Biochemical characterization of poliovirus type 1 temperature-sensitive mutants

VIROLOGY

139.403-407

(1984)

SHORT COMMUNICATIONS Biochemical

Characterization

of Poliovirus

Type 1 Temperature-Sensitive

Mutants

CHRISTINE BELLOCQ,’ HENRI AGUT, SYLVIE VAN DER WERF, AND MARC GIRARD Uniti

de V+obgie

lUWire,

Institut Received

April

Pa&eur,

25 Rue du Dr. Roux,

18, 1981, accepted

August

7572.4, Paris

Cenkc

15, France

8, 1984

Four temperature-sensitive mutants selected upon chemical mutagenesis of the poliovirus type 1 Mahoney strain [H. Agut, T. Matsukura, C. Bellocq, M. Dreano, J. C. Nicolas, and M. Girard, Ann Viral. (Inst. Pasteur), 132E, 445-460 (1981)]. were analyzed by Tl oligonucleotide mapping. Three mutants (ts 203, ts 221, and ts 035) had Tl fingerprints identical to that of wild-type virus while mutant ts 24’7 exhibited two differences on oligonucleotides that mapped in the region of the genome coding for the replicase (polypeptide 4b) and for the protease (polypeptide 7c) of the virus. Cells were infected with each of the four mutants separately, labeled with [8SSlmethionine, and the labeled polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. Mutants and wild-type virus polypeptides showed a similar electrophoretic pattern except for the replicase and the protease of ts 247 which showed abnormal apparent molecular weights. The labeled proteins were subjected to two-dimensional ieoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Polypeptides 4b (replicase) and 2 (the common precursor to polypeptides 7c and 4b) of ts 247 and ts 035 exhibited distinct charge alterations when compared to the corresponding wild-type polypeptides. These alterations were also found on polypeptide 6a in the case of ts 247 and on polypeptide 6b in the case of ts 035, both polypeptides resulting from an alternate cleavage of polypeptide 2. 0 1984 Academic

Press. Inc.

Following chemical mutagenesis of poliovirus type 1 (Mahoney strain), we isolated four temperature-sensitive mutants (ts) of poliovirus by replica plating at 37” (permissive temperature) and 39” (nonpermissive temperature). These mutants (ts 203, ts 221, ts 247, and ts 035) exhibited approximately a l@-fold lower plaquing efficiency on HeLa cells at 39” as compared to 37” (1). Preliminary characterization of the phenotypic properties of these mutants has been described (I). Virions from mutants ts 203, ts 221, and ts 247 were much more heat sensitive (at 45’) than wildtype virions. On the other hand, virions from ts 035 were as heat resistant as willd-type virions. Mutants ts 221, ts 247, and ts 035 displayed an RNA (-) phenotype at 39”, whereas ts 203 behave as RNA (+). We furthermore found evidence r Author addressed.

to whom

requests

for

reprints

should

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403

for genetic recombination between ts 035 and each of the three other mutants. Recombination frequency was the highest between ts 035 and ts 203 (2). We concluded from these results that ts 203 was most probably a capsid mutant, ts 035 a replicase mutant, and that ts 221 and ts 247 could be pluri mutants. In order to map the mutations more precisely, the RNA of wild-type virus and of the four ts mutants was analyzed by Tl oligonucleotide mapping (3). Digestion of viral RNA with RNase Tl and labeling of the Tl resistant oligonucleotides with [y-?P’JATP in the presence of T4 polynucleotide kinase was as described by Pedersen and Haseltine (4), except that treatment with phosphatase was omitted and labeling was performed simultaneously with the RNase Tl digestion. This method was chosen because of its reproducibility and ease although it gave less uniformly labeled Tl oligonucleotides than in wivo labeling. Oligonucleotide numbering was, according to Lee et al. (5), not taken into 0042-6822/84 Copyright All rights

$3.00

Q 1984 by Academic Press. Inc. of reproduction in any form reserved.

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A

FIG. 1. RNase Tl fingerprints of wild-type RNA was digested with 25 units of RNase Tl 1 hr at 37” with 10 &i of [@aPjATP in the Biochemicals). The Tl oligonucleotides were gel electrophoresis. Electrophoretic migration V for 6 hr and in the second dimension from shows the change in migration of oligonucleotide tide 8.

B RNA (A) and ts 247 RNA (B). A 300-ng amount of (PL Biochemicals) and simultaneously 5’ labeled for presence of 5 units of T4 polynucleotide kinase (PL then separated by two-dimensional polyacrylamide in the first dimension was from left to right at 750 bottom to top at 700 V for 16 hr. The arrow (B) 10 and the triangle, the absence of oligonucleo-

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account unidentified spots that most likely corresponded to partial digestion products. Three of the mutants, ts 203, ts 221, and ts 035 exhibited oligonucleotides fingerprints similar to that of wild-type virus while mutant ts 247 showed two oligonucleotide changes (Fig. 1). The first change was the complete disappearance of oligonucleotide 8 (triangle in Fig. 1B) and the second was the altered migration of oligonucleotide 10 in the first dimension (arrow in Fig. lB), revealing a change in charge. In this particular experiment oligonucleotide 9, which usually migrates close to oligonucleotide 10, was poorly labeled and therefore not resolved in the wild-type oligonucleotide fingerprint (Fig. 1A). As deduced from the poliovirus nucleotide sequence (6, 7), oligonucleotide 8 is located at position 7125 on the genome, i.e., in the sequence coding for the replicase, polypeptide 4b (polypeptide 3D according to the L434 nomenclature (8)). whereas oligonucleotide 10 is located at position 5644, in the region coding for the protease, polypeptide 7c (polypeptide 3C). To characterize the ts mutants further, we analyzed the virus-specific polypeptides synthesized in infected cells maintained at permissive temperature (37”) or transferred to nonpermissive temperature (39”). Infected cells were labeled with pS]methionine and cytoplasmic extracts were prepared as described by Lowe et al. (9). Labeled proteins were analyzed by electrophoresis on a 10-W% SDS-polyacrylamide gel (10). At the permissive temperature (Fig. 2), the polypeptides synthesized by mutants ts 203, ts 221, and ts 035 exhibited the same electrophoretic pattern as those of wild-type virus. In the case of mutant ts 247, however, two major alterations were detected, affecting polypeptides 7c and 4b. These polypeptides migrated respectively slower and faster than their wild-type counter parts (Fig. 2, lane E). Identity of wild-type and ts 247 polypeptides 7c has been confirmed by immunoprecipitation using specific anti-7c antibodies (data not shown). Furthermore, the altered polypeptides consistently appeared as weaker bands, suggesting that although functional at 37”, their overall

synthesis was reduced or their turnover increased. No shutoff of host-cell protein synthesis could be detected at the nonpermissive temperature in the case of mutants ts 221, ts 247, and ts 035, i.e., the RNA (-) mutants and viral proteins could not be detected in the mutant-infected cell extracts (data not shown). On the opposite, analysis of the proteins synthesized in cells infected with ts 203, an RNA (+) mutant, revealed a normal host cell

ABCDEFGH 92,5K

-

69K

-

46K

-

30K

VPI VP2 VP3

14,3K

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FIG. 2. Analysis of viral polypeptides synthesized in HeLa cells infected with wild-type or mutant viruses. HeLa cells seeded at 5 X 106 cells per 35mm petri dish were infected at a multiplicity of 100 PFU/cell and incubated at 3’7’. Four hours after infection, the medium was replaced with methioninefree Eagle’s medium and 30 min later, the cells were labeled for 1 hr with 30 &i/ml of PShnethionine. The cells were lysed at 4” with 0.1 ml of 0.01 M Tris-HCl, pH 7.4, 1 m&f EDTA, 0.5% Nonidet-P40. Cell debris and nuclei were pelleted for 2 min at 10,000 g and cytoplasmic extracts were collected. The extracts (10s cpm) were denatured in 2% SDS, 5% 2mercaptoethanol at 100” for 2 min and applied onto a lo-18% linear gradient SDS-polyacrylamide gel (JO) and subjected to electrophoresis for 7 hr at 130 V. The gel was dried and autoradiographed for 24 hr. [86S)-labeled wild-type purified virions (lane A) and rqlabeled protein standards (Amersham) (lane B) were used for molecular-weight determinations. Mock-infected cells (lane H) were included as a control. The cytoplasmic extracts analyzed were from cells infected with ts 221 (lane C), ts 203 (lane D), ts 247 (lane E), ts 035 (lane F), or wild-type virus (lane G). Arrows point to the positions of wildtype polypeptides 4b and 7c.

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protein synthesis shutoff at 39” and a viral protein profile similar to that of wild-type virus. We further analyzed the viral polypeptides of the mutants by two-dimensional polyacrylamide gel electrophoresis (9, 11, 12) using isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. The polypeptides of mutants ts 203 and ts 221 exhibited no alteration IEF

in charge when compared to those of wildtype virus. On the other hand two-dimensional analysis of mutants ts 247 and ts 035 revealed charge alterations of their polypeptides as compared to wild type (Fig. 3). In the case of mutant ts 247, polypeptides 4b, and NCVPZ (polypeptide 3CD), the common precursor to ‘7~ and 4b, exhibited an altered p1 (Fig. 3C). In addition, polypeptide 6a (polypeptide 3C), cl

@--+

i’ 4b . VP0

-0Ga

VP0

.

.

6b-

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FIG. 3. Two-dimensional IEF SDS-polyacrylamide gel electrophoresis of poliovirus polypeptides. Labeling of cells and preparation of cytoplasmic extracts were as in Fig. 2. Cytoplasmic extracts were added with 3.5% (v/v) bmercaptoethanol, 9 M urea, and 0.1 mg/ml of RNase A, then incubated for 15 min at 3’7”. Samples were subjected to IEF as described (9) except for the use of pH 3.5-9.5 ampholines (LKB). Following electrophoresis the gels were frozen, extruded, and equilibrated for 30 min in 2.3% SDS (w/v), 5% 2-mercaptoethanol (v/v), 10% glycerol (w/v), and 0.0625 M Tris-HCl, pH 6.8 (II). A 1% agarose solution in the same buffer was used to seal the first-dimension gels to the second-dimension slab gel. The second-dimension electrophoresis was performed on a l&18% (A, C) or 15% (B)~SDS-polyacrylamide gel. Analyses of wild-type (wt) virus-infected cell extracts (A) and of mixtures of infected-cell extracts: wt + ts 035 (B), wt + ts 247 (C), were performed;

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which results from an alternate cleavage pathway of NCVPB, showed an analogous charge alteration. It was indeed confirmed that polypeptides 4b and 7c had an altered apparent molecular weight. Furthermore, polypeptide 7c, which is always poorly labeled and thus only detected on overexposed autoradiographs, showed no change in charge. In the case of mutant ts 035, polypeptides 4b and NCVPZ as well as polypeptide 6b (polypeptide 3D’), the other product of the alternate cleavage of NCVPZ, exhibited an altered pL As illustrated here, such biochemical analysis proved, within certain limits, invaluable for the characterization of viral mutants. In the case of mutants ts 203 and ts 221, no conclusion could be reached using the techniques described in this communication. As mentioned in the accompanying paper, however, other results suggest that ts 203 may be a morphogenesis mutant. Two-dimensional analysis of ts 035 polypeptides points to an alteration of the replicase (4b), in agreement with the RNA (-) phenotype of this mutant. Regarding mutant ts 247, both Tl oligonucleotide mapping and analysis of viral polypeptides point to the location of mutations in the replicase (4b) and the protease (7~). It remains however to be determined whether the structural defects revealed here by the biochemical analysis of the RNA or of the proteins, do correspond to functional defects. In particular, it is difficult to conciliate the RNA (-) character of ts 247 and the alteration of its capsid as revealed by the enhanced thermolability of the virions. An explanation could be found by assuming a pleiotropic effect of the mutation of the protease. Another explanation could be the presence of yet another mutation located in the Pl region of the genome coding for the capsid polypeptides. To dissociate the effects of the different mutations which were detected,

407

we are presently analyzing ts 247 revertants and ts 247 X ts 035 recombinants by Tl oligonucleotide mapping as well as by 2D analysis of viral polypeptides. Such biochemical analysis should help to establish a relationship between the structural mutations and their biological effect on the growth of the virus. ACKNOWLEDGMENTS We thank Andrew King, David MacCahon, and John Newman for their cordial hospitality and for initiating one of us (C.B.) to BD-analysis of proteins. We are grateful to Eckard Wimmer for the gift of anti-% antibodies. This work was supported in part by NATO Grant RG/175/89 and by DGRST Grant 81E 214. REFERENCES 1. AGUT, H., MATSUKURA, T., BELLOCQ, C., DR~ANO, M., NICOLAS, J. C., and GIRARD, M., Ann. Viral (Inst. Pasteur), 132E, 445-469 (1981). 2. AGUT, H., BELLOCQ, C., VAN DER WERF, S., and GIRARD, M., Virology 139, 393-402 (1984). 3. DE WACHTER, R., and FIERS, W., And Biochem 49.184-196 (1972). .& PEDERSEN, F., and HASELTINE, W., J. ViroL 33, 349-365 (1980). 5. LEE, Y. F., KITAMURA, N., NOMOTO, A., and WIMMER, E., J. Gen ViroL 44, 311-312 (1979). 6. KITAMURA, N., SEMLER, B. L., ROTHBERG, P. G., LARSEN, G. R., ADLER, C. J., DOWER, A. H., EMINI, A., HANECAK, R., LEE, J. J., VAN DER WERF, S., ANDERSON, C. W., and WIMMER, E., Nature (London) 391, 547-553 (1981). 7. RACANIELLO, J. R., and BALTIMORE, D., Proc Nat1 Acd Sci USA 78.4887-4891 (1981). 6. RUECKERT, R. R., and WIMMER, E., J. Viral 50, 957-959 (1984). 9. LOWE, P. A., KING, A. M. Q., MCCAHON, D., BROWN, F., and NEWMAN, J. W. I., Proc N&l Acad 5’ci USA 78,4448-4452 (1981). 10. HANECAK, R., SEMLER, B. L., ANDERSON, C. W., and WIMYER, E., Proc NatZ Ad S$ USA 79,3973-3977 (1982). 11. O’FARRELL, P. H., J. Biol Chm. 250, 4007-4021 (1975). 12 WiEGERS, K. J., and DERNICK, R., J. Gen V&l ’ 52.61-69 (1981).