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Formation of a coat protein subunit from R17 bacteriophage
VIROLOGY 48, 288-290 (1972)
Formation of a Coat Protein Subunit from R17 Bacteriophage
of the samples into pH 7, 0 .1 ionic strength sodium phosphate buffer resulted in material with a typical protein spectrum (X mn = 276 am, y m, a = 249 nm, and A959/duo = 1 .5) . This material sedimented with s,o,, = 10 .9 X 10 -" in the model E analytical ultracentrifuge (Fig . 1) . Artificial top component (ATC) of R17 has been prepared by Samuelson and Kaesberg (10) . This material is the intact capsid minus the RNA . Treatment of
The most probable path for the assembly of large protein particles is through stepwise aggregation of smaller subunits (1) . Evidence for this mechanism is found in diverse biological sources, including tobacco mosaic virus (,2, 3), tail of bacteriophage lambda (4), and phycobilisomes of blue-green and red algae (5, 6) . To study this process further an isometric virus was disassembled into protein subunits . 1117 bacteriophage was selected because its capsid is relatively simple, consisting of about 7 .80 molecules of coat protein and 1 molecule of A protein (7) . The only other component of this virus is a single strand of RNA (8) . By the term "subunit" is meant a protein species composed of two or more coat protein molecules and smaller than the empty capsid (41S) . The capsid of R17 was found to be resistant to many possible methods of disassembly. Recently, the disaggregation of succinatc dchydrogenase was accomplished by freezing the enzyme in the presence of sodium perchlorate (9) . Application of this technique to R17 resulted in a high yield of an 11S subunit . This subunit was formed by suspending the virus in 1 lbl sodium thiocyanate, pH 7 .1, freezing in liquid nitrogen, and thawing six times . Sedimentation velocity experiments with schheren optics in the analytical ultracentrifuge showed . a major boundary scdimenting at, 11S in the 1 1VI NaSCN, pH 7, solution . Neither freezing and thawing nor 1 M NaSCN alone produced any 118 species . Furthermore, the treatment of R17 bacteriophage with 0 .2 Dl NaSCN, pH 7, and freeze-thawing completely destroyed its plaque-forming ability, but with the addition of 0 .2 na NaSCN alone or freezing alone at pH 7, its infectivity was maintained . Removal of the 1 M NaSCN by dialysis and treatment with ribonuclease (Worthington Biochemicals, 4400 U/mg) did not further disassemble the 1IS component . Precipitation with 33% saturated ammonium sulfate and redissolving in 1 A NaSCN, pH 7, three times and final dialysis
Fm . 1 . Sedimentation velocity pattern obtained with schlieren optics for the ammonium sulfatepurified 11S subunit . Experiments were performed at 59,780 rpm and 25 ° . Picture was taken 16 min after full speed was reached . Sedimentation is from left to right .
ATC with 1 M NaSCN and freeze-thawing in liquid nitrogen also produced the 11S protein species . When NaCIO 4 was substituted for NaSCN, the 11S subunit was still formed. When 1 M NaCl was used, freeze-thawing in liquid nitrogen failed to produce the 118 species . These results suggest that hydrophobic forces are involved in holding the 11S protein subunits together in the capsid . Both thiocyanate and perchlorate disrupt water structure and thus weaken hydro288
Copyright Q 1972 by Academia Prew, Sic .
Bra, 2 . Quehterlony double-diffusion studies . The medium used was 1 .5% Bacto-Agar. In plates a, e . c, and e, .1 0 ionic strength sodium phosphate buffer, pH 7 .0 was used ; in plate, d, 0 .l ionic strength sodium phosphate buffer, pIl 6 .0 . All phase samples and subunit samples were approximately 1 mg/ml and in p1l 7,0 buffer, a, Ab, rabbit antiserum to whole R17 phage . S is R17 subunit and is Rd7 phagc treated with I Al NaSCN with repeated freeze-thawing . DS, the, sample in S dialyzed inn, 0 .1 ionic strength buffer, pH 7 .0 . AS, the sample in S purified by airunonium sulfate fractionation free o£ 1,11 RN?. . R17, the -whole 1117 phago . Note lines of identity between S, AS, and DS and the very close relationship of R._7 to S and 1JS, b, ATC, artificial top component preparation . STC, ATC preparation treated with 1 hd NaSCN and repeatedly freeze-thawed . Note lines of identity between STO and S subunits . . MS2 is M92 subunit and is a sample of whole MS2 phagc treated with 1 dd NaSCN and repeatedly frcezcthawed . f2 is f2 subunit and is a sample of whole f2 phage treated with 1 31 NaSCN and repeatedly freeze--tha.wed . Note lines of identity between MS2, AS, DS, and £2 subunits . d, Tmanunodiffasion study of subunit preparations performed in pH 6 .0 agar . Note further disaggregation and apparent reaggregation, probably to artificial top component . In all cases where precipitin lines meet, there is total identity between S, MS2, DS, and f2 snharnits . e, Right-angle immunodiffusien study . AntiR17 is rabbit antiserum to whole R17 phage . R17sub is R17 subunit and is the 8 .17 whole phage preparation treated with 1 d1 NaSCN and repeatedly freeze-thawed . 1117-AS-Subb is the 11,17-Sub preparation freed from all RNA by ammonium sulfate purification . The tangent of the angle the precipitin line makes with the antigen, well is the same within experimental error for both subunits, 1177-AS-Sub and R17 -Sub, indicating they have the same diffusion coefficient . 259
290
SHORT COMI1UNICATIONS
phobic interactions between protiens ; chloride ions do not produce this effect (11) . Cowpea chlorotic mottle virus (.12) was completely and rapidly disassembled by merely treating it at pH 7 with 1 M NaCl. Apparently the stability of various viruses is a complex phenomenon . Freezing and thawing MS2 and f2 bacteriophages (Miles Laboratories) in the presence of 1 _h7 NaSCN also caused these viruses, both closely related to 1117, to form 11S subunits as shown by sedimentation velocity experiments in I . M NaSCN . The f2 subunit is probably the same species obtained by Zclazo and Haschemeyer (13, 14) . The major consequence of the difference in the procedure we followed and that previously reported for the IIS subunit from f2 (13) may well be in the relatively gentle effect of the thiocyann .te anion with freezing on the coat protein . We have established that there is no observable effect of the dissociation on the immunological properties of the coat protein . It is important to demonstrate that any subunit obtained is not denatured or an artifact of the treatment, and that it is a true structural component and perhaps an assembly step for the virus . In the presence of rabbit antiserum to purified 1117, the antigenic behavior of the 11S subunit was very similar to that of the whole virus (Fig. 2) . This relationship between the surface structure of the whole virus and the 11S subunit indicates that the subunits and the virial capsomeres are closely associated . The antigenic characteristics of the 11S subunit from MS2 and f2 likewise arc similar to those of the 11S subunit from R17 . Right-angle immunodiffusion plates allowed calculation of a diffusion coefficient of 3 .6 X 10 -7 em-!sec for the subunit (1G) . This value was the same within experimental error for both purified and unpurified subunits, The identity of both the sedimentation and diffusion coefficients for purified and unpurified R17 suggests that the purification procedure did not alter the size of the subunit . An approximate subunit weight of 2 .8 X 10° was calculated by the Svedberg equation . When pH 6.0 was used for these antigen-antibody reac-
tions, , additional precipitation lines, both faster and slower, were observed . ACKNOWLEDGMENTS This research was supported in part by Grant GB-21315 from the National Science Foundation . R17 bacteriophage was prepared in large batches by the New England Enzyme Center, Tufts University School of Medicine, Boston, Mass . Our original sample of the virus was a gift from Dr . James Eley . We thank Drs, Donald Roufa and Martia Kessel for their advice and expertise in microbiology . REFERENCES 1 . KnsxsEm, D . J ., Bacteriol . Rev . 33, 302-345 (1(69) . 2 . DURHAM, A . C . H ., FINCH, J . T ., and KLUG, A ., Nature New Biol . 229, 37-42 (1971) . 3 . DuREAM ; A . C . H ., and KLUG, A ., Nature New Biol . 229, 42-46 (1971) . 4 . BLEVrss, M ., and E,AwcEnmtoox, K . B ., Can . J . ,'Ticrobiol . 17, 947-954 (1971), 5 . MACCOLL, R ., LEE, J . J ., and BERxs, D . S ., Bioclaem . J . 122, 421-426 (1971)6 . EDWARDS, M . R ., and GANTT, E ., J . Cell Biol . 50, 896--900 (1971) . 7 . OsaoRy, M ., WEINER, A. M ., and WERER, K ., Fur . J . Biochem . 17, 63-67 (1970) . 8 . GES'TELA\D, F . F ., and BOEDTKER, H .,,T . Mol . Bid . 8, 496-507 (1964) . 9 . HAYSTEIN, W . G ., DAVIS, K . A ., GnALAMROR, M . A ., and HATEFH, Y ., Biochemistry 10, 2517-2524 (1971) . 10 . SAMUELSON, G ., and KAESDERO, P ., J . Mat . Bid . 47, 87-91 (1970) . 11 . HATEFI, Y ., and HANSTEIN, W . G ., 1'ror; . Nat . Acad . Sci . U . S . A . 62, 1129-1136 (1969) . 19 . BANCROFT, J . B ., WAGNER, G . W ., and BRACEER, C . E ., Virology 36, 14(i-149 (1968) . 13 . ZELAZO, P . 0 ., and H .ASCREMEFER, IL. H ., Biochemistry 8, 3587-3592 (1969) . 14 . ZELAZO, P . 0 ., and HASCREIIEYER, R. H ., Science 168, 1461-1462 (1970) . 15 . ALLISON, A . C ., and HuMrnpEY, J . H ., Immunnlogy 3, 9-106 5 (1960) . ROBERT MACCOLL DONALD S . BERNS NORMAN L . K04EN' Division of Laboratories and Research New York State Department of Health Albany, New York 1 .2201 Accepted January 10, 1972 ' A summer student on a fellowship from the Brown_Hazcn Fund, Research Corporation
Formation of a Coat Protein Subunit from R17 Bacteriophage
of the samples into pH 7, 0 .1 ionic strength sodium phosphate buffer resulted in material with a typical protein spectrum (X mn = 276 am, y m, a = 249 nm, and A959/duo = 1 .5) . This material sedimented with s,o,, = 10 .9 X 10 -" in the model E analytical ultracentrifuge (Fig . 1) . Artificial top component (ATC) of R17 has been prepared by Samuelson and Kaesberg (10) . This material is the intact capsid minus the RNA . Treatment of
The most probable path for the assembly of large protein particles is through stepwise aggregation of smaller subunits (1) . Evidence for this mechanism is found in diverse biological sources, including tobacco mosaic virus (,2, 3), tail of bacteriophage lambda (4), and phycobilisomes of blue-green and red algae (5, 6) . To study this process further an isometric virus was disassembled into protein subunits . 1117 bacteriophage was selected because its capsid is relatively simple, consisting of about 7 .80 molecules of coat protein and 1 molecule of A protein (7) . The only other component of this virus is a single strand of RNA (8) . By the term "subunit" is meant a protein species composed of two or more coat protein molecules and smaller than the empty capsid (41S) . The capsid of R17 was found to be resistant to many possible methods of disassembly. Recently, the disaggregation of succinatc dchydrogenase was accomplished by freezing the enzyme in the presence of sodium perchlorate (9) . Application of this technique to R17 resulted in a high yield of an 11S subunit . This subunit was formed by suspending the virus in 1 lbl sodium thiocyanate, pH 7 .1, freezing in liquid nitrogen, and thawing six times . Sedimentation velocity experiments with schheren optics in the analytical ultracentrifuge showed . a major boundary scdimenting at, 11S in the 1 1VI NaSCN, pH 7, solution . Neither freezing and thawing nor 1 M NaSCN alone produced any 118 species . Furthermore, the treatment of R17 bacteriophage with 0 .2 Dl NaSCN, pH 7, and freeze-thawing completely destroyed its plaque-forming ability, but with the addition of 0 .2 na NaSCN alone or freezing alone at pH 7, its infectivity was maintained . Removal of the 1 M NaSCN by dialysis and treatment with ribonuclease (Worthington Biochemicals, 4400 U/mg) did not further disassemble the 1IS component . Precipitation with 33% saturated ammonium sulfate and redissolving in 1 A NaSCN, pH 7, three times and final dialysis
Fm . 1 . Sedimentation velocity pattern obtained with schlieren optics for the ammonium sulfatepurified 11S subunit . Experiments were performed at 59,780 rpm and 25 ° . Picture was taken 16 min after full speed was reached . Sedimentation is from left to right .
ATC with 1 M NaSCN and freeze-thawing in liquid nitrogen also produced the 11S protein species . When NaCIO 4 was substituted for NaSCN, the 11S subunit was still formed. When 1 M NaCl was used, freeze-thawing in liquid nitrogen failed to produce the 118 species . These results suggest that hydrophobic forces are involved in holding the 11S protein subunits together in the capsid . Both thiocyanate and perchlorate disrupt water structure and thus weaken hydro288
Copyright Q 1972 by Academia Prew, Sic .
Bra, 2 . Quehterlony double-diffusion studies . The medium used was 1 .5% Bacto-Agar. In plates a, e . c, and e, .1 0 ionic strength sodium phosphate buffer, pH 7 .0 was used ; in plate, d, 0 .l ionic strength sodium phosphate buffer, pIl 6 .0 . All phase samples and subunit samples were approximately 1 mg/ml and in p1l 7,0 buffer, a, Ab, rabbit antiserum to whole R17 phage . S is R17 subunit and is Rd7 phagc treated with I Al NaSCN with repeated freeze-thawing . DS, the, sample in S dialyzed inn, 0 .1 ionic strength buffer, pH 7 .0 . AS, the sample in S purified by airunonium sulfate fractionation free o£ 1,11 RN?. . R17, the -whole 1117 phago . Note lines of identity between S, AS, and DS and the very close relationship of R._7 to S and 1JS, b, ATC, artificial top component preparation . STC, ATC preparation treated with 1 hd NaSCN and repeatedly freeze-thawed . Note lines of identity between STO and S subunits . . MS2 is M92 subunit and is a sample of whole MS2 phagc treated with 1 dd NaSCN and repeatedly frcezcthawed . f2 is f2 subunit and is a sample of whole f2 phage treated with 1 31 NaSCN and repeatedly freeze--tha.wed . Note lines of identity between MS2, AS, DS, and £2 subunits . d, Tmanunodiffasion study of subunit preparations performed in pH 6 .0 agar . Note further disaggregation and apparent reaggregation, probably to artificial top component . In all cases where precipitin lines meet, there is total identity between S, MS2, DS, and f2 snharnits . e, Right-angle immunodiffusien study . AntiR17 is rabbit antiserum to whole R17 phage . R17sub is R17 subunit and is the 8 .17 whole phage preparation treated with 1 d1 NaSCN and repeatedly freeze-thawed . 1117-AS-Subb is the 11,17-Sub preparation freed from all RNA by ammonium sulfate purification . The tangent of the angle the precipitin line makes with the antigen, well is the same within experimental error for both subunits, 1177-AS-Sub and R17 -Sub, indicating they have the same diffusion coefficient . 259
290
SHORT COMI1UNICATIONS
phobic interactions between protiens ; chloride ions do not produce this effect (11) . Cowpea chlorotic mottle virus (.12) was completely and rapidly disassembled by merely treating it at pH 7 with 1 M NaCl. Apparently the stability of various viruses is a complex phenomenon . Freezing and thawing MS2 and f2 bacteriophages (Miles Laboratories) in the presence of 1 _h7 NaSCN also caused these viruses, both closely related to 1117, to form 11S subunits as shown by sedimentation velocity experiments in I . M NaSCN . The f2 subunit is probably the same species obtained by Zclazo and Haschemeyer (13, 14) . The major consequence of the difference in the procedure we followed and that previously reported for the IIS subunit from f2 (13) may well be in the relatively gentle effect of the thiocyann .te anion with freezing on the coat protein . We have established that there is no observable effect of the dissociation on the immunological properties of the coat protein . It is important to demonstrate that any subunit obtained is not denatured or an artifact of the treatment, and that it is a true structural component and perhaps an assembly step for the virus . In the presence of rabbit antiserum to purified 1117, the antigenic behavior of the 11S subunit was very similar to that of the whole virus (Fig. 2) . This relationship between the surface structure of the whole virus and the 11S subunit indicates that the subunits and the virial capsomeres are closely associated . The antigenic characteristics of the 11S subunit from MS2 and f2 likewise arc similar to those of the 11S subunit from R17 . Right-angle immunodiffusion plates allowed calculation of a diffusion coefficient of 3 .6 X 10 -7 em-!sec for the subunit (1G) . This value was the same within experimental error for both purified and unpurified subunits, The identity of both the sedimentation and diffusion coefficients for purified and unpurified R17 suggests that the purification procedure did not alter the size of the subunit . An approximate subunit weight of 2 .8 X 10° was calculated by the Svedberg equation . When pH 6.0 was used for these antigen-antibody reac-
tions, , additional precipitation lines, both faster and slower, were observed . ACKNOWLEDGMENTS This research was supported in part by Grant GB-21315 from the National Science Foundation . R17 bacteriophage was prepared in large batches by the New England Enzyme Center, Tufts University School of Medicine, Boston, Mass . Our original sample of the virus was a gift from Dr . James Eley . We thank Drs, Donald Roufa and Martia Kessel for their advice and expertise in microbiology . REFERENCES 1 . KnsxsEm, D . J ., Bacteriol . Rev . 33, 302-345 (1(69) . 2 . DURHAM, A . C . H ., FINCH, J . T ., and KLUG, A ., Nature New Biol . 229, 37-42 (1971) . 3 . DuREAM ; A . C . H ., and KLUG, A ., Nature New Biol . 229, 42-46 (1971) . 4 . BLEVrss, M ., and E,AwcEnmtoox, K . B ., Can . J . ,'Ticrobiol . 17, 947-954 (1971), 5 . MACCOLL, R ., LEE, J . J ., and BERxs, D . S ., Bioclaem . J . 122, 421-426 (1971)6 . EDWARDS, M . R ., and GANTT, E ., J . Cell Biol . 50, 896--900 (1971) . 7 . OsaoRy, M ., WEINER, A. M ., and WERER, K ., Fur . J . Biochem . 17, 63-67 (1970) . 8 . GES'TELA\D, F . F ., and BOEDTKER, H .,,T . Mol . Bid . 8, 496-507 (1964) . 9 . HAYSTEIN, W . G ., DAVIS, K . A ., GnALAMROR, M . A ., and HATEFH, Y ., Biochemistry 10, 2517-2524 (1971) . 10 . SAMUELSON, G ., and KAESDERO, P ., J . Mat . Bid . 47, 87-91 (1970) . 11 . HATEFI, Y ., and HANSTEIN, W . G ., 1'ror; . Nat . Acad . Sci . U . S . A . 62, 1129-1136 (1969) . 19 . BANCROFT, J . B ., WAGNER, G . W ., and BRACEER, C . E ., Virology 36, 14(i-149 (1968) . 13 . ZELAZO, P . 0 ., and H .ASCREMEFER, IL. H ., Biochemistry 8, 3587-3592 (1969) . 14 . ZELAZO, P . 0 ., and HASCREIIEYER, R. H ., Science 168, 1461-1462 (1970) . 15 . ALLISON, A . C ., and HuMrnpEY, J . H ., Immunnlogy 3, 9-106 5 (1960) . ROBERT MACCOLL DONALD S . BERNS NORMAN L . K04EN' Division of Laboratories and Research New York State Department of Health Albany, New York 1 .2201 Accepted January 10, 1972 ' A summer student on a fellowship from the Brown_Hazcn Fund, Research Corporation