Vol. 23, No. 2 Printed in U.S.A.

VIROLOGY, Aug. 1977, p. 439-442

Copyright ( 1977 American Society for Microbiology

Two Infectious Forms of Bacteriophage OX174 HISAO FUJISAWA' AND MASAKI HAYASHI* Department of Biology, University of California, San Diego, La Jolla, California 92093 Received for publication 11 April 1977

Infectious particles with S values of 114 and 132 were isolated from cells infected with bacteriophage OX174. Electron micrographs of the 132S particle revealed a spherical structure with a diameter of about 40 nm. The 114S particle had spikelike projections and a diameter of about 32 nm. The 132S particles could be converted to 114S particles in vitro. However, pulse and pulse-chase experiments indicated no precursor-product relationship between these two particles in vivo. The icosahedral 114S virion particle of bacteriophage OX174 consists of a circular singlestranded DNA molecule enclosed by a protein coat. Weisbeek and Sinsheimer (3) have reported isolating, from Escherichia coli cells infected with 4X174, infectious particles having S values of 140 and 114. The 140S particle contains the protein products of phage genes F, G, H, and J in the same proportions as those found in the 114S phage particle. In addition, the 140S particle contains D protein, which is not present in the 114S particle. Under appropriate conditions, the 140S particle can be converted in vitro to the 114S phage particle. This conversion process is apparently irreversible, since addition of 114S particles to cell extracts containing D protein does not lead to alteration of the S value of the added phage particles. Previously we described the presence in phage-infected cells of a particle with a sedimentation value of 132S (1, 2). Our 132S particle has virtually the same properties as those reported by Weisbeek and Sinsheimer (3) for their 140S particle, implying that these two particles are identical. The similarity between the compositions of the 132S and 114S particles and the fact that the 132S particle can be converted in vitro to 114S phage particles led us to characterize the 132S particle in more detail to elucidate the relationship between these two particles during phage development. Labeling patterns of 132S and 114S particles during a pulse-chase experiment are shown in Fig. 1. E. coli HF4704 was infected with a lysisdefective mutant of 4X174 (N11) and pulsed with [3H]thymidine between 20.0 and 20.5 min after infection. The culture was then chased with an excess amount of unlabeled thymidine, and portions of the culture were removed 0.5 and 25 min after adding unlabeled thymidine.

Extracts of the infected cells were prepared and analyzed by sedimentation in sucrose gradients. Radioactivity incorporated after the short chase (Fig. la, 0.5 min) into the 50S com-


I0 x


Q. C.


10 20 fraction number


FIG. 1. Sedimentation profile of [3H]DNA from extracts of cells infected with 4X174. Mitomycintreated E. coli HF4704 (thyA, hcr-) was infected at 370C with a lysis-defective mutant of 4X1 74 (Nil) at a multiplicity of infection of 7. [3H]thymidine (5 jaCil ml, 1 iuglml) was added to the culture 20 min after infection and chased at 20.5 min after infection by the addition of unlabeled thymidine (200 jug/ml) and thymine (50 mglml). Samples were taken at 21 min (a) and 45.5 min (b) after infection. Cell extracts were prepared and centrifuged through 5 to 30% sucrose gradients in an SW50.1 rotor at 49,000 rpm for 65 min at 4TC. In this and subsequent figures, direction of sedimentation is from right to left. Me' Present address: Department of Botany, Faculty of Sci- dia, buffer, and preparation of extracts have been ence, Kyoto University, Kyoto, Japan. detailed in previous papers (1, 2). 439




plex and RFII, which both are phage precursors (1), moves into the 132S and 114S peaks after the long chase (Fig. lb, 25 min). Radioactivity in the 132S peak is not chased into the 114S peak even after prolonged chase periods beyond 25 min. The specific infectivity, defined as the ratio of PFU to radioactivity (counts per minute), of the 132S peak is about the same as that of the 114S peak after either the short or the long chase period (Table 1). Sedimentation patterns identical to those shown in Fig. 1 were obtained with extracts from cells infected with wild-type phage (data not shown). These observations indicate that the 132S particle is not a precursor of the 114S particle in vivo.







The effects of various treatment on the sedimentation behavior of purified 132S particles are shown in Fig. 2. In the presence of buffer containing 1 mM Mg2+, the 132S particle is very efficiently converted to infectious 114S particles (Fig. 2b). The specific infectivity of the 114S particles obtained is identical to that of the 132S particles from which they were derived. Either Ca2+ or Mn2+ (1 mM, final concentration) can substitute for Mg2+ in the conversion of 132S particles to infectious 114S particles (data not shown). Dilution of 132S particles into 20 mM Tris buffer results in loss of infectivity and formation of defective particles sedimenting around 60S (Fig. 2c). These results are similar to those obtained by Weisbeek and Sinsheimer (3). The 132S particle is stable in buffer containing 0.1 M NaCl (compare Fig. 2a and c). In the presence of Zn2 , the 132S particle is converted to material that sediments with an S value slightly greater than 114 (Fig. 2d). The particles formed from 132S particles in the presence of Zn2+ are not infectious. The infectivity of 114S phage particles is not affected by Zn2+, TABLE 1. Specific infectivity of 132S and 114S







Time after chasing (mn)



0.5 25

16.9 9.2

14.9 10.5

(c) a

Specific infectivity is expressed as the ratio of

PFU to radioactivity.


1.0 K

114S particles





20 10 fraction number


FIG. 2. Conversion of 132S particles. The 132S particles labeled with [3H]thymidine and isolated as described in Fig. 1 were diluted 10-fold with (a) 0.1 M NaCl-20 mM Tris-hydrochloride (pH 7.4), (b) 0.1 M NaCl-1 mM MgCI2-20mM Tris-hydrochloride (pH 7.4), (c) 20 mM Tris-hydrochloride (pH 7.4), and (d) 0.1 M NaCl-1 mM ZnCl2-20 mM Tris-hydrochloride. (pH 7.4). Samples were kept at room temperature for 3 min and sedimented through 5 to 30% sucrose gradients as detailed in Fig. 1. No change in the sedimentation of [H]thymidine-labeled 114S particles was observed after identical treatment with the buffers listed above. The position of 114S particles is shown by the arrow in each panel.

132 S particles



4 2 3 mM of Zn


FIG. 3. Effects of Zn2+ on infectivities of 114S and 132S particles. The 114S and 132S particles were isolated as described in Fig. 1 and diluted 100-fold with buffer containing 0.1 M NaCl, 20 mM Trishydrochloride (pH 7.4), and various concentrations of ZnC12. Diluted particles were kept at room temperature for 5 min and assayed for plaque-forming ability with E. coli CR63.1 (sus+) as an indicator.

FIG. 4. Electron micrographs of 14S and 132S particles. The 114S, 132S, and 114S particles derived from 132S were negatively stained with 0.5% phosphotungstic acid (pH 7.4). The magnification bar represents 100 nm. The 114S particles (a) and 132S particles (b) were isolated from infected cells as described in Fig. 1. The 114S particles derived from 132S particles (c) were prepared by floating grids, which had adsorbed 132S particles in 0.1 M NaCl-1 mM MgCl2-10 mM phosphate buffer (pH 7.4) for 1 min at room temperature. 441



whereas 132S particles are readily inactivated by this cation (Fig. 3). Electron micrographs (Fig. 4) of the 114S phage particle reveal a structure having spikelike projections and a diameter of about 32 nm. The 132S particle appears spherical in shape and has a diameter of about 40 nm. The 114S particles derived in the presence of Mg2+ from 132S particles are indistinguishable by electron microscopy from 114S phage particles isolated directly from infected cells. We have previously proposed that D protein serves a scaffolding function during virion assembly (2; H. Fujisawa and M. Hayashi, submitted for publication). We also suggested that the D protein is in the outside layer of the 132S particle, since the infectivity of this particle is not affected by antiserum directed against 114S phage particles (2). The electron micrographs are consistent with the suggestion concerning the location of D protein in the 132S particle. Indications are that the 132S particle is not simply a 114S phage particle fortuitously associated with D protein. The 114S phage particle cannot be converted to the 132S particle after incubation in infected cell extracts containing D protein (3). Exposure of 132S particles to buffers of low ionic strength or to buffer containing Zn2+ leads to loss of infectivity, whereas 114S phage particles are unaffected by such treatment (Fig. 3). Evidently, the conformation of the virion particle undergoes an alteration upon removal of D protein from the 132S parti-


cle. "Incorrect" removal of D protein from the 132S particle leads to defective particles. These observations suggest an active role for D protein interaction with virion components during phage morphogenesis. The 114S phage particle exhibits greater stability than the infectious 132S particle and is assumed to be the mature phage particle. Our pulse-chase experiments (Fig. 1) indicate that the 132S particle is not a precursor in vivo of the 114S particle. It is possible that the 132S and 114S particles are produced from a common precursor (such as the 50S complex [1]) by two pathways. Alternatively, the 132S particle may be the mature virion made in infected cells, and the 114S particle may be formed only after lysis or purification procedures. We are grateful to F. Fujimura for invaluable help with the manuscript. This work was supported by grants from the National Science Foundation (BMS74-10710) and from the National Institute of General Medical Sciences (GM12934).

LITERATURE CITED 1. Fujisawa, H., and M. Hayashi. 1976. A viral DNA synthesizing intermediate complex isolated during assembly of bacteriophage OX174. J. Virol. 19:409415. 2. Fujisawa, H., and M. Hayashi. 1977. Functions of gene C and gene D products of bacteriophage 4X174. J. Virol. 21:506-515. 3. Weisbeek, P. J., and R. L. Sinsheimer. 1974. A DNAprotein complex involved in bacteriophage 4X-174 particle formation. Proc. Natl. Acad. Sci. U.S.A. 71:3054-3058.

Two infectious forms of bacteriophage phi X 174.

JOURNAL OF Vol. 23, No. 2 Printed in U.S.A. VIROLOGY, Aug. 1977, p. 439-442 Copyright ( 1977 American Society for Microbiology Two Infectious For...
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