VIROLOGY 174,472-478 (1990)

Quantized Viral DNA Packaging Revealed by Rotating Gel Electrophoresis

TODD LANE, PHILIP SERWER,* SHIRLEY I. HAYES,* AND FREDERICK EISERLING’

Microbiology Department and Molecular Biology institute, University of California at Los Angeles, California 90024-1489; and *Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Cur/ Drive, San Antonio, Texas 78284-7760

Received May 23, 1989; accepted October 6, 1989

Two classes of missense mutations in the bacteriophage T4 gene coding for the major head protein produce phage with different length heads. The pt (petite) mutations produce phage with normal, intermediate, and isometric heads, whereas pfg (petite and giant) mutations also produce greatly elongated (giant) heads. DNA from petite, normal, and giant particles was clearly resolved by discontinuous rotating gel electrophoresis, and several new species of headful length DNA were found. These results confirm the idea that the major stop points for head length regulation are at Q = 13, 17, and 21, and also show that minor stop points exist at Q = 16, 18 and 20. The existence of these well-defined classes of DNA that correlate with capsid structure suggest that a structural relationship between the scaffold protein and the capsid protein determines head length and thus DNA length. o 1990Academic PESS, IIIC.

INTRODUCTION

Bacteriophage T4 packages DNA by a “headful” mechanism: size of the capsid determines the amount of DNA packaged (Streisinger et a/., 1967). The length of the T4 head is precisely regulated. Attempts to un- derstand the problem of head length regulation in T4 are part of the larger problem of the determination of the shape and size of viral and cellular structures. Mu- tations in the major capsid protein gene, 23, alter the head size and the amount of DNA packaged (Eiserling et a/., 1970; Doermann et al., 1973). Head size variants were described years ago (Mosig, 1963; Mosig er a/., 1972), when the only methods of measuring DNA length were genetic mapping (Mosig, 1963), rate zonal centrifugation, or electron microscopy (Kim and David- son, 1974). Accuracy was no better than 5-l OYO, in- sufficient to test theories of head size determination. Recent developments in electrophoretic methods of DNA separation (Anand, 1986) have made possible more accurate length measurements of large, intact vi- ral molecules. We have applied the method of rotating gel electrophoresis (Serwer, 1987; Southern et al., 1987) to the DNA contained within normal and head size variants produced by phage 14 gene 23 muta- tions, and have found that there is a strong preference for certain DNA lengths. This preference can be ex- plained by the predominance of a subclass of those lattices included in the theory of icosahedral surface lattice geometry developed by Caspar and Klug (1962), as modified for bacteriophage T4 by Moody (1965) and Aebi er a/. (1974). The predominance of head length

’ To whom requests for reprints should be addressed.

sizes which constitute regularly spaced points along the lattice suggests that head length is determined by an interaction between two structures of unequal heli- cal spacings that come into register at certain posi- tions, like a mechanical vernier.

MATERIALS AND METHODS

Preparation of bacteriophage

Phage were prepared by the multicycle lysis inhibi- tion technique described by Doermann er a/. (1973). The ptg mutations produce isometric (ISO), intermedi- ate (INT), normal (NOR) and giant particles (Doermann et al., 1973; Doermann and Pao, 1987). Mutant ptg

phage were produced by a single cycle lysis inhibition technique that enhanced production of giant particles. This method follows Doermann et a/., (1973) except that in the single cycle technique, Escherichia co/i B at a titer of about 4 X 1 O’/ml are infected at a multiplicity of infection (m.0.i.) of 5 PFU per cell and incubated at 30” with vigorous aeration. At 8 min postinfection the cells are superinfected at an m.o.i. of 5. At 150 min postinfection the pregnant bacteria are harvested by centrifugation and lysed. These details are important because variations in culture conditions affect the rela- tive frequencies of head size classes.

Sucrose gradient analysis

The particle size distribution in phage preparations was analyzed by centrifugation in isokinetic sucrose gradients (Doermann and Pao, 1987) prepared using a Biocomp (Fredricton N.B. Canada) gradient maker (25- 50% sucrose). Approximately 5 X 10” particles in 0.1

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472

QUANTIZED VIRAL DNA PACKAGING

ml were layered on each 12-ml, 4-5” gradient and cen- trifuged at 35,000 rpm in the SW41 rotor of a Beckman ultracentrifuge for 1 hr at 5”. Thereafter the uv absor- bance profile was determined using an lsco gradient fractionator.

Rotating gel electrophoresis

The phage particle preparation, stored in 0.2 /VI NaCI, 0.01 MTris-HCI, pH 7.4, 0.001 NI MgCI,, was brought to 0.14 hll NaCI, 0.003 M MgC&, and 7 fig/ml DNAase I (Cooper Biomedical) and incubated at 30” for 30 min. To stop digestion, a 0.1 X vol of 0.2 M EDTA, pH 7.4 (0.06X vol for giants; 0.1 X otherwise) was added. To first inactivate the DNAase and then release DNA from capsids, DNAase-treated particles were added to at least a 5X amount of 0.5% Sarkosyl NL97 in 0.1 ILINaCI, 0.01 11/1Tris-HCI, pH 7.4,O.OOl ILIEDTA (NET) and this mixture was heated to 75” for 15 min. A portion was added to a :X vol of 55% sucrose, 400 lg/ ml bromphenol blue in 0.01 M sodium phosphate, pH 7.4, 0.001 M EDTA. For electrophoresis, 30 ~1 was lay- ered in a sample well of 1.5% agarose (Seakem LE, Marine Colloids Division of the FMC Corporation) gel in 0.01 M sodium phosphate, pH 7.4, 0.001 n/l EDTA. The gel had been poured on a circular disc for RGE by procedures previously described (Serwer, 1987). Pi- peting was performed slowly (cl00 &ec) with a mi- cropipet that had an inner diameter of 0.2 cm. After waiting 1 hr for buffer discontinuities to diffuse, electro- phoresis was started at 2 V/cm, 21’ without moving the gel. Ten minutes later, back and forth rotation of the gel was started and continued throughout electro- phoresis at 3 V/cm by procedures previously described (Serwer, 1987). The total angle of rotation was 128”; the time between rotations was either 15 (Fig. 2) or 80 set (Fig. 3); the time during rotation was 1.5 sec. After either 48 (Fig. 2) or 35 hr (Fig. 3) of electrophoresis, the gel was stained with 1 pg/ml ethidium bromide for 1 hr, destained for at least a week in distilled water at room temperature, and then photographed (Serwer, 1987). The destaining significantly increased the visibility of the bands in Fig. 3.

Purification of giants

Giant particles were partially purified from prg Ng 19 1 c by sucrose gradient centrifugation. Phage par- ticles were concentrated by centrifugation and resus- pended in M9 medium (pH5) without glucose and cas- amino acids. The low pH keeps the tail fibers retracted and improves separations. This same medium was also used to prepare all sucrose solutions. Linear gradi- ents (45.5 ml, 25-75%) were poured in Beckman VTi- 50 tubes using a Biocomp gradient maker. A 2-ml sam-

v-

I ,tg Ngl91c In t A )t E920g N l-

I ,t 21-34~ C

int

(A+C)

FIG. 1. Sucrose gradient profiles of T4 head length mutants. De- scriptions of the mutants are in (Doermann et a/., 1973; Doermann and Pao, 1987; Eiserling et al., 1970); sedimentation is from right to left. (A) ptg Ngl9 1 c, (B) pt E920g, (C) pt 2 1-34c, (D) mixture [l : 1 of plaque forming units (pfu)] of ptg Ngl91 c and pt E920g, (E) mixture ofpt E920g andpt21-34c, (F) mixture ofptg NglSlc andpt21-34~.

ple containing approximately 2 X 10” PFU was loaded on each gradient and centrifuged at 30,000 rpm in the VTi-50 rotor of a Beckman ultracentrifuge for 20 min at 25”. The rapidly sedimenting fraction was removed using a syringe, was concentrated by centrifugation, and resuspended in phosphate buffer. Electron micros- copy showed 40% giant phage.

RESULTS

The results that emerged from the study below are the following: (1) several new intermediate-length T4 DNA molecules were discovered, (2) one mutant phage was found that contained DNA shorter than nor- mal length, yet could form plaques, (3) DNA from “gi- ant” T4 particles of at least one ptg mutant formed dis- crete bands at regular spacings.

Sucrose gradient analysis and electron microscopy were the first steps in determining the head size distri- bution of intact bacteriophage from phage T4 pt and ptg mutants (Doermann et al., 1973; Doermann and Pao, 1987; Eiserling et al., 1970). Figure 1 shows the sucrose gradient profiles of the three mutants used in this study. Head sizes were verified by electron micros- copy (for examples, see Doermann et al., 1973). Muta- tion pt 2 1-34~ produces isometric, intermediate, and normal heads, pt E920g produces isometric and nor-

474 LANE ET AL.

mat heads, and ptg NglSlc produces intermediate, normal, and long (giant) heads (Doermann et al., 1973). A mixture of pt E920g and ptg Ngl91 c (Fig. 1 D) shows a narrow distribution of normal size particles (indicating that normal size particles from both mutants are the same size), whereas the profile of the mixture of pt 21- 34c and pt E920g (Fig. 1E) shows a slightly broader peak which indicates that the “normal” particles from these two mutants may not be of identical size. In the mixture of pt 21-34~ and ptg Ngl9lc (Fig. 1F) the peaks of normal and Intermediate particles cannot be resolved, indicating significant heterogeneity in the head sizes of these classes of particles. However, nei- ther sucrose gradient analysis nor electron microscopy were sufficient methods to resolve clearly these size differences.

The theory of icosahedral virus structure of Caspar and Klug (1962) defines a triangulation number, T, that describes the icosahedral surface lattice and sets other structural parameters. Analysis of images of fro- zen-etched particles by Branton and Klug (1975) show that, for phage T2, the surface lattice is T = 13 I (laevo); this has been confirmed and extended to phage T4 (Bashong et al., 1988). If an icosahedron is extended along its fivefold axis, that extension can also be de- scribed by icosahedral surface lattice theory and the length assigned a number, Q, that defines the quan- tized values of the elongation (also see Fig. 4). On the basis of theoretical calculations of Aebi (1983) for phages T2 and T4, the DNA length in isometric parti- cles (Q = 13) should be 0.69 and intermediate particles (Q = 17) about 0.85 that of normal particles (Q = 21). By use of either genetic techniques (Mosig, 1963) or electron microscopy (Eiserling et a/., 1970; Mosig et al., 1972) the length of DNA molecules in isometric par- ticles was estimated at 0.67 of that in normal phage. Figure 2 shows the results of applying rotating gel elec- trophoresis to DNA released from the particles of Fig. 1. On these gels DNA from normal T4D is apparently 178 kilobasepairs (kbp) compared to the bacterio- phage X standard. Nucleotide mapping data (Clark et al., 1980; Kim and Davidson, 1974; Kutter and Ruger, 1983; Streisinger et al., 1967) indicates that the actual length of T4D DNA is 170 kbp. The difference is proba- bly caused by the extensive glycosylation of T4 DNA. Scaling the lengths to the 170-kbp length of wild type, isometric particles contain 1 16 and intermediate parti- cles contain 144 kbp of DNA. Dividing the difference in DNA tength between isometric and normal particles, 54 kbp, by the difference in their Q values, 8, we obtain an increase in DNA length of 6.75 kbp for each Q num- ber above 13.

The sizes of each class of DNA produced by the mu- tants are summarized in Table 1, with probable Q num-

FIG. 2. Rotating gel electrophoresis (RGE) of phage particles from lysates of T4 pt and pfg mutants. The particles were taken from the following lysates: (lanes labeled “C”) pt 21.34c, (lanes “A”) pfg Ngl91 c, (lanes “B”)pt E920g. The sample for lane labeled “X” con- tains a collection of concatemeric bacteriophage h DNAs formed by joining mature X DNA (48.5 kbp [Sanger et al., 19821) end to end, thereby forming integral multimers, i.e., monomer, dimer. etc. (X lad- der; see Ref. [Anand, 19861). The sample for lanes labeled “S” con- tains a mixture of the mature DNAs of bacteriophages T7 (39.9 kbp [Dunn and Studier, 1983]), T5 (120 kbp [Son ef a/.. 1988]), and T4 (170 kbp [Kutter and Ruger, 19831). A preparation of DNA from par- tially purified giants was subjected to electrophoresis after dilution with 2 parts of NET, dilution by 0.5 parts NET, and no dilution (lanes labeled “G”). The origins of electrophoresis are indicated by the ar- rowheads; the direction of electrophoresis is indicated by the arrow. All lanes are from two sections of the same gel.

bers assigned on the basis of DNA length. The DNA length from “normal” particles produced by mutants ptg Ng191c and pt E920g was indeed normal; how- ever, the DNA of particles from pt 2 l -34c, surprisingly, was approximately 7 kbp shorter than wild type. The DNA length in isometric particles produced by the mu- tants agreed well with published estimates (Mosig, 1963; Eiserling et a/., 1970; Mosig et a/., 1972). Mutant pt 2 1-34~ produced two classes of “intermediate” par- ticles; the DNA length from one class agreed well with that calculated for Q = 17, the other (a minor compo- nent) was about 6 kbp shorter. The DNA from the “in-

QUANTIZED VIRAL DNA PACKAGING 475

TABLE 1

LENGTH AND PROBABLE Q No. OF DNA SPECIES FROM THREE HEAD LENGTH MUTANTS

lenga et al., 1976; lshii and Yanagida, 1975; Yanagida, 1977). Then the increase in head length per unit incre- ment of Q can be expressed as

Mutant DNA size (kb)a Probable Q no. AL = gsina,

T4D 170 21 pr 2 l -34c 116 13

138 16 144 17 163 20

ptgNgl9lc 144b 17 151 18 170 21

pf E920g 116 13 1296 15 144b 17 170 21

where g is the lattice constant and cy is the pitch angle. Assuming that the shape of the midlength cross sec- tion of the cylindrical part of the head is circular, then the volume increase per unit increment of Q is

(U. Aebi, personal communication), where T is the tri- angulation number (T = 13). The increase in volume per unit increment of Q, using g = 12.9 nm, is 1.34 X 10-l’ cm3.

a Values of DNA length have been corrected from apparent sizes as described in the text.

b Minor component.

Assuming that the n/r, of a DNA basepair is 650 Da, then the mass of the measured increment of 6.75 kbp is 7.29 X 10-l’ g. The packing density is then 0.54 g/ cm3. This value corresponds well with the published

termediate” particles of ptg Ng 191 c was about 7 kbp longer than would be expected from a Q = 17 particle.

Values of head length, Q, are correlated with the DNA length found in T4 particles on a surface lattice in Fig. 4. Only a few Q numbers are represented by DNA species, and, except for one very minor species, the Q numbers represented are within 1 unit of a line defined by the Q numbers 13, 17, and 2 1, each with an incre- ment of 4 Q numbers. The Q numbers at which termi- nation of head elongation occurs are allele specific; each different mutant places fivefold vertices at only a few selected sites. Since the DNA length from the small particles varies by multiples of 7 kbp, approxi- mately the length expected by an increase in Q number of one, it is unlikely that DNA molecules shorter or longer than the headful length are packaged in phage particles, This suggests that head length termination can occur after each increase of one Q number but that termination is more likely to occur after an increase of four Q numbers. This 4-unit increment is equivalent to the addition of a single row of hexamers to the cylindri- cal part of the elongating head.

I234

>

A reviewer has suggested that the density and mo- lecular separation of the additional packaged viral DNA could be estimated by calculating the volume incre- ment per unit increase in Q, then comparing this value to our data on the incremental amount of DNA pack- aged. This can be done for the central part of the cap- sid, exclusive of the icosahedral caps. Data needed for these calculations are the lattice constant of expanded heads (12.5 to 12.9 nm) and the consensus value for the pitch angle (14”) of the rows of hexamers that make up the cylindrical part of the capsid (Aebi, 1977; Bij-

FIG. 3. The length distribution of DNA in T4 giants. DNA from a partially purified preparation of T4 giants was subjected to RGE after (1) dilution with two parts of NET, (2) dilution with 0.5 parts of NET, (3) no dilution. Lane 4 contains a X ladder. The arrowheads indicate the origins of electrophoresis; the vertical arrow indicates the direc- tion of electrophoresis. The horizontal arrow indicates a peak in the distribution of lengths for the T4 giants.

476 LANE ET AL.

NORMAL

INTERMEDI

ISOMETRIC

FIG. 4. Surface lattice arrangements predicted for bacteriophage T4 head length variants. Trle models show the isometric (Q = 13) intermediate (Q = 17) and normal (Q = 21) length heads. A flattened portion of the surface lattice shows the relative position of the fivefold vertices for capsids with Q numbers between 13 and 29. The lines labeled m and n indicate the pathway between fivefold vertices of a capsid with a triangulation number(T) of 13. The heavy lines indicate the edges of a facet of a Q = 13 head. The heavy line with Q = 13, 17, 21, etc., shows the preferred locations of the flvefold vertices that lead to quantized DNA packaging.

packing density of 0.52 g/cm3 and thus the interhelical distance of 2.64 nm for the ordered region of bacterio- phage T4 heads (Earnshaw eT al., 1978). These inter- helical distances are only somewhat smaller than the 2.8-nm spacings observed in columnar hexagonal liq- uid crystals of DNA (L.ivolant et a/., 1989; Franklin and Gosling, 1953). We note that these calculations are very sensitive to the value of the lattice constant and the assumptions made about the shape of the phage cross section. However, they do support the idea that, as the size of the phage head is increased, the addi- tional DNA is packaged at the same density.

We also used rotating gel electrophoresis to analyze the length distribution of DNAfrom “giant” (elongated- head) particles produced by ptg Ng 19 1 c (Fig. 3). Giant particles constitute approximately 3% of the virus parti- cles produced by this mutant and the DNA from these particles has an average length 2.5 times that of the DNA from normal T4D (Doermann er a/., 1973). We were able to resolve a number of discrete bands of gi- ant DNA species over a length of 265 to 510 kbp. These bands were arrayed in doublets: the two bands within the doublets were separated by approximately 19 kbp which roughly corresponds to a difference of three Q numbers and the distance between the second band of each doublet and the first band of the next dou-

blet was approximately 26 kbp which corresponds to a difference of four Q numbers. The length of the giant DNA species roughly corresponded to particles with Q = 33 to Q = 69 in alternating steps of three and four Q numbers.

It is evident from this analysis that the giant DNA length of at least one ptg mutant is quantized. How- ever, there is evidence that the length of giants may not be regulated by the same mechanism as the smaller particles. Giant particles account for only about 3% of the phage produced byptg Ng 19 1 c and consist of par- ticles of several different lengths; therefore giant parti- cles of a specific length make up a very small fraction of the total phage produced. Smaller particles of each specific length make up large fractions of the total phage produced, and in fact each termination site for the smaller particles is much more highly selected than all of the giant termination sites taken together. The ter- mination sites for the smaller particles lie upon or adja- cent to the surface lattice line running through Q num- bers 13, 17, and 21; judging from the alternating incre- ment of giant length distribution the termination sites for giants may not all lie along on the same line. Eluci- dation of the mechanism of giant length regulation re- quires the study of giants from additional ptg mutants.

DISCUSSION

The head length variants are produced by mutations in the major head protein gene 23. Doermann and co- workers mapped 54 separate ptg mutations and deter- mined the DNA sequence and predicted amino acid substitution for half of them (Doermann et a/., 1987; Mooney et a/., 1987). All the ptg mutations and pt 2 l- 34c map in 10 loci arranged in three clusters in gene 23 (Doermann et al., 1987). Cluster 1 runs from amino acid residue 66 to 97, cluster 2 runs from amino acid 268 to 287, and cluster 3 runs from amino acid 457 to 46 1. These three regions of gp 23 have been shown to have protein sequence homology with gp24 (Yasuda et a/., 1989). Mutant ptg Ngl91 c is the change of asp287 to asn, pt 21-34~ ala275 to thr, and pt E920g thr457 to ala and ala485 to val. Doherty found in his studies of second-site suppressors of the head length mutant ptg Bu 19-80 (Doherty, 1982a, 1982b) that in- teractions between the proteins of the shell and those of the core are involved in head length regulation. Doh- erty examined these interactions by isolating and map- ping suppressors of the ptg mutant 19-80. All of the suppressors of ptg 19-80 that were subsequently iso- lated turned out to be double mutants, with one muta- tion mapping in gene 24 (the vertex protein) and the other mutation mapping in gene 22 (the core protein). These suppressors were determined to be allele spe-

QUANTIZED VIRAL DNA PACKAGING 477

cific; none of the suppressors of ptg 19-80 altered the phenotype of any other ptg mutation when combined together. These indications of possible interactions be- tween gp22, gp23, and gp24 during head length deter- mination are consistent with the operation of a vernier mechanism of head length regulation described below.

Although a variety of positions have been identified on the surface lattice of the T4 capsid as possible ter- mination points for head growth, we have shown that for the mutants studied here, there is a strong selection for preferred stop sites at intervals of four Q numbers. The regular spacing of the termination event along the surface lattice suggests that head length is determined by an interaction- between two structures of unequal helical spacings that come into register at certain sizes like a mechanical vernier. The two components would be the core, composed of six helical strands of gp 22, plus gp 67, and gp 68 (Keller eta/., 1988), and the gp 23 shell. The elongation of an icosahedron along a fivefold axis produces a five start helix (Aebi et al., 1974). Some structural data on these spacings and on the symmetry mismatch between the sixfold helical core and the fivefold helical shell have been presented (Paulson and Laemmli, 1977; Engel et al., 1982). A vernier model predicts that as each helical row of hexamers is added to the elongating capsid there is a point at which the shell and the core helices are nearly in register. Such a mechanism might operate in bacteriophage T4 in the following manner: after initiation and formation of the proximal cap of the capsid, two complete rows of hex- amers add to the elongating capsid, bringing the shell and the core into precise register. Termination involv- ing the vertex protein gp 24 would then occur, at the spacing of Q = 21. Following this same model, the points at which the core and shell are nearly in register would lie on the surface lattice of the capsid along the line defined by the Q numbers 13, 17, and 21. The different head length mutations described by Doer- mann and Pao (1987) would affect head length regula- tion by slightly altering the “vernier mark” on the shell protein, thus causing termination to occur at points where the shell and core are normally close to being in register, or every four Q numbers. The new observa- tions that DNA length is remarkably quantized provides good evidence that the T4 capsid performs an essen- tial measuring function during DNA packaging.

ACKNOWLEDGMENTS

We thank M. Glngery and Drs. D. Rees and D. Eisenberg for many helpful discussions. This work was supported by grants from the Na- tional Science Foundation (DMB-8705427 to F.E.) and the National Institutes of Health (GM24365 and All 6117 to P.S.).

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