JOURNAL OF VIROLOGY, June 1976, p. 894-903Copyright C 1976 American Society for Microbiology

Vol. 18, No. 3Printed in U.SA.

Identification of Gene Products Required for In VitroFormation of the Internal Peptides of Bacteriophage T4

JUDITH GOLDSTEIN GIRI, JOHN E. McCULLOUGH, AND SEWELL P. CHAMPE*Waksman Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey 08903

Received for publication 24 December 1975

In vitro formation of both bacteriophage T4 internal peptides (II and VII) frompreexisting precursor protein was shown to require the product of T4 gene 21.The proteolytic factor was detectable in extracts of cells infected with certainphage mutants blocked in early steps of head assembly but could not bedemonstrated in extracts of T4 wild-type infected cells. This finding suggeststhat the proteolytic factor is inactivated during normal phage assembly. Theproduct ofT4 gene 22 appears to be the precursor of peptide VII but not of peptideII.

During assembly of the head of bacterio-phage T4, several different phage-coded pro-teins undergo proteolytic cleavage (5, 9, 11, 12).This cleavage process is coupled to head assem-bly since conditionally lethal mutants of anyone of seven genes (genes 20, 21, 22, 23, 24, 31,and 40), which fail to make normal heads underrestrictive conditions, exhibit none of the pro-tein modifications observed with wild-typephage (12). One of the proteins cleaved duringhead assembly, p22 (the product of gene 22),functions in an early step of head assembly butis not found associated with the finished phageparticle in any recognizable form (9, 10, 12).This protein is apparently degraded to low-mo-lecular-weight fragments unlike other headproteins, the cleaved forms of which, represent-ing more than 80% of the molecule, are incorpo-rated into the mature phage particle.

Previous studies on the two acid-soluble pep-tides contained in the T4 head suggest thatthese peptides are fragments of one or anotherof the proteins that undergo assembly-associ-ated modification (4, 6, 12). The formation ofthe internal peptides in infected cells requiresthe same genes that are required for cleavage(6, 12). Since, owing to their acid solubility andunique chromatographic properties, the inter-nal peptides can be easily detected in infectedcells, their formation provides a convenient andspecific assay for the occurrence of assembly-associated cleavage.

In an earlier report, we demonstrated the invitro formation of one of the T4 internal pep-tides, peptide II, from a preexisting precursorprotein (8). This peptide is formed upon incuba-tion of substrate protein, prepared from cellsinfected with a phage mutant blocked in cleav-age, with a suitable source of the proteolytic

factor. The required proteolytic factor wasfound to be contained in extracts of cells in-fected with certain T4 mutants but not in ex-tracts of uninfected cells, which implies thatthe activity is phage induced.

In the present paper, using this in vitro assayfor cleavage, we examined extracts of cells in-fected with various T4 mutants for the presenceof cleaving activity and for ability to supply theprecursors of the internal peptides. The resultsidentify the T4 gene product required for cleav-age and suggest the identity of the gene prod-uct which is the precursor of one of the internalpeptides.

MATERIALS AND METHODSThe temperature-sensitive T4 mutants,

tsA3(20-), tsN8(21-), and tsL84(40-), are describedby Edgar et al. (7). The mutant IP" is a triple mu-tant, constructed by Lindsey Black (2), which lacksall three internal proteins. The multiple mutant,IP" in combination with the 21- mutant amN90 andthe 23- mutant amHll, was obtained from MichaelShowe. Anti-p22 serum was generously supplied byLouise Onorato. All other phage mutants, proce-dures for preparing cell extracts and substrate pro-tein, in vitro incubation conditions, and methods ofchromatographic analysis are described in a pre-vious communication (8). Minor exceptions or addi-tions to the methods are noted in the figure legends.

Protein concentrations of extracts were deter-mined by the standard Folin-Ciocalteau method us-ing bovine serum albumin as a standard. The pro-tein concentrations given in the text refer, in allcases, to the final concentration in the in vitro reac-tion mixture.

RESULTSGene dependence of II*-forming activity. In

a previous report (8), we demonstrated the invitro formation of a peptide, II*, which is indis-tinguishable from T4 internal peptide II. The in

894

INTERNAL PEPTIDE FORMATION IN PHAGE T4 895

vitro- and in vivo-formed peptides co-chromato-graph on Sephadex G50 and on Dowex 50-X2and share a number of other properties, in-cluding an identical T2-T4 chromatographicspecies specificity. In these earlier experi-ments, the formation of peptide II* was effectedby incubating [14C]lysine-labeled substrate pro-tein from 21--infected cells with an unlabeledextract of 23--infected cells. Both native andacid-denatured substrate were found to yield11*. An extract of uninfected Escherichia coliwas found to be lacking the activity required forII* formation, which implies that the activity isphage induced.To identify the T4 gene required for II* for-

mation, we examined extracts of cells infectedwith various phage mutants for the presence ofthe activity. Figure 1 shows chromatographicanalyses on Dowex 50 of the acid-soluble invitro incubation products formed upon incuba-tion of denatured ['4C]lysine-labeled substrateprotein (the same preparation in all cases) withvarious cell extracts. In each case, the 14C-la-beled material was prechromatographed onSephadex G50 with 3H-labeled marker peptideII. The material eluting with the marker waspooled and rechromatographed on Dowex 50 asshown. The presence of 14C-labeled materialthat eluted from Dowex 50 in coincidence withmarker peptide II was taken as evidence for II*-forming activity in the extract. The presence ofother components varies from one experimentto another, since it depends on the efficacy ofthe prefractionation on Sephadex.

Figure lb shows, in confirmation of our pre-viously reported finding (8), that uninfectedcells do not possess II*-forming activity. Theactivity was also absent from cells infected witha mutant of an early T4 gene (gene 44), asshown in Fig. lc, consistent with the conclusionthat the activity is the product of a late-phagegene. Of the other mutants tested, all of whichare blocked in an early step of head formation,those of genes 20, 23, 24, and 31 clearly showedactivity. The activity of the 22- mutant wasdepressed but still detectable. The only mutantfor which activity could not be detected is the21- mutant amN90 (Fig. le).As shown in Fig. la, an extract of T4 wild-

type infected cells also lacks II*-forming activ-ity. This was noted by us previously (8) and isconsistent with the finding of Onorato andShowe (14) and Bolin and Cummings (3) thatextracts of wild-type-infected cells cannot de-grade p22 in vitro. The possible implications ofthis finding will be discussed later.

It is conceivable that the depressed activity ofthe 22- mutant and the absence of activity ofthe 21- mutant shown in Fig. 1 could be due to

entrapment of the active factor in aberranthead structures known to be formed by thesem,utants. We tested this possibility by using22-23- and 21-23- double mutants which, dueto the presence of the 23- amber mutation,make no head-related structures. It was found,however (not shown), that the double mutantsexhibited essentially the same 11*-forming ac-tivity as the single 22- and 21- mutants of Fig.1. This result implies that p22 is required foroptimal activity of p21 and has been noted byothers as well (3, 13, 14). As a possible explana-tion, Onorato and Showe (14) suggest that p21may be stabilized in vitro by complexing withp22.The only other known T4 gene participating

in the early steps ofhead assembly not tested inthe experiment of Fig. 1 is gene 40, which isdefined only by temperature-sensitive muta-tions. To test the effect of a 40- temperature-sensitive mutation on the induction of II*-form-ing activity, cells were infected at the restric-tive temperature (42 C) with tsL84, and theresulting extract was used in the in vitro reac-tion at the standard incubation temperature of37 C. As controls, temperature-sensitive mu-tants of genes 20 and 21 were assayed in thesame experiment. As seen in Fig. 2, the 40- tsmutant, like the 20- ts mutant, is able toinduce II*-forming activity. The 21- ts mutant,however, does not induce the activity, consist-ent with the result of Fig. le using the 21-mutant amN90.Although the experiments of Fig. 1 and 2

were performed using one standard substratepreparation, the extracts used as the source ofcleaving activity were necessarily separatepreparations and could vary in cleaving activ-ity for reasons other than the nature of thephage mutant used. For most experiments inwhich different extracts were employed, theextracts were adjusted to a comparable proteinconcentration. Probably a more valid criterionof similarity is the fraction of the total sub-strate label solubilized. These values for thevarious extracts employed in the experimentsof Fig. 1 and 2 are given in Table 1, from whichit is seen that they do not differ greatly fromone preparation to another.

In vitro formation of VII*. In our previouslyreported experiments demonstrating the in vi-tro formation of peptide II* (8), we were unableto detect an in vitro-formed component corre-sponding to the second of the T4 internal pep-tides, peptide VII. Subsequent studies haveshown, however, that a component correspond-ing to VII is formed in vitro if more concen-trated extracts are employed. This is illustratedin Fig. 3, which is an analysis of the acid-

VOL. 18, 1976

896 GIRI, McCULLOUGH, AND CHAMPE

0~~~~~~~~~~~H0N ~~~~~~~~~~~3-x I, ~~~~~~~~~~~~~~~~~~~~~~~x

10 IX03H

FIG. 1. II-frmn aciiyi xrcso elsifce ihvrosT mbrmtnscddntrdcorepndntoa50m2utrfifce el.Teetacswda oreo h rtoyiatrwr

5 f

pH7.5, contaii 0

23com-1bl ) (g)24camiN65)0he31tailsofH

ar

310 U-~~~~3H11-3H

2

5

0 gI~~~~~~~~~~~~~~~~

10 20 30 10 20 30 10 20 30FRACTION NUMBER

FIG. 1. 11*-forming activity in extracts of cells infected with various T4 amber mutants. Acid-denatured.,[a4C]lysine-labeled substrate protein from amH29(21 )-infected E. coli B was divided into 0.5-mi portionscorresponding to a 5.0-mi culture of infected cells. The extracts used as source of the proteolytic factor wereprepared friom 100 ml of infected cells concentrated approximately 200-fold into 0.05 M Tris-hydrochloride,pH 7.5, containing 0.0014 M 2-mercaptoethanol. Volumes were adjusted, if necessary, to give the extractscomparable protein concentrations. Other details of substrate and extract preparation methods are describedelsewhere (8). After incubating the substrate-extract mixture (0.5 ml of each) for 6 h at 37 C, dichloroaceticacid was added to a final concentration oflO0%, and the resulting precipitate was removed by centrifugation.The acid-soluble supernatant material was fractionated by gel filtration through a column of Sephadex G50together with 3H-labeled internal peptide 11. The 14C2-labeled material eluting with the 3H marker was pooled,lyophilized with about 20 pAg of internal peptides added as carrier, and rechromatographed on Dowex 50-X2as shown.

soluble incubation products formed using an 3H-labeled marker peptide VII (Fig. 3a) in-extract of approximately five times the concen- cludes a component that co-chromatographedtration employed earlier (8). The '4C-labeled with the marker on Dowex 50 (Fig. 3b). Thismaterial that elutes from Sephadex with the component, like the marker peptide VII, is

J. VIROL.

INTERNAL PEPTIDE FORMATION IN PHAGE T4

10 20 30 10 20 30 10 20 30FRACTION NUMBER

FIG. 2. Effect of20-, 40-, and 21- temperature-sensitive mutations on II*-forming activity. The experimentwas performed in an identical manner as the experiment ofFig. 1 except that the infected cells used as thesource ofthe proteolytic factor were grown at the nonpermissive temperature for temperature-sensitive mutants(42 C).

TABLE 1. Protein concentrations and proteolyticactivities ofextracts employed in experiments ofFig.

1 and 2% Sub-

Protein strate *Gene Mutant concn counts/ tIforns-

(mg/ml) min 81U- tionbilized

Fig. 1am+ 10 15.5 0

Uninfected 10 15.9 044 amN82 10 11.0 020 amN50 10 10.7 +21 amN90 11 10.4 022 amE209 10 7.3 +23 amHll 10 17.8 +24 amN65 10 17.4 +31 amN54 10 14.3 +

Fig. 220 * tsA3 7.5 13.0 +40 tsL84 7.5 9.1 +21 tsN8 7.5 10.7 0

insensitive to degradation by chymotrypsin(Fig. 3c), and gave similar fragments as VIIupon digestion with trypsin (Fig. 3d). This invitro-formed component will be designatedVII*.

Although the dependence on extract concen-tration for II* and VII* formation was not di-rectly compared, detectable formation of VII*appears to require at least four times the con-centration required for maximal yield of II*.Maximal yield of II* was obtained with an ex-tract concentration of about 3 mg/ml of protein.The experiment of Fig. 4 shows that extracts

of both 21--infected cells and T4 wild-type-in-fected cells are inactive in VII* formation, asis true also for II* formation. (The '4C-labeledmaterial that eluted with marker II from Seph-adex in this experiment did not co-chromato-graph with the marker on Dowex 50.)Precursors of the T4 internal peptides. Of

the phage proteins known to be cleaved duringT4 assembly, a likely candidate for the precur-sor ofthe internal peptides is p22, the product ofgene 22, since this protein appears to functionas a core for head assembly (15) and is exten-sively degraded (12). If this. is the case, anamber mutation in gene 22 'should eliminatethe precursor of both internal peptides if theregion of p22 from which the peptides are de-rived is C-proximal to the site of chain termina-tion.

I0

IT0

x

(-)

(-

5

0

0x

a-

VOL. 18, 1976 897

898 GIRI, McCULLOUGH, AND CHAMPE

0

'o

a-

o 0

x x

I ()a.)

0

x

Ia-u-

40 50 60 70 40 50 60 70 80FRACTION NUMBER

FIG. 3. In vitro formation of peptide VII*. The extract used as the source of the proteolytic factor was

prepared from 23-e- (amHllamH26)-infected cells and was adjusted to give a concentration of20 mg/ml ofprotein in the in vitro reaction mixture. Other details are as described in the legend ofFig. 1. (a) SephadexG50 chromatography of the acid-soluble incubation products. Fractions of 2.0 ml were collected, 0.5 ml ofwhich was assayed for radioactivity. (b) Dowex 50 chromatography of the pooled remaining part offractions50 to 55 from (a). Fractions of 4.0 ml were collected, 1.0 ml of which was assayed for radioactivity. Theremaining portions offractions 64 to 69 were pooled and concentrated by lyophilization and redissolved in 2%ammonium bicarbonate. One-half of this material was digested with trypsin (0.05 mg/ml for 10 h), and theother half was digested with chymotrypsin (0.05 mglml for 10 h). (c) Dowex 50 chromatography of thechymotrypsin digestion products offractions 64 to 69 from (b). (d) Dowex 50 chromatography of the trypsindigestion products offractions 64 to 69 from (b).

As shown in Fig. 5, substrate protein pre-pared from the 22- mutant amE209 failed toyield VII*, whereas VII* was formed if themore C-proximal mutation amB270 was used.The tentative conclusion is that peptide VII isderived from p22 and from a region of thisprotein more N-proximal than the amB270 siteand more C-proximal than the amE209 site.Not shown in this experiment, but indicatedbelow in Table 2, is the fact that amE209 doesnot eliminate the precursor of peptide II*.

It is to be noted that, in Fig. 3a, 4a, and 5b,the peak of 14C-labeled material that includesVII* is slightly shifted from the position of 3H-labeled marker peptide VII, indicating that thispeak contains components in addition to VII*.All components in this peak are, however, ap-

parently of p22 origin, since no "4C-labeled ma-

terial was detectable in this region if amE209-infected cells were used as the substrate (Fig.5a). This non-VII* material contained in theSephadex peak was identified as the singlecomponent, labeled VIII* in Fig. 6, whichelutes from Dowex 50 some 18 fractions afterpeptide VII*. This component was missed inearlier experiments, since the elution fromDowex 50 was generally terminated shortlyafter VII* emerged. No component correspond-ing to VIII* was found in vivo.To confirm further the above indication that

p22 is the precursor of VII, we used partiallypurified p22 (prepared by precipitation of a

crude infected cell extract with anti-p22 serum)as the substrate in the in vitro reaction. Chro-matographic analysis of the resulting acid-solu-ble incubation products (Fig. 7) showed the

[ D I

(c) CHYMOTRYPSIN TREATED (b)

4-~~~~~~~~~4

a~ ~ ~ ~ ~ ~~~~~I

3 .6

ii

iI2 4

2

0 0~~~~~000(d) TRYPSIN TREATED (b)

3 614C

2 4H

I ~I 2

01--

IoI

EJ. VIROL.

DOWEX 50 - X2

INTERNAL PEPTIDE FORMATION IN PHAGE T4 899

5

4

3

2

1

0

4

3

2

40 50 60

FRACTION NUMBER

5

4

3

5

4

3

2

00

5 5

0 0

E EI uI U

4

3

2

0

5

4

3

2

o

23- | ( b) 6K (am Hll) ME *

a/ -5

- 4

- 1p' - 3

14c_*_ 9 2~~~

b1 0

_21 (d)(am N90),

'0

4IH _

0,

.o 0

_-am- If )

Do_

14

-,~ 0 00.0L .J

I

I1-50 55 60 65

FRACTION NUMBER

5

04

Ea3 u

I

-2

- 1

5

4

3

2

FIG. 4. VIP-forming activity of various infected cell extracts. The experimental conditions were the sameas described in the legend ofFig. 3. The right-hand panels are the profiles obtained by rechromatographingthe pooled fractions indicated in the left-hand panels. (a, b) amH11(23-)-infected cell extract, (c, d)amN90(21 -)-infected cell extract, (e, t) am+-infected cell extract.

TABLE 2. Formation ofpeptide II* using substrate protein from T4 amber-infected cellsCounts/min x 10-3

Substrate from Total sub- Dichloroa-mutant (gene): strate pro- cetic acid- % Soluble Peptide II PeptideP II* % II*/II

tein soluble

am' (in vivo) 7,500 470 6.2 21.6 100amN50 (20) 5,200 540 10.5 7.5 35amH29 (21) 5,500 720 13.1 5.2 24amE209 (22) 5,300 580 10.9 9.9 46amHll (23) 5,000 880 16.0 12.0 56amN65 (24) 6,100 680 11.1 4.4 20amN54 (31) 6,500 640 9.9 5.8 27amN82 (44) 5,500 420 7.7 0.13 0.6Uninfected E. coli 9,400 900 9.6 0.13 0.6

a Yields of peptide II* were measured after sequential fractionation of the dichloroacetic acid-solublematerial on Sephadex G50 and Dowex 50-X2 and are corrected for slight variations in counts per minute oftotal substrate protein.

DOWEX 50-X2SEPHADEX G50

23- (om Hll) (a)

i POOL I

-m 14cP-o]'0 d. H-~0 o0-'0 -'H

0.0

.

o.o' N_0)

0

Eau

VOL. 18, 1976

I1

21 (am N90) ( c ) _..

1

900 GIRI, McCULLOUGH, AND CHAMPEI

(a) 22i(omE209)

1, 0,0IOoPz1°>° 0 0 14

0, 1C '0I

0.o0'

0 ,

(b) 22-(om B270)

o 14C/ 0-0-0

_I A_ °'l

50FRACTION NUMBER

60

FIG. 5. Elimination of the precursor of VII* by anamber mutation in gene 22. The source and concen-tration ofthe extract were as described for the experi-ment ofFig. 3. The figure shows chromatography onSephadex G50 of the acid-soluble incubation prod-ucts formed using: (a) substrate protein fromamE209(22-)-infected cells, and (b) substrate pro-tein from amB270(22-)-infected cells.

presence of VII* but no trace of II*. The im-mune precipitate used as substrate in this ex-periment corresponded to about 3% of the labelin the crude lysate, but, when analyzed bySDS-acrylamide gel electrophoresis, was foundto contain several components in addition top22. The heterogeneity of this preparation thusprevents a rigorous proof that p22 is the pre-cursor of VII, although the experiment of Fig. 7did show conclusively that p22 cannot be theprecursor of peptide II. Decisive proof that p22is the precursor of VII will require a demonstra-tion that VII can be derived from pure p22eluted from gel slices, an experiment currentlyin progress.

In an attempt to identify the precursor ofpeptide II with a particular phage gene product,substrate protein prepared from cells infectedwith various amber mutants was assayed forability to yield II*. Table 2 shows that, for mu-tants of genes 20, 21, 22, 23, 24, and 31, peptide11* is formed in each case in an amount thatvaries from 20 to 56% of the amount of II pro-duced in vivo by comparable T4 wild-type-in-fected cells. Thus, if peptide II* is derived fromany one of these gene products, it is derivedfrom a region more N-proximal than the site ofthe chain-terminating mutation. In the case ofthe 22- mutant amE209, the site of this muta-tion is likely close to the N-terminus, since itmaps about 3.5 recombination units from the C-proximal site of amB270 (16).

6

4

N

0XEuS

2

10

8

0

6x

Qu

T4

2

20 40 60 80

FRACTION NUMBER

FIG. 6. Detection ofan additional in vitro cleavage fragment ofp22. The experiment is the same as that ofFig. 3 a,b except that the material pooled from Sephadex chromatography included the entire middle '4C-labeled peak, and the rechromatography on Dowex 50 was extended to detect late-eluting components. Thefigure shows the rechromatography on Dowex 50.

10

5

0

0

S

E-

15

10

5

0

40

EI:

vr

O _

.,:Po > d00R

J. VIROL.

i. _-Jz

VIIT *

01 -lw-

%. %%%,.0 3 H

00 W/

INTERNAL PEPTIDE FORMATION IN PHAGE T4

DISCUSSION

The results presented in this paper and thosereported previously (8) demonstrate the in vitroformation of both T4 internal peptides, II andVII, from precursor proteins. A phage-inducedfactor required for the formation of both pep-

tides has been identified as the product ofphagegene 21. This finding is in agreement withother in vitro cleavage studies using differentapproaches: Onorato and Showe (14) and Bolinand Cummings (3) have reported that 21--in-fected cell extracts are unable to degrade p22,and Bachrach and Benchetrit (1) found the

30

25

° 20

a-

C.>

15

10

5

0

20

15

10

,o S

x

I l;o Ol

: u

20

15

10

5

n

I0x

C-)

30 40 50 60 70 80 90o "40 50 60 70-FRACTION NUMBER

FIG. 7. In vitro formation of VII* from partially purified p22. Partially purified p22 was prepared asfollows: 50 ml ofE. coli B was infected with the quintuple amber mutant IP° 21-23- and labeled with 50 pACiof[14C]lysine from 10 to 30 min after infection, at which time the cells were collected by centrifugation andresuspended in 2.5 ml of CHC13-saturated phosphate buffer (0.01 M, pH 7.0). The lysate was treated withDNase (40 pg/ml) in the presence of10-3 M MgS04 for 10 min at 25 C, sonicated for 3 min, and centrifugedfor 4 h at 50,000 x g in an SW50 rotor. The resulting supernatant was made 0.15 M in NaCl, and anti-p22serum (obtained from L. Onorato) was added in small amounts until a precipitate would form upon standingfor2 h at 4 C. The precipitate was removed by centrifugation, and serum precipitation ofthe supernatant wasrepeated twice. The pooled precipitates were resuspended in 3.0 ml ofsaline (0.15 M NaCl) and washed withsaline four times. The washed immune precipitate contained about 3% of the label present in the originallysate and was shown by SDS-gel electrophoretic analysis to exhibit a strong band ofp22, with other minorcomponents as well. This material was acid-denatured, as described previously (8), resuspended in 1.0 ml ofTris-hydrochloride (0.1 M, pH 7.8), and incubated with 1.0 ml ofan extract of23-e--infected cells at a finalprotein concentration of6.0 mglml. The figure shows: (a) Sephadex G50 chromatography of the acid-solubleincubation products, (b) Dowex 50 chromatography of the pooled material eluting in (a) with the 3H-labeledmarker, and (c) Dowex 50 chromatography ofa tryptic digest ofthe material eluting with the 3H marker in (b).Digestion with trypsin was performed as described in the legend of Fig. 3.

901VOL. 18, 1976

902 GIRI, McCULLOUGH, AND CHAMPE

same gene 21 dependence for the in vitro cleav-age of an internal protein. Similarly, Laemmliet al. (13) have shown that incubation of par-tially purified polyheads prepared from 20--in-fected cells results in cleavage of several of theconstituent proteins including p22, but thatcleavage does not occur if the polyheads arederived from 20-21--infected cells. Whether p21is itself a protease or functions to activate aphage-induced or host protease is not known.However, of possible relevance to this questionis the suggestion from the experiments of Bolinand Cummings (3) that p21-dependent cleavageactivity requires an additional unknown com-ponent.From the fact that all T4 cleavage reactions

so far examined require p21, it might be in-ferred that a single proteolytic activity is in-volved. Tsugita et al. (17), in fact, have foundthat p22, p23, and two internal proteins arecleaved between glutamyl-alanyl residues, cre-ating N-terminal alanine and C-terminal glu-tamate. However, peptides II and VII have N-terminal residues identified as glycine and ly-sine, respectively (4). These peptides could beN-terminal fragments of their precursorscleaved by an enzyme with Glu-Ala specificity.Alternatively, they could result from internalcleavage by other proteases of different specific-ities.In our earlier experiments demonstrating the

in vitro formation of peptide II* (8), we wereunable to detect formation of a product corre-sponding to internal peptide VII. The presentresults show, however, that the formation ofVII* can be effected if a more highly concen-trated source of cleaving activity is employed.This difference in efficiency of formation of thetwo internal peptides may reflect the differingnatures of their precursors, but could, alterna-tively, indicate that the formation of peptideVII* requires factors not required for the forma-tion of peptide II*.Our finding that extracts of T4 wild-type-in-

fected cells are unable to effect the formation ofeither internal peptide is consistent with thefinding of Onoroto and Showe (14) and Bolinand Cummings (3) that such extracts are una-ble to degrade p22. The reason for this apparentdisappearance of activity in a wild-type infec-tion is not understood. Conceivably, the proteo-lytic factor could be masked by entrapment inmature phage particles. Onorato and Showe(14), in fact, report that p22-degrading activityis associated with the "crummy" heads made by23- temperature-sensitive mutants, a findingconsistent with an entrapment mechanism.However, attempts by them and by us to

J. VIROL.

release cleaving activity from mature phageparticles have been negative. Alternatively,the proteolytic factor may itself be inactivatedby cleavage in a wild-type infection and, likethe precursors of the cleaved proteins, neveraccumulate to a high level unless head assem-bly, and thus cleavage, is blocked.Two kinds of evidence lead to the conclusion

that p22 is the precursor of peptide VII but notof peptide H. (i) Preparations of partially puri-fied p22 include the precursor of VII* but notthe precursor of II*. (ii) An N-proximal ambermutation in gene 22 (amE209) eliminates theprecursor of VII* but not the precursor of II*.Neither of these experiments conclusivelyproves that p22 is the precursor of peptide VII,but the fact that partially purified p22 yieldsVII* but not II* shows that the two internalpeptides are derived from different precursors,contrary to what had been previously assumed.This implies that some protein, in addition top22, is cleaved in the interior of a proheadduring T4 head morphogenesis. This proteincannot be identified with any of the knowncleaved proteins, including the three internalproteins, since the cleavage fragments of theseproteins have amino acid compositions quitedifferent from the composition ofpeptide II (17).The only information bearing on the identity

of the precursor ofpeptide II is the map locationof the genetic determinant of this peptide. Us-ing interspecies crosses between T2 and T4 andthe fact that the two phages have chromato-graphically different forms of peptide II, Stern-berg and Champe (16) mapped the locus in orvery close to gene 21. We are thus led to con-sider that p21 may itself be cleaved to yieldpeptide II. If this cleavage inactivates the p21function it could, of course, explain the absenceofp21-dependent cleaving activity in extracts ofT4 wild-type-infected cells.Only recently has p21 been detected by acryl-

amide gel electrophoresis (L. Black, C. Hsiao,and C. Castillo, personal communication).Thus, the hypothesis that p21 is the precursorof peptide II should now be directly testable byassaying the ability ofthe gel band identified asp21 to yield peptide II* upon incubation with anextract containing the phage-induced cleavingfactor. If the result is negative, the samemethod could be applied to all of the otherphage proteins resolved by gel electrophoresis.Our attempts to reconstruct the process of

assembly-associated proteolytic cleavage in vi-tro have revealed that the responsible proteo-lytic activity is detectable only in extracts ofT4mutant-infected cells (other than 21- mutants)in which cleavage is blocked in vivo. The ab-

INTERNAL PEPTIDE FORMATION IN PHAGE T4 903

sence of cleaving activity in extracts of wild-type-infected cells, for which in vivo cleavageoccurs efficiently, suggests that the cleavageprocess is designed to be self-terminating. Thiscould be necessary to prevent proteolytic de-struction of mature phage particles whose sub-units may have residual, slowly cleavablebonds.

ACKNOWLEDGMENTSWe would like to thank Louise Onorato and Michael

Showe for many useful discussions.This investigation was supported by Public Health Ser-

vice grant GM-17020 from the National Institute of GeneralMedical Sciences and by grant GB-33564 from the NationalScience Foundation. J.G.G. received support from PublicHealth Service training grant GM-00507 from the NationalInstitute of General Medical Sciences.

LITERATURE CITED1. Bachrach, U., and L. Benchetrit. 1974. Studies on

phage internal proteins: cleavage of a precursor ofinternal proteins during morphogenesis of bacterio-phage T4. Virology 59:51-58.

2. Black, L. W. 1974. Bacteriophage T4 internal proteinmutants: isolation and properties. Virology 60:166-179.

3. Bolin, R. W., and D. J. Cummings. 1975. Structuralaberrations in T-even bacteriophage. VII. In vitroanalysis of the canavanine-mediated inhibition ofproteolytic cleavage. J. Virol. 16:1273-1281.

4. Champe, S. P., and H. Eddleman. 1967. Polypeptidesassociated with morphogenic defects in bacteriophageT4, p.55-70. In J. S. Colter and W. Paranchych (ed.),The molecular biology of viruses. Academic PressInc., New York.

5. Dickson, R. C., S. L. Barnes, and F. A. Eiserling. 1970.Structural proteins of bacteriophage T4. J. Mol. Biol.53:461-474.

6. Eddleman, H., and S. P. Champe. 1966. Components inT4-infected cells associated with phage assembly. Vi-rology 30:471-481.

7. Edgar, R. S., G. H. Denhardt, and R. H. Epstein. 1964.A comparative study of conditional lethal mutationsof bacteriophage T4D. Genetics 49:635-648.

8. Goldstein, J., and S. P. Champe. 1974. T4-induced ac-tivity required for specific cleavage of a bacterio-phage protein in vitro. J. Virol. 13:419-427.

9. Hosoda, J., and R. Cone. 1970. Analysis ofT4 proteins.I. Conversion ofprecursor proteins into lower molecu-lar weight peptides during normal capsid formation.Proc. Natl. Acad. Sci. U.S.A. 66:1275-1281.

10. Hosoda, J., and C. Levinthal. 1968. Protein synthesis inEscherichia coli infected with bacteriophage T4D. Vi-rology 34:709-727.

11. Kellenberger, E., and C. Kellenberger-van der Kamp.1970. On a modification of the gene product P23 ac-cording to its use as subunit of either normal capsidsofphage T4 or of polyheads. FEBS Lett. 8:140-144.

12. Laemmli, U. K. 1970. Cleavage of structural proteinsduring assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

13. Laemmli, U. K., J. R. Paulson, and V. Hitchins. 1974.Maturation of the head of bacteriophage T4. V. Apossible DNA packaging mechanism: in vitro cleav-age of the head proteins and structure of the core ofthe polyhead. J. Supramol. Struct. 2:276-301.

14. Onorato, L., and M. K. Showe. 1975. Gene 21 protein-dependent proteolysis in vitro of purified gene 22product ofbacteriophage T4. J. Mol. Biol. 92:395-412.

15. Showe, M. K., and L. W. Black. 1973. Assembly core ofbacteriophage T4: an intermediate in head formation.Nature (London) New Biol. 242:70-75.

16. Stemnberg, N., and S. P. Champe. 1969. Genetic deter-minant of an internal peptide of bacteriophage T4. J.Mol. Biol. 46:377-392.

17. Tsugita, A., L. W. Black, and M. K. Showe. 1975.Protein cleavage during virus assembly: characteri-zation of cleavage in T4 phage. J. Mol. Biol. 98:271-275.

VOL. 18, 1976