For many animal and plant viruses, a virus-coded proteinase activity is vital for the synthesis of infectious virus (for review, see (1) ). These virus-coded proteinases are appealing targets for antiviral therapy. Human adenoviruses encode a proteinase activity that is required for the maturation of infectious virions. Of the 12 major polypeptides from which adenovirus virions are assembled, six are proteolytically processed. Weber (2) isolated a temperature-sensitive mutant, H2ts-1 (ts-1), ()of human adenovirus serotype 2 (Ad2) that lacks proteinase activity at the nonpermissive temperature. Virions of ts-1 assemble at the nonpermissive temperature, but contain precursors in place of the mature components present in wild-type virus. Such immature virions attach to cells, but fail to initiate a productive infection(3, 4) . The mutation in ts-1 was identified as a single base pair change in a 204-codon open reading frame (L3 23-kDa protein) at the 3`-end of the L3 family of late messages(5) . The nucleotides in the L3 23-kDa open reading frame were cloned into plasmids that permitted efficient expression in Escherichia coli(6) .

Recently, we developed a specific, sensitive, and quantitative assay for the adenovirus proteinase and used it to characterize the activity in disrupted wild-type virus. ()The assay is based upon the observation that the adenovirus proteinase will cleave small peptides with sequences that correspond to the sequences on the amino-terminal side of the cleavage sites in virion precursor proteins. For example, the substrate (Leu-Arg-Gly-Gly-NH)-rhodamine is cleaved to Leu-Arg-Gly-Gly-NH-rhodamine by the adenovirus proteinase; this is accompanied by a 3500-fold increase in fluorescence that is proportional to the amount of proteinase.

We had previously shown that when wild-type Ad2 virus was incubated with (Leu-Arg-Gly-Gly-NH)-rhodamine, significant hydrolysis of the substrate was observed, and that when ts-1 virus was incubated with (Leu-Arg-Gly-Gly-NH)-rhodamine, no hydrolysis of the substrate was observed(7) . Little or no hydrolysis was observed with purified recombinant L3 23-kDa protein (recombinant endoproteinase (rEP)) expressed in E. coli. However, when ts-1 virus and rEP were incubated together with (Leu-Arg-Gly-Gly-NH)-rhodamine, significant hydrolysis of the substrate occurred. This implied that cofactors may be required for maximal activity. The first cofactor we discovered was the viral DNA. If disrupted wild-type virus is treated with DNase, proteinase activity is lost, but can be restored upon addition of Ad2 DNA(7) . A second cofactor was shown to be a plasmin-sensitive virion protein (7) that turned out to be the 11-amino acid peptide from the C terminus of the precursor to virion protein VI, pVIc(7, 8) .

Here, we present our purification procedure for a recombinant L3 23-kDa protein expressed in E. coli. Although others have published purification procedures for a recombinant form of the L3 23-kDa protein from E. coli(9, 10) and insect cells(8) , none of the purification procedures utilized a quantitative assay, so there is, for example, no report of increases in specific activity or even yields. We also present our procedure for the purification and identification of pVIc as a cofactor for proteinase activity. We then show that the cofactors stimulate proteinase activity not by decreasing K, which changes by no more than 2-fold, but by increasing k, which increases >6000-fold. By measuring initial velocities as a function of pH, we show that the enzyme activity is clearly different from that of papain. Moreover, the pK values of the amino acids that affect catalysis are different in the presence and absence of Ad2 DNA.

EXPERIMENTAL PROCEDURES

Materials

Cells and Viruses

Assay for Proteinase Activity

Protein concentration was determined by the bicinchoninic acid protein assay (Pierce) and/or for rEP with a calculated molar absorbance coefficient at 280 nm of 26,510(11) . The concentration of pVIc was determined by titration of its cysteine residue with Ellman's reagent and confirmed by quantitative amino acid analysis. The cysteine titration was done by adding 10 µl of pVIc stock solution to 0.99 ml of Ellman's buffer (0.1 M NaHPO (pH 7.3) and 1 mM EDTA containing 0.33 mM 5,5`-dithiobis(2-nitrobenzoic acid)) and then monitoring the increase in absorbance at 412 nm. The moles SH/mol of pVIc was calculated using a molar extinction coefficient at 412 nm of 14,150 (12) for thionitrobenzoate.

SDS-Polyacrylamide Gel Electrophoresis

Expression of the Recombinant L3 23-kDa Protein in E. coli

Figure 1: Purification of adenovirus rEP. Samples of lysates from bacteria induced with IPTG to express rEP (lane a), the 10,000 g supernatant of the lysate (lane b), the flow-through fraction from the DEAE chromatography step (lane c), the rEP activity pool from the S-Sepharose chromatography step (lane d), rEP purified by passage over a zinc-iminodiacetic acid-Sepharose column (lane e), and molecular mass markers (lane f) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue.

Purification of the Recombinant L3 23-kDa Protein Expressed in E. coli

The lysate was clarified by centrifugation at 10,000 g for 10 min. This fraction, named the supernatant, was loaded onto a 2.5 25-cm TSK-DEAE column equilibrated in 50 mM Tris (pH 8.0), 15 mM NaCl, and 5 mM -mercaptoethanol. The column was washed with 300 ml of the equilibration buffer. rEP was located in the flow-through fractions by SDS-PAGE and by activity assays. The fractions with rEP were pooled and named DEAE FT.

The DEAE FT pool was fractionated on a 1.6 15-cm S-Sepharose Fast Flow column equilibrated in 50 mM Tris (pH 8.0), 15 mM NaCl, and 1 mM DTT. After loading, the column was washed with 200 ml of 50 mM Tris (pH 8.0), 15 mM NaCl, and 1 mM DTT to remove the Triton X-100. Then, a 150-ml linear gradient of 15-400 mM NaCl in 50 mM Tris (pH 8.0) was applied to the column. The fractions containing rEP were identified and pooled and named SSEPH. rEP consistently eluted from this column at an ionic strength of 0.1 M NaCl.

The SSEPH pool was applied to a 1.6 5-cm chelating Sepharose column charged with zinc. Charging with zinc was accomplished by a thorough washing of the resin with 0.05 M EDTA and 1 M NaCl, followed by washing with water to remove the EDTA, and then followed by washing with 0.2 M ZnCl in 5 mM HCl. Next, the column was equilibrated in 25 mM HEPES (pH 8.0) and 0.1 M NaCl. The column was loaded with the SSEPH pool and washed with the equilibration buffer until the absorbance at 280 nm was <0.02. Then, a series of solutions were applied in steps, each step containing 25 mM HEPES (pH 8.0) and the indicated constituents: 1 M NaCl; 0.1 M NaCl; 35 mM imidazole and 0.1 M NaCl; 0.1 M NaCl; 35 mM imidazole and 0.1 M NaCl; 0.1 M NaCl; 35 mM imidazole and 0.1 M NaCl; and 0.1 M NaCl. rEP eluted in 25 mM HEPES (pH 8.0), 0.1 M NaCl, and 0.01 M EDTA. After dialysis against 20 mM HEPES (pH 8.0), 5 mM NaCl, and 0.1 mM EDTA, the purified enzyme was stored at -70 °C.

Purification of the Protein Cofactor pVIc

The flow-through fraction from the second Centricon-30 centrifugation was evaporated to dryness, dissolved in 0.1% trifluoroacetic acid, and applied to a C column (Aquapore OD-300 7µ, 2.1 100 mm). The activity was eluted by a linear gradient of 0-30% acetonitrile in 0.1% trifluoroacetic acid at a rate of 1%/min. Assays of fractions from each peak were performed in the presence of Ad2 DNA and rEP.

RESULTS

Comments on the Purification of rEP

Complementation of the ts-1 Mutation by rEP

Figure 2: Processing of Ad2 ts-1 precursor proteins by rEP. Twice banded Ad2 ts-1 virions were disrupted by two cycles of freeze/thaw followed by heat treatment. Reactions of 0.18 ml contained 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10 mM octyl glucoside, 3 10 virions, and 0.25 µM rEP. After the indicated times at 37 °C, aliquots were removed from the reactions, and the proteins were fractionated by SDS-PAGE on 8-25% gradient gels. Lanes a and h, wild-type Ad2 virions; lane g, Ad2 ts-1 virions; lanes b-f, Ad2 ts-1 virions incubated with rEP for 6, 4, 2, 1, and 0.5 h, respectively. The precursor proteins of the ts-1 virion and their mature counterparts in the wild-type (wt) Ad2 virion are labeled. The proteins were visualized by silver staining.

Comments on the Purification and Identification of the Second Cofactor, pVIc

Figure 3: Initial purification of the second cofactor activity. Virus was disrupted by treatment with 10% pyridine. The pellet and supernatant (Sup) were obtained after centrifuging disrupted virus. The pellet was solubilized, placed in a Centricon-30, and centrifuged to yield retentate-1 and flow-through-1. Ammonium acetate to 1 M was added to retentate-1, and the solution was recentrifuged to yield retentate-2 and flow-through-2. Assays were performed in the presence of 3 nM rEP and in the absence or presence of 778 ng/ml Ad2 DNA.

The final step in the purification of the second cofactor was reverse-phase chromatography. The flow-through fraction from the second Centricon-30 centrifugation was applied to a C column, and the cofactor activity was eluted with a linear gradient of 0-30% acetonitrile (Fig. 4). Each peak from the column was assayed for proteinase activity. Only the three peaks marked with arrows showed proteinase activity in the presence of rEP and Ad2 DNA. A profile of the proteinase activity in each of the three peaks is shown in the inset in Fig. 4. Of the total cofactor activity applied to the C column, 7% was recovered in peak a, 22% in peak b, and 14% in peak c. The final yield was >49%.

Figure 4: Final step in the purification of the second cofactor activity. The flow-through fraction from the second Centricon-30 centrifugation was applied to a C column, and the activity was eluted by a linear gradient of 0-30% acetonitrile in 0.1% trifluoroacetic acid. Inset, assays of fractions from peaks a, b, and c were performed in the presence of 778 ng/ml Ad2 DNA and 2.2 nM rEP. mAU, milli-absorbance units.

Identification of the Second Cofactor as pVIc

Figure 5: Amino acid sequence of the second cofactor and comparison with the amino acid sequence of the precursor to adenovirus protein VI. The amino acid sequences of the proteins in peaks a-c in Fig. 4(inset) were determined in a gas-phase sequencer. The question mark at position 6 indicates a variable yield of lysine, as described under ``Results.'' The amino acid at position 10 was presumed to be a cysteine. The two adenovirus proteinase consensus cleavage sequences in pVI are underlined, and the location of the second cofactor sequence beginning at position 240 is in boldface. The amino acid sequence of pVI was from Roberts et al.(14) .

The proteins in peaks a-c of Fig. 4were also subjected to time-of-flight mass spectrometry. In peak a, there was one major species with an M of 1350. Thus, there was mostly pVIc present. In peak b, there were two species present at a ratio of 2:1, one with an M of 1350 and the other with an M of 2700. Thus, there was mostly pVIc present, but also some pVIc dimer. In peak c, there was one major species with an M of 1350.

Identification of the Proteins in the Other Peaks

Reconstitution of Proteinase Activity in Vitro with Purified Components

Figure 6: Reconstitution of proteinase activity in vitro with purified components and titration with the second cofactor (A) and with Ad2 DNA (B). A, assays in 400 µl contained 1.42 µg/ml Ad2 DNA, either 2 nM (open circles) or 4 nM (closed circles) rEP, and the indicated volumes of cofactor activity purified as described in the legend to Fig. 3. B, assays in 400 µl contained 2 nM rEP, 10 µl of cofactor activity purified as described in the legend to Fig. 3, and the indicated concentrations of Ad2 DNA. Rates are expressed as the difference in rates between assays in the presence and absence of the cofactor (A) and Ad2 DNA (B).

Optimization of Assay Conditions

Figure 7: Optimization of assay conditions in the absence (closed circles) and presence (open circles) of Ad2 DNA: temperature (A), ionic strength (B), octyl glucoside (C), and dithiothreitol (D). In A, complexes between rEP and pVIc were formed by incubating 70 nM rEP and 208 nM pVIc in 0.9 ml of 0.1 mM TAPS (pH 8.5), 10 mM octyl glucoside, 1 mM EDTA, and 0.5 mM DTT for 5 min at 37 °C. The rEPpVIc complexes were then assayed at pH 8.5 using TAPS buffer. Complexes between rEP, pVIc, and Ad2 DNA were formed the same way, except that the reactions contained 14 nM rEP, 200 nM pVIc, and 140 ng/ml Ad2 DNA. The rEPpVIcAd2 DNA complexes were assayed at pH 8.0 using Tris buffer. After the preincubations, 0.1 ml of 0.1 M buffer, 30 µM (Leu-Arg-Gly-Gly-NH)-rhodamine, 10 mM octyl glucoside, 1 mM EDTA, and 0.5 mM DTT was added; the reactions were incubated at the indicated temperatures; and after 10 min, the increase in fluorescence was determined. In B-D, complexes were formed in 0.9 ml as described for A, except for the absence of the indicated variable. Then, 0.1 ml was added as described for A, except that it contained 10 times the final concentration of the indicated variable, and the increase in fluorescence at 37 °C was monitored as a function of time.

Based upon these and other observations, standard assay conditions were adopted that included 37 °C, 10 mM buffer at pH 8.5 for the absence of DNA and at pH 8.0 for the presence of DNA, and 10 mM octyl glucoside. Occasionally, a preparation of rEP was stimulated by DTT and/or EDTA, in which case, assays also included 0.5 mM DTT and/or 1 mM EDTA. EDTA at higher concentrations was inhibitory; with rEPpVIc complexes, half-maximal activity was lost at 50 mM EDTA (data not shown). Under standard assay conditions, the increase in fluorescence as a function of time with (Leu-Arg-Gly-Gly-NH)-rhodamine as the substrate was linear for >30 min.

K and k of rEP with the Cofactors

V as a Function of pH

Figure 8: Maximal velocity as a function of pH for rEPpVIc complexes (A) and for rEPpVIcAd2 DNA complexes (B). Complexes were formed by incubating 63 nM rEP and 200 nM pVIc (A) or 15.75 nM rEP, 200 nM pVIc, and 5.6 pM Ad2 DNA (B) for 5 min at 37 °C in 0.9 ml of 0.1 mM TAPS (pH 8.5) containing 1.1 mM EDTA, 0.55 mM DTT, and 11 mM octyl glucoside. Then, 0.1 ml was added containing components such that the final concentration of buffer was 10 mM and that of (Leu-Arg-Gly-Gly-NH)-rhodamine was 3 µM, and the ionic strength was 16 mM. The increase in fluorescence was then measured as a function of time. The buffers used were sodium citrate (pH 4.0 and 4.6), sodium acetate (pH 5.0 and 5.5), MES (pH 6), sodium cacodylate (pH 6.0-6.8), HEPES (pH 7-7.8), Tris (pH 8.0 and 8.5), CHES (pH 9.0 and 9.5), and CAPS (pH 10.0 and 10.5). The curves were fitted using the program Peakfit (Jandel Scientific) assuming merged gaussian peaks (dotted lines).

DISCUSSION

The rEP protein was purified to apparent homogeneity using three chromatographic steps with an overall yield of 66%. About 125 mg of rEP were induced by IPTG in a 4-liter culture of cells grown to an absorbance at 600 nm between 0.5 and 0.6 before induction by IPTG. rEP does not bind to DEAE probably because of its high isoelectric point, which was calculated to be 8.68. Also, the DEAE step was used to remove nucleic acids because they bind to the column at NaCl concentrations <0.3 M(15) . The S-Sepharose anion-exchange column gave the largest increase in specific activity, from 67.7 to 485 units/mg. A chelating Sepharose column charged with zinc was used because rEP contains eight free cysteines ()and three histidines. rEP bound quite tightly to this column as it had to be eluted with EDTA. The final step in the purification of rEP was dialysis against 0.5 mM EDTA. This was done to remove all traces of zinc, which is an inhibitor of enzyme activity. With (Leu-Arg-Gly-Gly-NH)-rhodamine as the substrate, the specific activity increased from 22.4 to 815 nmol of substrate hydrolyzed per s/mg of protein.

We were able to observe the processing of most of the virion precursor proteins by adding purified rEP to disrupted ts-1 virus. This established that rEP can find its cofactors and become activated and that activated rEP can cleave in vivo substrates. Furthermore, this indicated that rEP need not be post-translationally modified, e.g. glycosylated, to become activated and cleave in vivo substrates, unless such modifying enzymes and their substrates are in the virion. These data also allow one to conclude that the gene coding for the L3 23-kDa protein is indeed the gene whose inactivation gives rise to the ts-1 phenotype. The viral DNA was implicated as a cofactor because pretreatment of disrupted ts-1 virus with DNase prevented processing after the addition of rEP.

The purification of the second cofactor, pVIc, was very difficult, and, with hindsight, we now know why. We lost activity upon column chromatography because it was so basic, it stuck to glass. We lost activity upon dialysis because it was so small, it passed through the pores in the tubing. Consistent with the second cofactor being a small protein are the observations that boiling for 5 min did not irreversibly denature it; at high ionic strength, it passed through a Centricon-3 (3000-Da cutoff); and when incubated in 5 M urea, it rapidly regained activity upon dilution of the urea (data not shown). The amino acid sequence of the second cofactor is consistent with the biochemical data. The second cofactor was sensitive to plasmin because it contains one lysine and three arginines.

The purification data in Fig. 3implied that pVIc was bound to the viral DNA. At each step before the second Centricon-30 centrifugation, cofactor activity was not greatly stimulated by the addition of Ad2 DNA, but after, enzyme activity was greatly stimulated by the addition of Ad2 DNA. The second cofactor was not in the flow-through fraction after the first Centricon-30 centrifugation because it is a very basic protein that was probably bound to the viral DNA. High ionic strength would dissociate it from the viral DNA, and thus after the second Centricon-30 centrifugation, it was in the flow-through fraction. Cofactor activity in the flow-through fraction was greatly stimulated by the addition of Ad2 DNA.

The flow-through fraction from the Centricon-30 centrifugation gave numerous peaks on a reverse-phase C column. Three of the peaks contained cofactor activity. Sequencing of the three peaks indicated that there was a variable yield in lysine at position 6 and no amino acid was detected at position 10, where we expected a cysteine. Time-of-flight mass spectrometry analysis indicated that the major species in each peak had an M of 1350, consistent with the presence of a monomer of pVIc. Webster et al.(8) purified the cofactor by solubilizing virions in 4 M guanidine HCl and fractionating by fast protein liquid chromatography on a Superdex S-75 gel filtration column. Two peaks of complementing activity were detected by subsequent reverse-phase high pressure liquid chromatography. One peak was the monomer of pVIc, and the other peak was the disulfide dimer of pVIc.

We were able to reconstitute maximal proteinase activity with purified components: rEP, pVIc purified from wild-type virus, and Ad2 DNA. The cofactors stimulated proteinase activity by increasing k >6000-fold. The K changed by less than a factor of 3. Previous in vitro assays for the Ad2 proteinase activity were successful because they utilized Ad2 precursor proteins in an extract from ts-1-infected cells as substrate and disrupted wild-type virus (16) or rEP as the source of proteinase (6) or synthetic peptides as substrate and disrupted wild-type virus as the proteinase(17, 18) . Hence, in those assays, both cofactors were present.

Other proteinases require cofactors for activity, but none, so far, exhibits the requirements of the Ad2 proteinase. Several neutral proteinases need Ca for activity(19) . Some proteinases utilize ATP(20) . A serine proteinase anchored to the membrane of Plasmodium falciparum by a covalently attached glycosylphosphatidylinositol moiety is activated by phosphatidylinositol-specific phospholipase C(21) . Many proteinases are synthesized as zymogens and must be activated by proteolytic cleavage, e.g. the activation of trypsinogen to trypsin by enterokinase or trypsin (22) or the activation of plasminogen to plasmin by urokinase(23) . The assembly and activation of some of the proteins of the blood coagulation system require a negatively charged surface(24) . The virus-coded proteinases from the human immunodeficiency virus and avian sarcoma/leukosis viruses require themselves as cofactors as homodimers are the active form(25, 26, 27) . Although the E. coli RecA protein can facilitate the cleavage of the LexA protein bound to DNA, RecA apparently does so as an allosteric effector and not as a proteinase with an active-site nucleophile(28) .

The experiments on optimizing the assay conditions for the proteinase activity of rEPpVIc complexes in the absence and presence of the viral DNA revealed an unusual sensitivity to ionic strength. In the absence of the DNA, 10 mM NaCl inhibited 50% of the enzyme activity. In the presence of Ad2 DNA, 45 mM NaCl inhibited 50% of the enzyme activity. In contrast to these results, in disrupted virions, 300 mM NaCl was required for 50% inhibition of activity. The latter experiment implied that direct inhibition either of the binding of substrate to the active site or, once bound, of the rate of catalysis probably occurs at NaCl concentrations closer to 300 mM than to 30 mM. Thus, NaCl concentrations of 10-45 mM must inhibit enzyme activity by interfering with formation of an active complex, a complex already formed in a disrupted virus particle.

Although we settled upon standard assay conditions, occasionally they must be altered. We have found (data not shown) that with aged disrupted virus, when compared with newly isolated, disrupted virus, the degree of stimulation by low concentrations of DTT varied from zero with newly isolated, disrupted virus to 4-5-fold with aged disrupted virus. Purified rEP exhibits a similar pattern in that as it ages during storage, 0.5 mM DTT will stimulate more and more activity. This result can be interpreted as signifying the importance of the oxidation states of certain cysteine residues. Similarly, the presence of EDTA at concentrations <2 mM sometimes stimulated proteinase activity (data not shown). Perhaps some zinc, which is a potent inhibitor of enzyme activity, remained with the rEP after chromatography on a chelating Sepharose column charged with zinc.

The nature of the active site of the Ad2 proteinase is unclear. The inhibitor profile of wild-type virus does not correspond to profiles exhibited by classical serine or cysteine proteinases(17, 18, 29, 30) . Examination of the L3 23-kDa gene sequence led Webster et al.(17, 18) to propose that the Ad2 proteinase may be a member of a new subclass of cysteine proteinases described by Brenner(31) , by Bazan and Fletterick(32) , and by Gorbalenya et al.(33) . Based upon site-directed mutagenesis studies, two groups have argued that the enzyme is a cysteine proteinase and that Cys-104 is the active-site nucleophile (34, 35) .

The requirement for DNA as a cofactor for a proteinase activity is unprecedented. It is clearly required in the Ad2 virion because proteinase activity is lost upon treatment with DNase and restored upon addition of Ad2 DNA. In addition, the precursor proteins in disrupted ts-1 virus are processed upon incubation with rEP. However, no processing occurs if disrupted virions are pretreated with DNase. Reconstitution of proteinase activity in vitro with purified components indicates that Ad2 DNA affects k and not K. Webster et al.(36) found no stimulation of rEPpVIc complex activity by Ad2 DNA.

The experiments on V as a function of pH with rEPpVIc complexes in the absence and presence of Ad2 DNA indicated that the enzyme is quite different from the cysteine proteinase papain. Papain contains an active-site thiolate-imidazolium ion pair between His-159 and Cys-25(37) . The second-order acylation rate constant (k/K) as a function of pH conforms to a bell-shaped curve. The two ionizing groups with pK values near 4 and 8.5 probably correspond to His-159 and Cys-25, respectively, more appropriately to the formation and decomposition of the ion pair. An active-site thiolate-imidazolium ion pair in the adenovirus proteinase could have pK values in the absence of Ad2 DNA of 5.17 and 9.43, whereas in the presence of Ad2 DNA, the pK values could be 5.15 and 8.78. These pK values are similar to the normal pK values of 6.0 for histidine and 8.3 for cysteine. Our thiol protection experiment at pH 5.0 in vivo with disrupted virus is consistent with the presence of a thiolate-imidazolium ion pair.

The experiments on V as a function of pH implied that rEPpVIc complexes bind to Ad2 DNA. The profiles of rEPpVIc complexes in the absence and presence of Ad2 DNA are different: pK values of 5.2, 6.4, 6.9, 7.5, and 9.4 versus 5.2, 6.5, 7.4, and 8.8, respectively. This indicates that Ad2 DNA does affect the pK values of some of the amino acids involved in catalysis and therefore implies that the rEPpVIc complexes bind to Ad2 DNA. In addition, the results of measuring the V of rEPpVIc complexes in the presence of Ad2 DNA are similar to those obtained with enzyme activity in disrupted virus. The pK values for disrupted virus are 5.2, 6.2, 7.2, and 8.4. This implied that in the virion, rEPpVIc complexes are bound to the viral DNA.

FOOTNOTES

*

This work was supported by National Institute of Allergy and Infectious Diseases Grant AI26049 and in part by the Office of Health and Environmental Research, United States Department of Energy. Work performed by Suma Abraham (a Science and Engineering Research Semester Program participant) was supported by the Office of Science Education and Technical Information, Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 516-282-3373; Fax: 516-282-3407.

¶

Science and Engineering Research Semester Program participant supported by the Office of Science Education and Technical Information, Department of Energy.

()

The abbreviations used are: ts-1, temperature-sensitive mutant H2ts-1; Ad2, adenovirus serotype 2; rEP, recombinant endoproteinase; PAGE, polyacrylamide gel electrophoresis; IPTG, isopropyl--D-thiogalactopyranoside; DTT, dithiothreitol; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

()

McGrath, W. J., Abola, A. P., Toledo, D. L., Brown, M. T., and Mangel, W. F.(1996) Virology, in press.

()

W. J. McGrath and W. F. Mangel, unpublished observations.

ACKNOWLEDGEMENTS

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