Proc. Nati. Acad. Sci. USAVol. 84, pp. 714-718, February 1987Biochemistry

Adenovirus type 2 endopeptidase: An unusual phosphoproteinenzyme matured by autocatalysisPRADEEP K. CHATTERJEE AND S. J. FLINTDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544

Communicated by Walter Kauzmann, October 20, 1986 (received for review June 16, 1986)

ABSTRACT A 19-kDa protein, present in low copy num-ber in purified adenovirus type 2, has been characterized.Several criteria were used to establish that this protein isneither a degradation product of the known structural proteinsof the virion nor a minor, unusually modified, form of proteinVII. This 19-kDa protein, unlike other virion proteins, pos-sesses alkali-resistant phosphoamino acids. Analysis by partialproteolysis indicated that it is related to a 23-kDa phospho-protein present in H2ts-1 virions assembled in infected cellsmaintained at 39°C. Affinity labeling with [3H]diisopropylfluorophosphate showed that the 19-kDa protein contains theactive site for a serine protease. We, therefore, conclude thatthe 19-kDa protein is the active form of the adenovirus-encodedendopeptidase, defined by the H2ts-1 mutation, and is synthe-sized as a 23-kDa precursor that appears to mature byautocatalysis.

Several of the structural proteins comprising adenovirus type2 (Ad2) or 5 virions are synthesized in infected cells asprecursor proteins that undergo proteolytic cleavage duringvirion maturation (1-5). These include the precursors topolypeptides IIIa, VI, VII, VIII, and the terminal protein (6).A temperature-sensitive mutant ofAd2, H2ts-1, which fails toprocess these precursor polypeptides but produces nonin-fectious particles at the restrictive temperature, has beenisolated (7), and the mutation precisely mapped on theadenovirus genome (8-11): location of the H2ts-1 mutationidentified an open reading frame of predicted coding capacity23-kDa encoding the endopeptidase in the L3 region (11).Endopeptidase activity has been detected in extracts madefrom purified virions and cells infected with Ad2 (6, 12). Itappears that the protease has a cleavage specificity forGly-Ala peptide bonds and is inhibited by commonly usedserine protease inhibitors such as phenylmethylsulfonyl flu-oride or diisopropyl fluorophosphate (6). However, theprotease has been neither isolated nor characterized bio-chemically.

In this report we describe the identification of the virionendopeptidase as a low-copy-number virion protein with anunusual pattern of phosphorylation. We present evidencethat the protease itself is made as a precursor, which ispresent in H2ts-1 vinrons grown at the restrictive tempera-ture. It, therefore, appears likely that this protease precursorundergoes self-cleavage during virion assembly to generatethe mature form.

MATERIALS AND METHODSCells and Viruses. Wild-type Ad2 and H2ts-1 were grown in

HeLa cells maintained in suspension culture at a density of2-5 x 105 cells per ml. 32P-labeled Ad2 was grown andpurified as described in detail elsewhere (13). H2ts-1 virionswere grown and labeled at either 33°C or 39°C and purified as

described (14). [35S]Cysteine-labeled virions were isolatedand purified in the manner described for labeling with[35S]methionine or [3H]arginine (15), except that cysteine-free medium was used during labeling with [35S]cysteine (2,uCi/ml; 600 Ci/mmol; 1 Ci = 37 GBq; New EnglandNuclear).

Analysis of Viral Proteins. Virions were purified by cen-trifugation to equilibrium in cesium chloride gradients. Theviral DNA was then degraded chemically using diphenyl-amine in formic acid according to the procedure of Kunkeland Martinson (16) and described in detail (13, 14).NaDodSO4/polyacrylamide gels (17) were used to analyzethe phosphoproteins or [35S]cysteine-labeled proteins ofpurified virions. Alkali treatment ofgels was according to theprocedure of Cooper and Hunter (18). After alkali treatment,gels were neutralized and stained with Coomassie blue asdescribed (13). Partial proteolysis was according to theprocedure of Cleveland et al. (19).Treatment of Virions with [3H]Dlisopropyl Fluorophos-

phate. Purified virions were dialyzed overnight against 10mM Tris HCl, pH 6.8, containing 1 mM MgCl2. The dialyzed,native samples were treated directly with [3H]diisopropylfluorophosphate (2 Ci/mmol, Amersham) at a final concen-tration of =1 mM. Samples were Vortex mixed and incubatedat 370C for 20 min. Ten volumes of ethanol were added toprecipitate the proteins and the DNA. After centrifugation,the pellet was dissolved in NaDodSO4 sample buffer con-taining only 0.1% mercaptoethanol by incubation at 50°C for20 min. a-Chymotrypsin (from bovine pancreas; Sigma) wastreated in an identical fashion with [3H]diisopropyl fluoro-phosphate and served as a positive control. In some exper-iments both chymotrypsin and Ad2 virion proteins were alsoindividually treated with nonradioactive 1mM phenylmethyl-sulfonyl fluoride prior to treatment with [3H]diisopropylfluorophosphate.

RESULTSIdentification of a 19-kDa Ad2 Virion Protein as a Distinct

Species. During the course of our studies of the molecularstructure of the nucleoprotein core of adenovirus (13, 14, 20),we have undertaken the analysis of the phosphoproteinspresent in Ad2 virions. Wild-type Ad2 was grown in phos-phate-free medium containing 32P-labeled inorganic phos-phate. After purification of virions, viral DNA was degradedwith formic acid and diphenylamine (14, 16), the proteinswere precipitated with ethanol, and analyzed in 12% NaDod-S04/polyacrylamide gels. To investigate the nature of thephosphorylation of these phosphoproteins, such polyacryl-amide gels were also treated with 1 M NaOH according to themethod of Cooper and Hunter (18): the phosphate groups ofthe commonly occurring phosphoamino acids, phosphoser-ine and phosphotheronine, are hydrolyzed under these con-ditions, while those present in phosphotyrosine are resistantto such treatment (18). Typical results of such an analysis of

Abbreviation: Ad2, adenovirus type 2.

714

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Proc. Natl. Acad. Sci. USA 84 (1987) 715

virion phosphoproteins are shown in Fig. 1. The majorphosphoproteins observed were in good agreement withthose described (21, 22) and included a prominent species of19-kDa apparent molecular weight (Fig. 1, lane 1). This19-kDa species exhibited a slightly lower mobility than theabundant virion protein protein VII (compare lanes 1 and 5,Fig. 1). Moreover, protein VII contained only small quanti-ties of 32p (Fig. 1, lane 1), which was susceptible to alkalinehydrolysis (compare lanes 1 and 3, Fig. 1). By contrast, thesubstantial amount of 32p label in the 19-kDa protein waslargely resistant to alkaline hydrolysis. This property indi-cated that the 19-kDa species could not be a breakdownproduct of any of the higher molecular weight phosphopro-teins present in the virion.To investigate whether the 19-kDa phosphoprotein was the

precursor of the major core protein VII (1), incompletevirions and empty capsids commonly referred to as "topcomponents" (23) from a cesium chloride density gradientwere examined in parallel with mature virions: this fractionwas rich in protein preVII, but contained negligible quantitiesof 32P-labeled 19-kDa protein (Fig. 1, lanes 2, 4, and 6). Thus,the 19-kDa polypeptide cannot represent the protein VIIprecursor.To establish that the 19-kDa species was not a minor,

variant form of protein VII, virions were isolated fromAd2-infected cells that had been labeled with [35S]cysteineduring the late phase of infection: the amino acid sequencesof protein VII or its precursor (24) and the DNA sequence ofthe gene coding for them (25) have established that proteinVII and its precursor lack cysteine residues. Proteins ofpurified, [35S]cysteine-labeled Ad2 virions were compared tothose of [3H]arginine-labeled Ad2 virions and nuclear pro-teins from HeLa cells labeled with [35S]methionine, 20 hrafter infection with Ad2. As illustrated in Fig. 2, the 19-kDa

II [i

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FIG. 1. Analysis of phosphoproteins present in mature andincomplete Ad2 virions. Cells infected with Ad2 were labeled withinorganic [32P]phosphate in phosphate-free medium from 12 to 44 hrafter infection. Lanes 1 and 2 show an autoradiogram of phospho-proteins from purified virions and top components, respectively,analyzed in a 12% NaDodSO4/polyacrylamide gel. Lanes 3 and 4show the same gel re-exposed after treatment with 1M NaOH. Lanes5 and 6 show Coomassie blue stained gel of lanes 1 and 2,respectively, after alkali treatment.

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FIG. 2. Comparison of Ad2 virion proteins labeled with [3H]ar-ginine, [35S]cysteine, or [35S]methionine. Lanes 1 and 2 show[3H]arginine-labeled virion proteins and nuclear proteins of adeno-virus-infected cells labeled with [35S]methionine from 18 to 20 hr afterinfection, respectively. Molecular size standards were applied to lane3 and the molecular sizes in kDa are shown at the right. Lane 4 showsvirion proteins labeled with [35S]cysteine. The arrowhead marks theposition of the 19-kDa species in lane 4.

protein was labeled with [35S]cysteine. Moreover, the rela-tive intensity of labeling of this species compared to that ofprotein VI suggested that it is rather rich in cysteine residues,especially when its poor staining (Fig. 1, lane 5) and apparentfailure to be labeled with [3Hlarginine or [35S]methionine(Fig. 2, lanes 1 and data not shown, respectively) are takeninto account.

Analysis of Phosphoproteins of H2ts-1 Virions. 32P-labeledvirions were purified from cells infected with H2ts-1 virionsand grown at a restrictive temperature of 39°C as described(14). Under these conditions, the virion endopeptidase,defined by the H2ts-1 mutation, fails to process the precur-sors to proteins Ila, VI, VII, VIII, and the 55-kDa terminalprotein to their mature forms (3, 6, 7). A comparison of thephosphoproteins of such mutant virions to those obtainedfrom wild-type virus is shown in Fig. 3. Protein VII, identifiedfrom the Coomassie blue stain of this sample (Fig. 3, lane 5)and by partial proteolysis (data not shown), carried only asmall quantity of 32P radioactivity (13, 22) that was complete-ly labile to treatment with 1 M NaOH (Fig. 3, lane 3). Theprecursor to protein VII (preVII), present in H2ts-1 virionsgrown and labeled at a restrictive temperature (H2ts-1 39°Cvirions), also carried small quantities of 32P radioactivity (Fig.3, lane 2), similarly completely labile to 1 M NaOH treatment(Fig. 3, lane 4). As discussed, the 19-kDa protein (Fig. 3, lane1) retained almost all of its 32p following such a treatment(Fig. 3, lane 3). However, this phosphoprotein was notobserved in H2ts-1 39°C virions, which instead contained anew species, apparent molecular size 23 kDa, whose 32p labelwas similarly resistant to alkaline hydrolysis (Fig. 3, lanes 2and 4). The absence of a stained species at the position of the23-kDa phosphoprotein (compare lanes 6, 2, and 4, Fig. 3)

Biochemistry: Chattedee and Flint

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716 Biochemistry: Chatterjee and Flint

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FIG. 3. Comparison of the phosphoproteins of wild-type Ad2with those of H2ts-1 virions assembled at 39TC. Lanes 1 and 2 showan autoradiogram of a 12% NaDodSO4/polyacrylamide gel analysisof the phosphoproteins present in purified Ad2 and H2ts-1 virionsgrown at 390C, respectively. Lanes 3 and 4 show lanes 1 and 2,respectively, re-exposed after treatment with 1 M NaOH. Lanes 5and 6 show lanes 1 and 2 after staining with Coomassie blue afteralkali treatment.

suggested that the 23-kDa protein was, like the 19-kDaprotein species of wild-type virions, present in low copynumber. H2ts-1 virions grown and labeled with 32P at apermissive temperature produced a pattern of phosphopro-teins indistinguishable in these gels from that displayed bywild-type virions (data not shown, but see refs. 14).The 19-kDa Phosphoprotein of Wild-Type Virions Is Related

to the 23-kDa Phosphoprotein of H2ts-1 Virions Grown at39TC. Fig. 4 shows the results of protease V8 partial prote-olytic digestion of the 19-kDa phosphoprotein from wild-typeadenovirions and the 23-kDa phosphoprotein from H2ts-1virions isolated from infected cells grown at 39TC. It is clearthat the two proteins share at least one V8 proteolyticfragment, designated p, which contains the alkali-resistant32p radioactivity. This result suggested that the 23-kDaprotein might correspond to the precursor of the 19-kDaprotein in wild-type virions. To test this hypothesis, thepattern of V8 proteolytic fragments of the 23-kDa and the19-kDa phosphoproteins of H2ts-1 virions assembled at 39TCor 330C, respectively, were compared. The results shown inFig. 4B indicate that the 23-kDa phosphoprotein present inH2ts-1 virions assembled at 390C is indeed a precursor of the19-kDa phosphoprotein present in H2ts-1 virions assembledat 330C.

Affinity Labeling of the 19-kDa Protein by [3H]DiisopropylFluorophosphate. Results of several studies (6, 12) haveindicated that the virion endopeptidase that displays a spec-ificity for Gly-Ala or Ala-Gly peptide bonds is inhibited byserine-protease inhibitors, such as diisopropyl fluorophos-phate and phenylmethylsulfonyl fluoride. To test the hypoth-esis that the 19-kDa phosphoprotein species represented themature form of the virion endopeptidase, we thereforeaffinity-labeled the 19-kDa phosphoprotein with [3H]diiso-propyl fluorophosphate. Purified, native unlabeled Ad2virions were treated at room temperature with 1 mM[3H]diisopropyl fluorophosphate, and the virion proteins

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FIG. 4. (A) (Upper) Comparison of the partial proteolytic pat-terns of the 19-kDa phosphoprotein from wild-type Ad2 and the23-kDa phosphoprotein from H2ts-1 virions assembled at 390C.Lanes 1 and 2 show the products of V8 partial proteolysis of the19-kDa phosphoprotein, and lanes 3 and 4 show a similar analysis ofthe 23-kDa phosphoprotein from H2ts-1 virions assembled at 390C.(Lower) The same gel, re-exposed after treatment with 1 M NaOH,is shown. (B) Comparison of the 19-kDa and 23-kDa phosphoproteinspecies derived from H2ts-1 virions assembled at 330C and 390C,respectively. A NaDodSO4/polyacrylamide gel analysis of the prod-ucts of partial proteolysis by V8 protease of the 19-kDa species fromH2ts-1 virions assembled at 33°C (lanes 1 and 2) and the 23-kDaspecies from H2ts-1 virions assembled at 39°C (lanes 3 and 4) areshown. The autoradiogram is of the gel after treatment with 1 MNaOH.

were analyzed by electrophoresis in NaDodSO4/polyacryl-amide gels (17) after treatment at 50°C for 20 min in samplebuffer containing 0.1% 2-mercaptoethanol. Unlabeledchymotrypsin was treated identically and in parallel with[3H]diisopropyl fluorophosphate to serve as a positive con-trol. The species marked M in Fig. 5, lane 1, displayedelectrophoretic mobility identical to that of the 19-kDaspecies, while the band marked D appeared to represent adimer of 19-kDa protein, resulting from the limited treatmentof the samples with 2-mercaptoethanol. Migration ofchymotrypsin, under these conditions, predominantly as adimeric 26-kDa species (Fig. 5, lane 4) indicated that thedisulfide bonds linking the three polypeptide chains of thisenzyme were largely unaffected by the mild 2-mercaptoeth-anol treatment (see ref. 26). Under these conditions, no othervirion proteins were labeled with [3H]diisopropyl fluorophos-phate (Fig. 5, lane 1, and data not shown), strongly arguingthat the labeling observed was not the result of reactions ofnoncatalytic serine or threonine residues with the labeledligand. Such affinity labeling ofchymotrypsin and the 19-kDaprotein of Ad2 virions with [3H]diisopropyl fluorophosphatewas prevented if the samples were treated with nonradioac-tive phenylmethylsulfonyl fluoride prior to treatment with[3H]diisopropyl fluorophosphate (data not shown). Thus, the19-kDa protein in wild-type virions can be specifically labeledin vitro with the affinity label [3H]diisopropyl fluorophos-

Proc. Natl. Acad. Sci. USA 84 (1987)

I

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Biochemistry:ChatterjeeandFlintProc.Nati. Acad. Sci. USA 84 (1987) 717

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FIG. 5. Affinity labeling of native adenovirus proteins with

[3H]diisopropyl fluorophosphate. Purified Ad2 virions were dialyzed

overnight in Tris-HCI buffer, pH 6.8, and then treated with [3H]di-

isopropyl fluorophosphate. A fluorogram of the virion proteins

analyzed in 12% NaDodSO4/polyacrylamide gels is shown in lane 1.

Lane 4 shows an identical analysis performed in parallel with

a-chymotrypsin. Lanes 2 and 3 contain [3Hlarginine-labeled virion

proteins and [35S]methionine-labeled nuclear proteins from cells

infected with Ad2, respectively, as markers of protein VII and

protein preV11. M marks the position of the 19-kDa species, while D

has the apparent molecular size expected of a dimer of species M.

phate clearly indicating that the 19-kDa protein is a serineprotease (Fig. 5).

Copy Number of the 19-kDa Protease Within Virions.

Identification of the 19-kDa protein as the virion protease

allowed us to estimate its copy number from its relative

content of [35S]cysteine radioactivity and its known se-

quence. Sequence analysis has established that protein VI

has one cysteine residue per molecule (25) whereas the

19-kDa protease contains six cysteines per molecule in its

mature form (9, 11, 25). Protein VI is present at about 340

copies per virion (27). Assuming that protein VI and the

endopeptidase are made from the same pool of [355S]cysteineand that these proteins have similar half-lives, we estimate,

from the labeling intensities of the 19-kDa protease and

protein VI in gels such as the one shown in Fig. 2, that there

are about 30 copies of this protease per virion. This estimate

is likely to be on the higher side, as a small quantity of 35S

label was detected in protein VII following [355S]cysteinelabeling, even though protein VII contains no cysteineresidues.

DISCUSSION

The 19-kDa protease of Ad2 virions discussed in this report

was first observed as a phosphoprotein unusual in the

resistance of its phosphate groups to alkaline hydrolysis (Fig.

1). This property, shared by the 23-kDa protein present in

H2ts-1 virions assembled at a nonpermissive temperature,

indicates that these phosphoproteins cannot, despite their

low concentration, be degradation products of larger virion

phosphoproteins: the latter carry phosphate groups that are

much more sensitive to alkaline hydrolysis (13, 14). The

efficient labeling of the 19-kDa protein by incorporation of

[355]cysteine (Fig. 2) establishes that it is not a minor, variant

form of protein VII or its precursor (24, 25, 28). The specific

labeling of the 19-kDa protein present in native virions with

[3Hldiisopropyl fluorophosphate, a serine protease inhibitor,

identifies it as the virion protease, shown to be sensitive to

such inhibitors (6, 12). The low yield of affinity-labeled19-kDa protein (Fig. 5) is presumably the result of its low

concentration in Ad2 virions and/or limited accessibility of

the protease in the native virions used in the experimentshown in Fig. 5.

The 19-kDa protein is not present in H2ts-1 virions isolated

from cells grown at a nonpermissive temperature, but rather

is replaced by a 23-kDa protein (Fig. 3). Although this result

was unexpected, it is clear that the 23-kDa protein is theprecursor to the 19-kDa protease: the two proteins share acharacteristic product of partial V8 proteolytic digestion thatcarries alkali-resistant phosphate groups (Fig. 4), and bothcan be specifically labeled with [3H]diisopropyl fluorophos-phate (Fig. 5 and data not shown). Thus the protease, likeseveral other virion proteins, is synthesized as a precursor(23 kDa) whose cleavage occurs in assembled virions. TheH2ts-1 mutation, which results in substitution of a leucine fora proline residue at amino acid 117 of the 23-kDa protein (11),inhibits the activity of the virion endopeptidase (6, 7). Thefailure of maturation of the precursor to the protease inH2ts-1 virions assembled at a nonpermissive temperaturetherefore implies that this substitution alters the secondaryand tertiary structure of the protein sufficiently to preventcleavage by a second, as yet unknown protease, or byautocatalysis. As the 23-kDa protease can also be labeledwith [3H]diisopropyl fluorophosphate, it does not seem likelythat the active site of the enzyme is destroyed by the H2ts-1mutation. Although it is dificult to rule out the formerpossibility, the second seems more likely because it has beendeduced from the patterns of cleavage of precursors toproteins lIla, VI, VII, VIII, and to the terminal protein thatthe virion protease cleaves specifically between Gly-Ala orAla-Gly residues (6, 12). The 23-kDa protease precursorcontains the sequence Ala-Gly at amino acids 45 and 46 (11)and removal of the 45 N-terminal amino acids of thispolypeptide would generate a protein of the predicted mo-lecular size, 19 kDa. Numerous cellular proteases, includingchymotrypsin, are synthesized as precursors (zymogens) thatundergo autocleavage reactions as part of their maturationprocess (26). Similar autocatalytic cleavages have beensuggested to occur in the maturation process of the poliovirus(29) and the encephalomyocarditis virus (30) proteases.The 23-kDa protease precursor, exhibiting negligible cleav-

age, can be isolated from virions assembled in cells main-tained at 390C, but purified and manipulated at room tem-perature. The mutant protease precursor of such H2ts-1virions can be labeled with [3H]diisopropyl fluorophosphate(P.K.C., unpublished observations), suggesting that the ac-tive site is not destroyed under nonpermissive conditions. It,therefore, appears that the processing of the protease pre-cursor, probably by autocatalysis, occurs in a coordinatedfashion at a particular stage in virion assembly. The identi-fication of the virion polypeptide carrying endopeptidaseactivity, particularly the demonstration that this minor virionprotein can be efficiently labeled with [15S]cysteine (Fig. 2),will permit the entry of the protease precursor into virionsand its proteolytic processing during virion assembly andmaturation to be examined in more detail. It will also be ofinterest to establish the location of the protease within thevirion and identify the proteins with which it interacts, for itis clear that the enzyme plays a pivotal role in the productionof infectious virus particles (7).

We thank Ueng-Cheng Yang and Cathy Castiglia for helpfuldiscussions and Rhonda Lunt, Margie Young, and Seema Chatterjeefor expert technical assistance. This work was supported by a grantfrom the American Cancer Society. S.J.F. is the recipient of aResearch Career Development Award from the National Institutes ofHealth.

1 . Anderson, C. W., Baum, P. R. & Gesteland, R. F. (1973) J.Virol. 12, 241-252.

2. Galibert, F., Hdrrisse, J. & Courtois, G. (1979) Gene 6, 1-7.3. Boudin, M. L., D'Halluin, J.-C., Cousin, C. & Boulanger, P.

(1980) Virologyv 101, 144-156.4. H~risse, J., Courtois, G. & Galibert, F. (1980) Nucleic Acids

Res. 8, 2173-2192.5. Akusjarvi, G. & Persson, H. (1981) J. Virol. 38, 469-482.

Biochemistry: Chattedee and Flint

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718 Biochemistry: Chattejee and Flint

6. Tremblay, M. L., D'ery, C. V., Talbot, B. G. & Weber, J.(1983) Biochim. Biophys. Acta 743, 239-245.

7. Weber, J. (1976) J. Virol. 17, 462-471.8. Hassell, J. A. & Weber, J. (1978) J. Virol. 28, 671-678.9. Kruijer, W., van Schaik, F. M. A. & Sussenbach, J. S. (1980)

Nucleic Acids Res. 8, 6033-6042.10. Akusjarvi, G., Zabielski, J., Perricaudet, M. & Pettersson

(1981) Nucleic Acids Res. 9, 1-17.11. Liang, Y.-K., Akusjarvi, G., Alestrom, P., Pettersson, U.,

Tremblay, M. & Weber, J. (1983) J. Mol. Biol. 167, 217-222.12. Bhatti, A. R. & Weber, J. (1979) Virology 96, 478-485.13. Chattejee, P. K., Vayda, M. E. & Flint, S. J. (1986) J. Mol.

Biol. 188, 23-37.14. Chatteree, P. K., Yang, U.-C. & Flint, S. J. (1986) Nucleic

Acids Res. 14, 2721-2735.15. Chattejee, P. K., Vayda, M. E. & Flint, S. J. (1985) J. Virol.

55, 379-386.16. Kunkel, G. R. & Martinson, H. G. (1978) Nucleic Acids Res.

5, 4263-4271.17. Laemmli, U. K. (1970) Nature (London) 227, 680-685.18. Cooper, J. A. & Hunter, T. (1981) Mol. Cell. Biol. 1, 165-178.

Proc. Nadl. Acad. Sci. USA 84 (1987)

19. Cleveland, D. W., Fischer, S. G., Kirschner, M. W. & Laemm-li, U. K. (1977) J. Biol. Chem. 252, 1102-1106.

20. Chattejee, P. K., Vayda, M. E. & Flint, S. J. (1986) EMBO J.5, 1633-1644.

21. Russell, W. C. & Blair, G. E. (1977) J. Gen. Virol. 34, 19-35.22. Axelrod, N. (1978) Virology 87, 366-383.23. Prage, L., Pettersson, U., Hoglund, S., Lonberg-Holm, K. &

Philipson, L. (1970) Virology 42, 341-348.24. Sung, M. T., Cao, T. M., Coleman, R. T. & Budelier, K. A.

(1983) Proc. Natl. Acad. Sci. USA 80, 2902-2906.25. Alestrbm, P., Akusjarvi, G., Lager, M., Liang, Y.-K. &

Petterson, U. (1984) J. Biol. Chem. 259, 13980-13985.26. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358.27. van Oostrum, J. & Burnett, R. M. (1985) J. Virol. 56, 431-448.28. Sung, M. T., Lischwe, M. A., Richards, J. C. & Hosokawa,

K. (1977) J. Biol. Chem. 252, 4981-4987.29. Hanecak, R., Semler, B. L., Anderson, C. W. & Wimmer, E.

(1982) Proc. Natl. Acad. Sci. USA 79, 3973-3977.30. Palmenberg, A. C. & Rueckert, R. R. (1982) J. Virol. 41,

244-249.

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