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Intracellular crystallization of the DNA coliphage N4
DISCUSSION
AND
PI~ELIRIINAI:Y
REFERENCES 1. HANAFUSA, H., HANAFUSA, T., and RUBIN, H., Proc. Natl. Acad. Sci. U.S. 49, 572-580 (1963). 2. LEVINSON, W., and RUBIN, H., Virology 28, 533-542 (1966). 3. HANAFUSA, H., and HANAFUSA, T., hoc. NatZ. Acod. Sci. U.S. 55.532-538 (1966). I’. K. Virology 30, 4. ISHIZAKI, R., and Vow, 375-387 (1966). 5. DOUGHERTY, R. M., and DI STEFANO, H. S., Virology 27, 351-359 (1965). 6. HANAFUSA, H., HANAFUSA, T., and RUBIN, H., Proc. Natl. Acad. Sci. U.S. 51, 41-48 (1964). 7. RUBIN, H. Proc. Natl. Acad. Sci. U.S. 46, 11051119 (1960). ROBIN WEISS Department of Anatomy and Embryology University College London London, England Accepted May 22, 1967
Intracellular
Crystallization Coliphage
of the DNA N4
Bacteriophage N4, a virus active on Escherichiu coli K12 strains (I), consists of a double-stranded DNA core of 40.5 million daltons (2), a protein coat composed of over 2000 chemical subunits (S), a base plate and a small noncontractile tail with 4-6 prongs or spikes. Ths diameter of the particle measures 700 A, and the molecular weight of the phage is S3 X lo6 (4). N4infected bacteria, in mass culture, are lysis-inhibited (5) and disintegrate only after extended periods of incubation, yielding high titers (5 t,o 10 X 10” PFU/ml) of phage (1, 6). Since, during N4 replicat’ion, the syntheses of host macromolecular components continue practically unimpaired, while the cell division processes are brought to a rapid halt., the infected population consists of hypertrophic individuals ranging in dimensions from 1.5 X 6 to 1.8 X 9p. Working with E. coli male-specific RNA phages, several investigators have shown that, whenever the infected bacteria do not lyse before the virion concentration exceeds a critical density of about 2000-3000 phages per cell, condensation of the particles in paracrystalline arrays may be noted (7-g). Since in the late stages of N4 development t’he infected complexes contain over 3000
I:EPOI
723
infectious units per cell, an att’empt has been made to ascertain whether these conditions were sufficient to induce the coalescence of the viral progeny into paracrystalline formations similar to those observed with the structurally more simple RNA phages. The electron micrographs presented here show that this is the case. Apart from the electron-transparent crystals described by Cole (10) in Cl phage-infected streptococcal cells, this seems to be the first report on the intracellular crystallization of a morphologically complex bacteriophage. Cultures of E. coli K12S grown with aeration at 37” in Brain Heart Infusion broth (Difco) to a density of 1 to 3 X IO* cells per milliliter were infected at a multiplicity of about 10 N4 particles per cell. After 240 minutes of incubation the bacteria were chilled rapidly and centrifuged at 10,000 rpm for 5 minutes. The pellets were fixed in glutaraldehyde and postfixed in osmium tetroxide as described by Margaretten et al. (11). Araldite \vas used for embedding. Thin sections, cut, with an LKB ultramicrotome and stained with uranyl acetate and lead citrate (Is), were examined in an Akashi Tronscope electron microscope. As shown in Fig. 1 a-d, crystalline inclusions are readily recognizable in thin sections of N4-infected cells. The structures in these arrays are electron dense and must certainly be virus, since they do not appear in normal cells incubated under the same conditions. The center-to-center distances between closely0 adjacent particles are of the order of 660A, a value slightly smaller than that obtained for the diameter of purified N4 after negative staining but in excellent agreement with the phage diamet,er computed from light-scattering measurements (4). Since t,he dimensions of the particles are comparable t’o those of the extracellular virus, the crystalline arrays are probably made up of completely assembled N4. If this is so, the distinct molecular asymmetry introduced in the virus particles by the presence of the small but rather complex adsorption apparatus does not seem to constitute, by itself, an obstacle sufficient to prevent the coalescence of the phage
FIG.
1. (a-d) Electron
micrographs
of ultraihill
sections
of Escherichia
coli K12S cells 240 minL ttes
aft,er infection with N4 bacteriophage. (a and 1)) Bacteria containing viral particles grouped together in la.rge crystalline formations. (c) 9 paracrystalline inclusion of N4 coliphage in wh .ich hexagonally shaped phage head membranes are observed as well as partially and fully conden sed heads.
(d) Paracryst
alline
arrays
of virions
in a backrillm
22,000; (h) X 44,000; Cc) X 35,200; ((1) x 21,120.
lmdergoing
lysis.
Magnification:
(a) X
progeny into ordered three-dimensional inclusions. N4 crystalline aggregates are present in about 2 % of t,he thin sections examined, a frequency an order of magnitude lower than that reported for the paracr:\stalline arrays produced during RKX cohphage replication. Since the volume occupied by N4 virus mat,erial attains the same levels (7) reached by f2 viroplasm prior to lysis (.5%. and 3.5 % of the t,otal cell volume, rcsp&ivcly), while the absolute number of phage present is very different in the t,wo cases (2000-3000 I’I:U as compared to about 20,000), this last parameter ma?- be of paramount importance in governing the frequcnc>- with which conditions suikble for crystallizat’ion are provided in restricted areas of the cell. A more complete st#udy on the stages of X4 replicat’ion and on the sequence of cellular fine structure changes subsequent to infect,ion is underway.
This investigation 115.12i1.0./1211 from Ricerche.
was srlpported 1)~ yritl~t Consiglio Nazionale delle
REFERENCES 1. MOLINA, A. M., PESCE, A.: and SCHITO, C;. C., Boll. Ist. Sieroterap. Xi/an. 44, 329-337 (1965). 2. SCHIT~, (:. C., RIALDI, (i., a~ld PESCE, A., Biochim. Biophys. Ada 129, 491-501 (1966). Jlicrobiol. 14, i5-88 3. SCHITO, G. C., Giorn. (1966) 4 SCHITO, Ci. C., RIALDI, (i., aud PESCE, A., Biochim. Riophys. Ada 129, 482-190 (1966). 5. DOERMAN, A. H., J. Bucleriol. 55, 257-276 (1948). 6. SCHITO, G. C., Virology 30, 157-159 (1966). r. SCHWARTZ, F. &‘I., and ZIXDER, N. I)., Vi’irology 21, 2iGp278 (1963) 8. DE PETRIS, S., alld Nava, C;., Giorn. Microbial. 11, l-7 (1963). 9. FRANKLIN, R. M., and C~RAXB~ULAN, N., J. Racteriol. 91, 834-818 (1966). i0. COLE, R. M., T'irology 26, 509-511 (1965). 11. MARGARETTEN, W., ?Ilonca~, C., ROSENKILANZ, H. W., and ROSE, H. M., J. Bacterial. 91, 823-833 (1966). 12. REYNOLDS, X. S., J. Cell. Biol. 17, 208-212 (1963). GIllN
Znstit&
of Microbiology
CaRLO
SCHITO
l,:nicersil!/ of Genoa Jferlical Genoa, Zfaly =I ccepled Jfa~y 12, 196Y
Immunofluorescent Surface
Demonstration
Antigen
Transformed
School
in Cells
of
a Specific
Infected
by Polyoma
or
Virus
Habe! (I) and Sjiigren et al. (2) have reported that adult mice immunized with polyoma virus (I’V) arc resistant to transplantation with polyoma tumor cells, and this rcsistancc is specific for tumors rcsulting from I’\’ infcctJion. Evidence has been obtained that this phenomenon is due t>o new transplantatit~n unt)igen present in tumor cells determined by I’V but distinct from the ant’igcns of t,hc virus particles (3). Subsequently, specific virus-induced transplantation antigens wcrc also found in cells transformed in vice and in vitro by &her oncogenic viruses. However, progress in the analysis of these antigens has been hindered by t’he absence of serologic tests or other immunologic reactions which can bc conducted in vi&o or at the level of a single cell. WC have made attcmpt)s to apply the fluorescent antibody method t’o the dctcction of specific tranrsplantation antigen in polyoma-transformed ham&r cells, as well as mouse and rat cells infected with PV. Antiserum against the specific transplantation antigen of PIT @PTA) was obtained after pretrcatmcnt of adult C8Hh mice with homografts of 11/f transplantable salivary gland polyoma tumor. The tumor coiit’ains the polyoma specific transplnntation antigen according to transplantation test (1-3). Six to eight inoculations were given subcut’ancously at intervals of 2-6 weeks. The homografted tumors regressed uniformly after a period of temporary growth. This t,umor was free of PV when tested by inoculation of mouse kidney cell culture ,4PO (4) with tumor cells. Sera of A/f mice wit’h transplantable tumors or immunized C3HA recipients were found to contain no antiviral antibodies by hemagglutination irrhibit,ion (HI) and neutralization tests.
AND
PI~ELIRIINAI:Y
REFERENCES 1. HANAFUSA, H., HANAFUSA, T., and RUBIN, H., Proc. Natl. Acad. Sci. U.S. 49, 572-580 (1963). 2. LEVINSON, W., and RUBIN, H., Virology 28, 533-542 (1966). 3. HANAFUSA, H., and HANAFUSA, T., hoc. NatZ. Acod. Sci. U.S. 55.532-538 (1966). I’. K. Virology 30, 4. ISHIZAKI, R., and Vow, 375-387 (1966). 5. DOUGHERTY, R. M., and DI STEFANO, H. S., Virology 27, 351-359 (1965). 6. HANAFUSA, H., HANAFUSA, T., and RUBIN, H., Proc. Natl. Acad. Sci. U.S. 51, 41-48 (1964). 7. RUBIN, H. Proc. Natl. Acad. Sci. U.S. 46, 11051119 (1960). ROBIN WEISS Department of Anatomy and Embryology University College London London, England Accepted May 22, 1967
Intracellular
Crystallization Coliphage
of the DNA N4
Bacteriophage N4, a virus active on Escherichiu coli K12 strains (I), consists of a double-stranded DNA core of 40.5 million daltons (2), a protein coat composed of over 2000 chemical subunits (S), a base plate and a small noncontractile tail with 4-6 prongs or spikes. Ths diameter of the particle measures 700 A, and the molecular weight of the phage is S3 X lo6 (4). N4infected bacteria, in mass culture, are lysis-inhibited (5) and disintegrate only after extended periods of incubation, yielding high titers (5 t,o 10 X 10” PFU/ml) of phage (1, 6). Since, during N4 replicat’ion, the syntheses of host macromolecular components continue practically unimpaired, while the cell division processes are brought to a rapid halt., the infected population consists of hypertrophic individuals ranging in dimensions from 1.5 X 6 to 1.8 X 9p. Working with E. coli male-specific RNA phages, several investigators have shown that, whenever the infected bacteria do not lyse before the virion concentration exceeds a critical density of about 2000-3000 phages per cell, condensation of the particles in paracrystalline arrays may be noted (7-g). Since in the late stages of N4 development t’he infected complexes contain over 3000
I:EPOI
723
infectious units per cell, an att’empt has been made to ascertain whether these conditions were sufficient to induce the coalescence of the viral progeny into paracrystalline formations similar to those observed with the structurally more simple RNA phages. The electron micrographs presented here show that this is the case. Apart from the electron-transparent crystals described by Cole (10) in Cl phage-infected streptococcal cells, this seems to be the first report on the intracellular crystallization of a morphologically complex bacteriophage. Cultures of E. coli K12S grown with aeration at 37” in Brain Heart Infusion broth (Difco) to a density of 1 to 3 X IO* cells per milliliter were infected at a multiplicity of about 10 N4 particles per cell. After 240 minutes of incubation the bacteria were chilled rapidly and centrifuged at 10,000 rpm for 5 minutes. The pellets were fixed in glutaraldehyde and postfixed in osmium tetroxide as described by Margaretten et al. (11). Araldite \vas used for embedding. Thin sections, cut, with an LKB ultramicrotome and stained with uranyl acetate and lead citrate (Is), were examined in an Akashi Tronscope electron microscope. As shown in Fig. 1 a-d, crystalline inclusions are readily recognizable in thin sections of N4-infected cells. The structures in these arrays are electron dense and must certainly be virus, since they do not appear in normal cells incubated under the same conditions. The center-to-center distances between closely0 adjacent particles are of the order of 660A, a value slightly smaller than that obtained for the diameter of purified N4 after negative staining but in excellent agreement with the phage diamet,er computed from light-scattering measurements (4). Since t,he dimensions of the particles are comparable t’o those of the extracellular virus, the crystalline arrays are probably made up of completely assembled N4. If this is so, the distinct molecular asymmetry introduced in the virus particles by the presence of the small but rather complex adsorption apparatus does not seem to constitute, by itself, an obstacle sufficient to prevent the coalescence of the phage
FIG.
1. (a-d) Electron
micrographs
of ultraihill
sections
of Escherichia
coli K12S cells 240 minL ttes
aft,er infection with N4 bacteriophage. (a and 1)) Bacteria containing viral particles grouped together in la.rge crystalline formations. (c) 9 paracrystalline inclusion of N4 coliphage in wh .ich hexagonally shaped phage head membranes are observed as well as partially and fully conden sed heads.
(d) Paracryst
alline
arrays
of virions
in a backrillm
22,000; (h) X 44,000; Cc) X 35,200; ((1) x 21,120.
lmdergoing
lysis.
Magnification:
(a) X
progeny into ordered three-dimensional inclusions. N4 crystalline aggregates are present in about 2 % of t,he thin sections examined, a frequency an order of magnitude lower than that reported for the paracr:\stalline arrays produced during RKX cohphage replication. Since the volume occupied by N4 virus mat,erial attains the same levels (7) reached by f2 viroplasm prior to lysis (.5%. and 3.5 % of the t,otal cell volume, rcsp&ivcly), while the absolute number of phage present is very different in the t,wo cases (2000-3000 I’I:U as compared to about 20,000), this last parameter ma?- be of paramount importance in governing the frequcnc>- with which conditions suikble for crystallizat’ion are provided in restricted areas of the cell. A more complete st#udy on the stages of X4 replicat’ion and on the sequence of cellular fine structure changes subsequent to infect,ion is underway.
This investigation 115.12i1.0./1211 from Ricerche.
was srlpported 1)~ yritl~t Consiglio Nazionale delle
REFERENCES 1. MOLINA, A. M., PESCE, A.: and SCHITO, C;. C., Boll. Ist. Sieroterap. Xi/an. 44, 329-337 (1965). 2. SCHIT~, (:. C., RIALDI, (i., a~ld PESCE, A., Biochim. Biophys. Ada 129, 491-501 (1966). Jlicrobiol. 14, i5-88 3. SCHITO, G. C., Giorn. (1966) 4 SCHITO, Ci. C., RIALDI, (i., aud PESCE, A., Biochim. Riophys. Ada 129, 482-190 (1966). 5. DOERMAN, A. H., J. Bucleriol. 55, 257-276 (1948). 6. SCHITO, G. C., Virology 30, 157-159 (1966). r. SCHWARTZ, F. &‘I., and ZIXDER, N. I)., Vi’irology 21, 2iGp278 (1963) 8. DE PETRIS, S., alld Nava, C;., Giorn. Microbial. 11, l-7 (1963). 9. FRANKLIN, R. M., and C~RAXB~ULAN, N., J. Racteriol. 91, 834-818 (1966). i0. COLE, R. M., T'irology 26, 509-511 (1965). 11. MARGARETTEN, W., ?Ilonca~, C., ROSENKILANZ, H. W., and ROSE, H. M., J. Bacterial. 91, 823-833 (1966). 12. REYNOLDS, X. S., J. Cell. Biol. 17, 208-212 (1963). GIllN
Znstit&
of Microbiology
CaRLO
SCHITO
l,:nicersil!/ of Genoa Jferlical Genoa, Zfaly =I ccepled Jfa~y 12, 196Y
Immunofluorescent Surface
Demonstration
Antigen
Transformed
School
in Cells
of
a Specific
Infected
by Polyoma
or
Virus
Habe! (I) and Sjiigren et al. (2) have reported that adult mice immunized with polyoma virus (I’V) arc resistant to transplantation with polyoma tumor cells, and this rcsistancc is specific for tumors rcsulting from I’\’ infcctJion. Evidence has been obtained that this phenomenon is due t>o new transplantatit~n unt)igen present in tumor cells determined by I’V but distinct from the ant’igcns of t,hc virus particles (3). Subsequently, specific virus-induced transplantation antigens wcrc also found in cells transformed in vice and in vitro by &her oncogenic viruses. However, progress in the analysis of these antigens has been hindered by t’he absence of serologic tests or other immunologic reactions which can bc conducted in vi&o or at the level of a single cell. WC have made attcmpt)s to apply the fluorescent antibody method t’o the dctcction of specific tranrsplantation antigen in polyoma-transformed ham&r cells, as well as mouse and rat cells infected with PV. Antiserum against the specific transplantation antigen of PIT @PTA) was obtained after pretrcatmcnt of adult C8Hh mice with homografts of 11/f transplantable salivary gland polyoma tumor. The tumor coiit’ains the polyoma specific transplnntation antigen according to transplantation test (1-3). Six to eight inoculations were given subcut’ancously at intervals of 2-6 weeks. The homografted tumors regressed uniformly after a period of temporary growth. This t,umor was free of PV when tested by inoculation of mouse kidney cell culture ,4PO (4) with tumor cells. Sera of A/f mice wit’h transplantable tumors or immunized C3HA recipients were found to contain no antiviral antibodies by hemagglutination irrhibit,ion (HI) and neutralization tests.