 x lo7 6.7 X lo7 of the clones

that

In the presence of guanidineHCl 60 bug/ml 10.9 x lo7 3.8 are able to plaque

x lo7 at 40”

In the presence of guanidineHCI, 60 rig/ml (B) 1.0 x 10’ <0.5 x lo3 1.5 x lo4 in the presence

of 60 rg/ml

Frequency of presumptive recombinants + mutants” 1o-4 10.5 2

x 1o-5 x 1o-4

of guanidine-HCI

128

TOLSKAYA

ET

AL.

VPP

c VP3

icrc

*

Ir + 1 VP

1

‘I

ELECTROPHORE313

FIG. 4. Fingerprints of tryptic peptides the following figures, Tl denotes MgrNr, between these two parents.

of the capsid T3 denotes

the type 1 parent) were selected (Table 5). In this experiment, the efficiencies of plaque formation at 40” in the presence of 60 pg/ml of guanidine-HCl by the progeny of single infection with r153 and by the progeny of the mixed infection were of quite comparable values, 10F4 and 2 X 10m4, respectively. This indicated, in particular, a relatively high rate of reversion of the tsl53 mutation, which was significantly lower in some other experiments. Anyway, such figures by themselves could hardly be regarded as evidence for the generation of recombinants in this cross. However, when phenotypic proper-

proteins VP2, VP3, and VPl. In this and ~557, and R1/3 denotes the recombinant

ties of 12 clones derived from the plaques formed at 40” in the presence of guanidine by the progeny of the mixed infection were investigated, it was found that all of them exhibited a tr gr phenotype, and seven and five clones among them displayed antigenie properties of type 1 and type 3, respectively. The former should be considered revertants, whereas the latter should represent recombinants (cf. Fig. 3). Biochemical Evidence for Intertypic Recombination in Poliovirus To obtain direct proof that the viruses which were considered recombinants in

POLIOVIRUS

RECOMBINATION

(Romanova et al., 1980). The data of Fig. 4 not only confirmed these studies, but also suggested that the entire, or almost entire, capsid region, including its most 5’-distal sequence that encodes VPl, was derived from the type 3 virus by the recombinant. On the other hand, the fingerprints of the proteins encoded in central (polypeptide X) and 3’ end-adjacent (polypeptides 2 and 4) regions of the viral genome demonstrated that the corresponding nucleotide sequences of the recombinant originated from the type 1 parent (Fig. 5). The evidence for this notion was sufficiently compelling for X, but it appeared somewhat less so for polypeptides 2 and 4, inasmuch as these two noncapsid polypeptides were

the preceding section were actually recombinants, comparison of the fingerprints of proteins coded for by a recombinant and both its parents was undertaken. A recombinant obtained upon crossing MgS7r and 657 and selected under conditions delineated in Table 4 was chosen for such an analysis. The fingerprints of the capsid proteins VP2, VP3, and VP1 (this is the 5’ - 3’ order of the corresponding coding sequences in the viral RNA-cf. Fig. 2) are presented in Fig. 4. They show unequivocally that the recombinant inherited its capsid polypeptides from the type 3 parent. This conclusion was already formulated on the basis of antigenic studies (see above) and partial proteolysis mapping Tl

RI/3

ELECTROPHOREBIS

FIG. 5. Fingerprints

129

T3

4

of tryptic peptides of the noncapsid proteins X, 2, and 4.

TOLSKAYA

130

RI/3

+ T3

FIG. 6. Fragments of fingerprints of tryptic peptides of different mixtures of the noncapsid proteins 4 of parental polioviruses and their recombinant. Only regions with peptides of a high electrophoretic mobility and a low chromatographic mobility are given. 1, Electrophoresis; 2, chromatography.

closely related in poliovirus type 1 and type 3 strains used (cf. Romanova et al., 1981). Nevertheless, a small but reproducible difference did exist between the appropriate proteins of the two parents, e.g., in tryptic peptides with a high electrophoretic mobility but a low mobility upon chromatography (Fig. 5). A comparison of this particular region of the fingerprints of polypeptides 2 as well as 4 of the recombinant and the parents suggested that these noncapsid proteins of the recombinant originated from the type 1 virus. To substantiate this conclusion, fingerprints of several mixtures of polypeptides of appropriate viruses were investigated. The regions of interest of these fingerprints are shown in Fig. 6. It is seen that the mixture of polypeptide 4-derived peptides of the recombinant and MgrS7r gave a pattern that was indistinguishable from that of MgrS’7r alone (cf. Fig. 5). On the other hand, the mixtures of protein 4-derived peptides of ~557 with those of either MgrS7r or the recombinant exhibited a different pattern, which contained, in particular, at least one additional spot (cf. Figs. 5 and 6). Thus, the results of the fingerprinting analysis confirmed our previous contention based on the partial proteolysis mapping techniques that the recombinant capsid proteins were derived from the type 3 parent, whereas polypeptides 2 and 4 were

ET AL.

derived from the type 1 parent (Romanova et al., 1980). In addition, the data presented here demonstrated that the polypeptide X of the recombinant has the type 1 origin. Hence, the crossover should occur at, or in the vicinity of, the border between the capsid and noncapsid regions of the genome. DISCUSSION

One of the major conclusions that can be drawn from our results is that the recombination between the genomes of polioviruses belonging to different serotypes does occur. The genetic evidence for this notion is rather strong. It consists not merely in the significantly higher yields, in mixed as opposed to single infections, of viruses that are able to multiply under conditions nonpermissive for either parent, but, not less importantly, also in the predictable inheritance of nonselected markers by the recombinants. Moreover, we were not able to detect some of the recombinant phenotypes in the progeny of single infections altogether. Biochemical evidence presented in this and in a previous (Romanova et al., 1980) paper shows directly that different portions of the genome of a clone, which has been genetically characterized as a recombinant, are actually derived from two parents. Only the recombinants that have arisen through a crossover between the locus determining the virus antigenicity and the guanidine locus were selected and investigated in this study. The frequency of the appearance of such recombinants was rather low. This fact merits some general comments. The frequency of the generation of recombinant clones in intertypic crosses should not be a simple function of the distance between the loci to be recombined. The frequency should be influenced, in addition, by at least two other parameters: (i) the extent of homology between the appropriate regions of the parental genomes (a plausible assumption is that the recombination between highly diver-

POLIOVIRUS

RECOMBINATION

gent regions is hampered or even prohibited), and (ii) the viability of the recombinants formed (for example, chimeric proteins may frequently turn out to be nonfunctional when they derive amino acid sequences from divergent polypeptides). If these considerations are valid, recombination within the capsid region may be severely restricted, since it is the region where polioviruses of type 1 and type 3 djffer from one another to a high degree (Cumakov et uZ., 1979; Romanova et al., 1981; this work). Then most of the recombinants are expected to inherit the entire capsid region from one of the parents. This appears to be the case for the recombinant investigated biochemically here. In any case, the crossover resulting in the emergence of recombinants between the entire capsid region and guanidine locus (which is located roughly in the center of the genetic map) should occur within a relatively narrow zone and hence with a low probability. The intertypic recombinants may be profitably utilized in several areas of picornavirus research. For example, they can help in aligning biochemical and genetic maps. Such alignment, in turn, would be important for elucidation of both the function of some poorly investigated virus-specific proteins and the nature of genetic alterations caused by mutations. Although this has not as yet been done, the data available at the moment do warrant a comment on one such problem, the nature of the guanidine locus. This locus is of evident interest since its product appears to be involved in the replication of the viral genome (cf. Cooper, 1977). The identity of this product is unknown, however. A hypothesis has been put forward that it is a capsid protein and, if correct, this hypothesis should have wide implications (cf. Cooper, 1977). However, our data indicate that the entire, or almost entire, capsid region of the poliovirus genome could be separated, by recombination, from the guanidine locus. Therefore, we suggest that the g locus is contained

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in the noncapsid region of the genome (cf. also Tolskaya and Kolesnikova, 1977; Romanova et al., 1980). Intertypic recombinants may be helpful in solving several applied problems too. We would like to list only some of them: (i) recombinants between virulent and attenuated strains may contribute to understanding the nature of viral pathogenicity; (ii) natural intertypic recombinants may arise in mixedly infected organisms, and the properties of such newly evolving viruses may be at least partially predicted by laboratory studies of intertypic recombination; (iii) intertypic recombinants should perhaps be investigated with respect to their possible use as vaccine strains. Note oddeo! in proof. Recent electrofocusing studies of polypeptides induced by gr mutants of aphtovirus also suggest that the guanidine locus lies outside the capsid region of the viral genome [K. Saunders and A. M. Q. King (1982) J. Vied 42,389-3941. REFERENCES

BALAYAN, M. S., TOLSKAYA, E. A., VOROSHILOVA, M. K., and YUROVETSKAYA, A. L. (1964). Study of intratypic differences between type 2 polioviruses. I. Relationship between neurovirulence, antigenicity and other properties determined in cells in vitro. Virology 23, 125-140. COOPER, P. D. (1977). Genetics of picornaviruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. Wagner, eds.), Vol. 9, pp. 133-207. Plenum Press, New York. CUMAKOV, I. M., LIPSKAYA, G. Y., and AGOL, V. I. (1979). Comparative studies on the genomes of some picornaviruses: denaturation mapping of replicative form RNA and electron microscopy of heteroduplex RNA. Virology 92,259-270. GAUTSCH, J. W., LERNER, R., HOWARD, D., TERAMOTO, Y. A. O., and SCHLOM, J. (1978). Strain-specific markers for the major structural proteins of highly oncogenic murine mammary tumor viruses by tryptic peptide analyses. J. Vi’irol. 27,633.699. GORBALENYA, A. E., SVITKIN, Yu. V., and AGOL, V. I. (1981). Proteolytic activity of the nonstructural polypeptide p22 of encephalomyocarditis virus. B&hem. Biophys. Res. Commun S&952-960. HIRST, G. (1962). Genetic recombination with Newcastle disease virus, poliovirus and influenza. Cold Spring Harbor Symp. Quad. Biol 27, 303-309.

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TOLSKAYA, E. A., and KOLESNIKOVA, M. S., (1977). Genetic analysis of the poliovirus. W&n Acad Mm! Nauk SSSR No. 5, 3-10 (in Russian).