Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.

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1997, American Society for Microbiology

Independently Cloned Halves of Cytomegalovirus Assemblin,

A

_n

and A

_c

, Can Restore Proteolytic Activity to Assemblin

Mutants by Intermolecular Complementation

MATTHEW R. T. HALL†

AND

WADE GIBSON*

Virology Laboratories, Department of Pharmacology and Molecular Sciences, The Johns Hopkins

University School of Medicine, Baltimore, Maryland 21205

Received 17 July 1996/Accepted 15 October 1996

Herpesviruses encode an essential serine proteinase called assemblin that is responsible for cleaving the

precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), assemblin

undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage

converts the enzyme to an active two-chain form consisting of the subunits A

n

and A

c

. We have recently shown

that the recombinant A

n

and A

c

subunits can spontaneously associate within eukaryotic cells to yield active

two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct

functional conformations and led us to test whether they are capable of intermolecular complementation. This

was done by coexpressing inactive mutant (point, deletion, and insertion) forms of assemblin together with the

wild-type subunit (either A

n

or A

c

) corresponding to the domain of assemblin that was mutated. Results of

these experiments showed that both A

n

and A

c

are able to rescue the enzymatic activity of assemblin mutants.

I-site cleavage of the mutated assemblin occurred during complementation but was not absolutely required, as

shown by effective complementation of inactive assemblins with noncleavable I sites. We have also shown that

intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor

and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant

simian CMV assemblin). These results indicate that assemblin is able to form active multimeric structures that

may be of functional importance.

Herpesviruses encode a serine proteinase (5, 9, 15, 32, 39)

that is synthesized as a precursor (20, 27, 40) and is essential

for the production of infectious virus (10, 26). Both the

pre-cursor and its substrate, the prepre-cursor assembly protein (pAP)

(Fig. 1A), are needed during capsid assembly and have a direct

role in forming the icosahedral, outer capsid shell (7, 34, 35).

During the capsid assembly and maturation processes, the

pro-teinase cleaves pAP at its maturational (M) site, severing the

mature assembly protein (AP) from its carboxyl tail (Fig. 1A)

(20, 40). In addition, the precursor proteinase called pNP1

(Fig. 1A) or ACpra (11) in cytomegalovirus (CMV) undergoes

three major autoproteolytic cleavages: (i) one at its M site,

present due to the nested, overlapping relationship of the

pro-teinase and assembly protein precursors (21, 38); (ii) another

at its release (R) site near the middle of pNP1, which releases

the proteolytic amino half of the precursor, assemblin (NP1

n

),

from the nonproteolytic carboxyl half (NP1

c

) (Fig. 1A) (1, 8,

40); and (iii) a third at its internal (I) site (1, 2, 39), which

converts active single-chain assemblin into an active two-chain

form (15) composed of its amino half (A

n

) and its carboxyl half

(A

c

) (Fig. 1A).

It has recently been demonstrated that active two-chain

CMV assemblin can be formed from the independently cloned

and expressed subunits, A

n

and A

c

. This was shown in vitro by

expressing and purifying A

n

and A

c

from bacteria as separate

glutathione S-transferase fusion proteins, removing the

gluta-thione S-transferase portion, and then combining the

frag-ments and renaturing them together (24). We showed that

two-chain assemblin can also form spontaneously within cells

coexpressing the A

n

and A

c

subunits (14). This in vivo

forma-tion of two-chain assemblin occurs in plasmid-transfected

hu-man cells and in recombinant baculovirus (rBV)-infected

in-sect cells and was demonstrated for both the human CMV

(HCMV) and simian CMV (SCMV) enzymes and their

inter-typic pairs (e.g., HCMV A

n

plus SCMV A

c

) (14).

The ability of A

n

and A

c

to associate spontaneously within

cells to form two-chain assemblin indicated that they can

fold and function without first being synthesized as a

one-chain enzyme and suggested that each might be able to rescue

the enzymatic activity of mutant proteinases by

intermolec-ular complementation. To test this possibility, we have

co-expressed A

n

or A

c

with a variety of inactive mutant

assem-blins in two expression systems: Spodoptera frugiperda Sf9

insect cells infected with rBVs, and human embryonal

kid-ney (HEK) cells transfected with recombinant expression

plasmids. The assemblin mutants included insertion,

dele-tion, and point mutations that affect domains and residues

critical to enzyme activity: insertion between the highly

con-served domains 1 and 2 (CD1 and CD2); deletion of the

carboxyl end (40); and replacement of the nucleophilic

serine (5, 9, 15, 32, 39) (e.g., Ser118 in SCMV), the

abso-lutely conserved histidine (19, 39) (e.g., His47 in SCMV), or

amino acids surrounding the I site (e.g., Ala127 in SCMV).

Results of our experiments show that intermolecular

com-plementation of assemblin can occur, indicating that the

en-zyme can form multimers that may be of functional

impor-tance. This conclusion is consistent with two recent reports that

* Corresponding author. Mailing address: Department of

Pharma-cology and Molecular Sciences, The Johns Hopkins University School

of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Phone: (410)

955-8680. Fax: (410) 955-3023. E-mail: [email protected]

.edu

† Present address: Department of Biological Chemistry, The Johns

Hopkins Medical Institutions, Baltimore, MD 21205.

956

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[image:2.612.68.539.93.631.2]

FIG. 1. Cleavage products, landmarks, and constructions. (A) SCMV strain Colburn cleavage products. Shown are the protein fragments generated by cleavage at the I, R, and M sites in the proteinase and by cleavage of pAP at its M site. The computer-predicted molecular mass is shown for each cleavage product. The shaded boxes labeled N2, C2, N1, and C1 indicate the peptide epitopes recognized by the anti-N2, anti-C2, anti-N1, and anti-C1 antisera, respectively. The highly conserved domains (CD1, CD2, and CD3) of assemblin and its herpesvirus homologs are also indicated by shaded boxes. (B) SCMV strain Colburn plasmid constructs and rBVs used. Names of plasmids and rBVs are given at the left, and the expected protein products are given at the right. Dashes indicate that no rBV was made. M1is the

start methionine for the full-length (APNG1 open reading frame product), and assemblin forms of the proteinase as well as An. Mpand M281are the start methionines

for Acand the pAP (product of the APNG.5 open reading frame), respectively. A127, A249, and A557represent the P1 position (29) of the I, R, and M sites. The box

labeled pvc indicates the position of the 45-bp insertion in the inactive assemblin proteinase, LM3.S. A179IQT indicates the final carboxyl residues of the inactive

assemblin deletion mutant AW5, A179is the final assemblin-specific residue, and I, Q, and T were added in the cloning process. Landmarks depicted are the same as

for panel A.

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the active form of bacterially synthesized, purified HCMV

as-semblin is a dimer (6, 23).

MATERIALS AND METHODS

Cells and antisera. Propagation and assay of rBVs was done in from S. frugiperda Sf9 cells (CRL 1711; American Type Culture Collection, Rockville, Md.). Sf9 cells were grown in suspension at 288C in 100-ml spinner flasks con-taining supplemented Grace’s medium (catalog no. 350-1605AJ; GIBCO, Grand Island, N.Y.) with 10% fetal calf serum (HyClone, Logan, Utah), 50mg of gentamicin (catalog no. 600-5750AD; GIBCO) per ml, and 125 ng of amphoter-icin B (Fungizone; catalog no. 600-5295AE; GIBCO) per ml.

HEK cells (CRL 1573, line 293; American Type Culture Collection) for trans-fection assays were grown in 24-well plates (catalog no. 3047; Becton Dickinson Labware, Lincoln Park, N.J.) containing Dulbecco’s modified Eagle’s medium (catalog no. 12100-061; GIBCO) supplemented with 10% fetal calf serum (Hy-Clone), 100 U of penicillin-streptomycin (catalog no. 15140-122, GIBCO) per ml, and 10 U of nystatin (catalog no. 153400-52; GIBCO) per ml. Each well con-tained approximately 106_{cells and 1 ml of medium.}

Antisera were prepared by injecting rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin (catalog no. 77100G; Pierce, Rockford, Ill.). The peptides used to make polyclonal antisera anti-N1, anti-N2, and anti-C2 have been described previously (14, 30, 39) and are mimics of the proteinase se-quences represented as shaded boxes in Fig. 1. Antisera were used at a dilution of 1:40 in a solution containing 10 mM Tris, 0.9% NaCl, and 5% bovine serum albumin (catalog no. 126602; Calbiochem, San Diego, Calif.), pH 7.4.

Construction of plasmids.Standard techniques were used to construct, clone, and propagate the plasmids (28). The new plasmids encoding SCMV proteins described here were derived from AW4, BJ1 (14), and AW1 (40) (Fig. 1B), which are constructs of the RSV.5(neo) expression vector (22) that contain, respec-tively, the SCMV Colburn APNG1 gene (encoding the full-length proteinase), the assemblin coding region, and the APNG.5 gene (encoding the assembly protein precursor) (Fig. 1B). Plasmids encoding altered forms of assemblin are referred to by their plasmid construction number (e.g., BJ14). Mutants of full-length proteinase constructs are denoted by the suffix .L.

The construction of MH28 and MH29, encoding SCMV Anand SCMV Ac,

respectively, has been described elsewhere (14).

BJ14 encodes S118A, a proteolytically inactive assemblin mutant in which the Ser118 nucleophile (39) has been changed to Ala. It was made by replacing the small SalI-to-BsrGI fragment of wild-type BJ1 with the corresponding mutated fragment from a plasmid, AW48 (39), that encodes S118A.L.

BJ20 encodes H47A and BJ21 encodes H47Q, two proteolytically inactive assemblin mutants in which His47 has been replaced with Ala and Gln, respec-tively. They were made by replacing the small SalI-to-SstII fragment of wild-type BJ1 with the corresponding fragment from plasmids AW44 and AW45, respec-tively (39), which encode H47A.Land H47Q.L.

BJ12 encodes I125-D1292, an assemblin mutant in which five I-site residues have been deleted (i.e., I125NA2AD129). It was made by replacing the small CelII-to-KspI fragment in wild-type BJ1 with the corresponding fragment from plasmid MH9 (39), which encodes I125-D1292.L

MH15 encodes A127Q.L, a point mutation at the I site of pNP1 in which the P1 (for nomenclature, see reference 29) Ala127 was changed to Gln by site-directed mutagenesis, essentially as described elsewhere for the construct MH14 (14). The oligonucleotide used for mutagenesis was 59-CGAGATATAAACCA GGCCGATGGCG-39(the underlined portion encodes the Gln change).

BJ13 encodes A127Q, an Ala1273Gln point mutation at the I site of assem-blin. It was made by replacing the small CelII-to-KspI fragment of wild-type BJ1 with the corresponding fragment from MH15, which encodes A127Q.L (de-scribed above).

Three doubly mutated assemblin constructs were made. MH34 encodes H47A, I125-D1292, which combines the His473Ala point mutation and the I125-D1292 I-site deletion; MH35 encodes H47A, A127Q, which combines the His473Ala and Ala1273Gln point mutations; and MH36 encodes H47Q, A127Q, which combines the His473Gln and Ala1273Gln point mutations. These doubly mutated plasmids were made by replacing the small EcoRV-to-BamHI fragment of BJ20 (encodes H47A) or BJ21 (encodes H47Q) with the corresponding fragment of BJ12 (encodes I125-D1292) or BJ13 (encodes A127Q).

MH43 encodes LM3.S, assemblin with a 15-amino-acid insertion in its amino half. This mutation has been previously described in the full-length proteinase construct, LM3 (40), and was made by replacing the small SalI-to-SstII fragment of BJ1 with the corresponding mutated fragment of LM3. The .Ssuffix denotes the short, NP1nor assemblin form of the enzyme.

AW5 encodes the first 179 amino acids of assemblin, with the addition of Ile, Gln, Thr, and a stop codon at its carboxyl end; its construction has been de-scribed elsewhere (40).

Transfection assay.Calcium phosphate transfection of HEK cells (4) was done essentially as described previously (12, 40). The amount of DNA used in single plasmid transfections was 1mg for the pAP-encoding plasmid or 2 mg for plasmids encoding the proteinase or its mutated forms. In all multiple plasmid transfections, the amount of each plasmid encoding An, Ac, or a mutant form of

the proteinase was 0.5mg and the amount of plasmid encoding pAP was 1.0mg.

Each transfection was done with 0.2mg of a plasmid encoding the simian virus 40 (SV40) large T antigen added to enhance plasmid copy number (12).

Construction and assay of rBVs.rBVs were made with the Baculogold system (catalog no. 21001K; Pharmingen, La Jolla, Calif.) as instructed by the manu-facturer and are referred to by the construct numbers of their recombinant transfer plasmids. SCMV and HCMV genes were introduced into the BV ge-nome by homologous recombination mediated by sequences in the transfer plasmid, pVL1392 or pVL1393 (25).

Construction of the following rBVs has been described previously (14): MH30 encodes wild-type SCMV assemblin with an amino-terminal N9-MHWHWH-C9 peptide fusion added for chromatographic purification (31), BJ28 encodes SCMV An, MH51 encodes SCMV Ac, JB2 encodes SCMV pAP, MH50 encodes

HCMV An, and MH52 encodes HCMV Ac.

MH38 encodes a Ser1183Ala mutation in SCMV assemblin (S118A). This construct was made by removing the small CelII-BamHI fragment from the previously described BJ14 construct (see above) and using it to replace the corresponding wild-type fragment of MH30 (14).

MH40 encodes the same Ser1183Ala mutation as MH38 but in the full-length proteinase (S118A.L). This construct was made by replacing the small wild-type CelII-BamHI fragment of MH30 (14) with the corresponding fragment from AW48 (39).

rBVs were plaque purified twice (3, 33); high-titer stocks were prepared for each rBV (33) and stored at 48C protected from light. Sf9 cells ('2.53105

/well) in 24-well plates (Becton Dickinson) were infected at multiplicity of infection of 5 to 10 by adding 100ml of virus in single infections or 50ml of each virus in coinfections. Infected cells were harvested 3 days after infection with 70ml of 23 sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sam-ple buffer containing 4% SDS (catalog no. 161-0300; Bio-Rad, Melville, N.Y.), 20%b-mercaptoethanol, 20% glycerol, 50 mM Tris (pH 7.0), and 0.02% bro-mophenol blue. Samples were heated for 3 min in a boiling water bath and stored at2808C until analyzed.

SDS-PAGE and Western immunoassay.SDS-PAGE was done essentially as described by Laemmli et al. (17); the ratio of N,N9-methylenebisacrylamide to acrylamide was 0.735:28.

Western immunoassays were done essentially as described by Towbin et al. (36). A semidry transfer unit with an Immobilon P membrane (Millipore, Bed-ford, Mass.) was used. The buffer was 50 mM Tris–20% methanol, and the time of transfer was calculated by the following formula: gel width3height32.55 mA per 30 min. Membranes were blocked in 10 mM Tris buffer (pH 7.4) containing 0.9% NaCl and 5% bovine serum albumin, reacted sequentially with appropriate antiserum and [125

I]protein A (catalog no. IM 144; Amersham, Arlington Heights, Ill.), and exposed to X-ray film, usually with a calcium tung-state intensifying screen (18).

RESULTS

The SCMV genes, proteins, and landmarks referred to in

this report are diagrammed in Fig. 1. Both plasmid number

and protein name are indicated for reference.

SCMV A

n

, A

c

, assemblin, pAP, and AP are expressed in

BV-infected insect cells.

As shown before (14) and reported

here for reference (Fig. 2), insect cells infected with the rBVs

used in this work express the expected protein products. When

pAP was expressed alone, no cleavage to AP was detected (Fig.

2, lane 5); when it was expressed with wild-type assemblin, both

expected cleavages (i.e., pAP

3

AP and A

3

A

n

1

A

c

) were

detected (Fig. 2, lane 6). Expression of pAP together with the

independently expressed subunits, A

n

and A

c

, resulted in

pAP

3

AP cleavage, due to the spontaneous association of the

A

n

and A

c

subunits to form a two-chain proteinase (Fig. 2, lane

7) (14). All of these proteins were detected in SCMV-infected

human foreskin fibroblast cells: pAP was detected primarily in

the Nonidet P-40 (NP-40) cytoplasmic fraction (Fig. 2, lane 1);

AP, A

n

, and A

c

were found primarily in the NP-40 nuclear

fraction (Fig. 2, lane 2); and assemblin (A) was found to be

approximately evenly split between the two fractions.

SCMV and HCMV A

n

can complement SCMV S118A

mu-tant proteinase.

To determine whether the wild-type A

n

and

A

c

subunits of assemblin are able to rescue the proteolytic

activity of enzymatically inactive mutants by intermolecular

complementation, we expressed each subunit of HCMV (hA

n

and hA

c

) and of SCMV (sA

n

and sA

c

) with the S118A mutant

SCMV proteinase. This mutation eliminates the serine

nucleo-phile and renders the proteinase inactive (5, 39). We tested for

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complementation of both the mature (S118A) and precursor

(S118A.

L

) forms of the mutant, as indicated by restoration of

pAP

3

AP processing.

Results of this experiment are summarized in Fig. 3. As

expected, coexpression of pAP and wild-type assemblin

re-sulted in conversion of pAP

3

AP (Fig. 3, lane 4). When the

mutants S118A and S118A.

L

were expressed with SCMV A

_n

,

the wild-type subunit that contains the serine nucleophile

mu-tated in the corresponding A

n

domain of the mutants,

conver-sion of pAP

3

AP was readily detected (Fig. 3, lanes 8 and 12,

respectively). Additionally, self-cleavage of the mutant

pro-teinase S118A.

L

occurred at both its M (indicated by

pNP1

3

NP1) and R (evidenced by NP1

3

NP1

c

1

A) sites (Fig.

3, lane 12; also see Fig. 1A). Thus, wild-type SCMV A

n

can

rescue both the pAP and self-cleavage activities of the S118A

mutants whose lesion is in CD3 (Fig. 1A). The SCMV A

c

subunit did not restore proteolytic activity to either S118A

(Fig. 3, lane 9) or S118A.

L

(Fig. 3, lane 13), as indicated by the

absence of pAP

3

AP cleavage.

We have shown previously that the A

n

and A

c

subunits of

SCMV and HCMV can be interchanged and still yield active

two-chain proteinase (14). For example, a two-chain

protein-ase consisting of the heterologous subunit pair, HCMV A

n

/

SCMV A

c

, has nearly the same proteolytic activity as one

composed of the homologous pair (e.g., SCMV A

n

/SCMV A

c

)

(14). We tested the interchangeability of HCMV and SCMV

A

n

subunits in complementing the SCMV proteinase mutants

and found that HCMV A

n

is able to substitute for SCMV A

n

in complementing the S118A mutant, as evidenced by

pAP

3

AP (Fig. 3, lane 6, open circle), but the efficiency of

heterologous complementation was far below that of the

ho-mologous SCMV A

n

(Fig. 3; compare lanes 6 and 8).

Comple-mentation of the mutant precursor enzyme, S118A.

L

, by the

HCMV A

n

subunit was below the limits of detection in this

assay system (Fig. 3, lane 10; no pAP

3

AP and no pNP1

3

NP1

1

A

1

NP1

c

). The finding that the cross-species HCMV

A

c

subunit does not complement the S118A mutants (Fig. 3,

lanes 7 and 11) was expected because the SCMV A

c

subunit

did not complement (Fig. 3, lanes 9 and 13).

SCMV A

n

and A

c

subunits complement other assemblin

mutants.

To examine the intermolecular complementation of

assemblin in greater detail, we used a set of plasmids encoding

inactive assemblins that had been previously made and

char-acterized in transient transfection assays of HEK cells (39).

Expression and detection of assemblin, of its A

n

and A

c

sub-units, and of pAP and AP in transfected HEK cells has been

described elsewhere (14, 40).

[image:4.612.133.223.70.272.2]

In addition to the S118A mutation described above that

inactivates the enzyme by eliminating the nucleophile,

muta-tion of the absolutely conserved histidine in CD2 (His47 in the

SCMV A

n

domain) (19, 39) or deletion of the carboxy-terminal

70 residues of the A

c

domain of SCMV assemblin (construct

AW5) (40) also renders the enzyme inactive. Thus, when the

SCMV assemblin mutant H47A, H47Q, S118A, or AW5 was

coexpressed with pAP in transfection assays, no pAP

3

AP

processing was detected (Fig. 4, lanes 3 to 6). However, when

the three constructs that contain changes in their A

n

domain

(i.e., H47A, H47Q, and S118A) were expressed together with

the SCMV A

n

subunit, each yielded pAP

3

AP cleavage (Fig. 4,

lanes 7, 8, and 10). Thus, point mutations of the essential His47

in SCMV assemblin were also complemented by the SCMV A

n

subunit. Consistent with the results obtained for the S118A

mutants expressed in insect cells (Fig. 3, lanes 9 and 13), the

SCMV A

c

subunit did not complement either the His47 or

S118A mutant in these transfection assays (i.e., no pAP

3

AP)

(Fig. 4, lanes 11, 12, and 14). Conversely, when the deletion

FIG. 2. Expression and detection of rBV-expressed proteins. A Western

[image:4.612.356.512.426.635.2]

im-munoblot probed with a mixture of the anti-N2, anti-N1, and anti-C2 sera is shown. Insect cells were infected with rBVs as described in Materials and Meth-ods and harvested 3 days later. Lysates were separated by SDS-PAGE in a 14% polyacrylamide gel and subjected to Western immunoassay as described in Ma-terials and Methods. pAP was expressed in the preparations contained in lanes 5 to 7; proteins of interest are indicated to the right, abbreviated as in Fig. 1A; the asterisk denotes one of several modified forms of pAP that are detected (12). Mock and w.t. BV are lysates of noninfected Sf9 cells and wild-type BV-infected cells, respectively. NP-40 cytoplasmic and nuclear fractions (Col. Cyto. and Col. Nuc.) of SCMV strain Colburn-infected human foreskin fibroblasts are shown for reference.

FIG. 3. Intermolecular complementation of the inactive S118A mutated forms of the full-length proteinase and assemblin expressed in insect cells. Shown is a fluorogram of a Western immunoblot probed with a mixture of anti-N2, anti-C2, and anti-N1. Samples were harvested and processed as described for Fig. 2; proteins were separated by SDS-PAGE in a 10% polyacrylamide gel and analyzed by Western immunoassay. SCMV pAP was expressed in the prepara-tions in lanes 4 and 6 to 13. Mock and w.t. BV are lysates of noninfected and wild-type BV-infected Sf9 cells; the prefix h or s indicates HCMV or SCMV origin of protein. Proteins of interest are labeled on the left, abbreviated as in Fig. 1A; the asterisk is explained in the legend to Fig. 2; open circles indicate the position of AP. Col. Cyto. and Col. Nuc. are described in the legend to Fig. 2.

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mutant AW5, which lacks 43% of its A

c

domain (i.e., Leu180

to Ala249), was coexpressed with the SCMV A

c

subunit,

pAP

3

AP cleavage was detected (Fig. 4, lane 13). As expected,

activity of AW5 was not rescued by the A

n

subunit (Fig. 4, lane

9).

We also tested the ability of the A

n

subunit to complement

an insertion mutation. Previous experiments had shown that

LM3, a full-length proteinase containing a 15-amino-acid

in-sertion between CD1 and CD2 in its A

n

domain, has only

minimal activity (40). LM3.

S

contains the same insertion in

assemblin and is also essentially inactive (i.e., no pAP

3

AP

cleavage) (Fig. 5, lane 6). The insertion also makes the mutant

proteinases larger than the wild-type forms (Fig. 5; compare

D

3A in lane 6 with A in lane 4). Coexpression of LM3.

S

with

the A

n

subunit resulted in rescue of the mutation and cleavage

of pAP to AP (Fig. 5, lane 7). As expected, coexpression of

LM3.

S

with the noncomplementing A

_c

subunit did not restore

cleavage activity (Fig. 5, lane 8).

Several other inactive SCMV assemblin mutants were

sim-ilarly tested in complementation assays. The results of these

assays are presented in Table 1. Three inactive glycine point

mutations in assemblin (obtained from Carlos Lopez and

El-cira Villarreal, Lilly Research Laboratories), one (G116V) in

the A

n

domain and two (G149V and G216V) in the A

c

domain,

were not complemented by the appropriate wild-type subunit

(e.g., A

n

did not complement G116V). These Gly mutants

were originally made to test the possibility that they are

con-formationally critical residues, and we suspect that their

inabil-ity to be complemented by the A

n

or A

c

subunit is due to the

fact that they are. But this has not been proven, and other

explanations are possible. We also tried to demonstrate

blin multimer formation directly by testing whether one

assem-blin mutant could complement another. This was done with the

pairs shown in Table 1; we found that (i) two mutant

assem-blins (S118A and H47A), both having changes in their A

n

domain, did not complement one another, whereas (ii) two

mutants with changes in their A

n

domain (H47A) and (S118A)

showed complementation and rescue of their proteolytic

activ-ity when coexpressed with an assemblin mutant having an A

c

domain change (AW5); (iii) no evidence of complementation

was detected between the A

c

domain mutant G216V and

ei-ther of the A

n

domain mutants, S118A or H47A, consistent

with the noncomplementability of the Gly mutants with the A

c

subunit.

[image:5.612.384.489.69.189.2]

A possible explanation for intermolecular complementation

in these experiments could be homologous recombination

be-tween the plasmids to generate a wild-type assemblin coding

sequence during the transfection. However, because each

mu-tant would have a similar chance of rescue by genetic

recom-bination with the complementing plasmid (particularly the

G116V and S118A mutations, which are within 6 bp), the

failure of several inactive assemblin mutants (e.g., G116V,

G129V, and G216V) to be complemented provides strong

ev-idence that the intermolecular complementation observed

does not result from homologous recombination. The fact that

FIG. 4. Intermolecular complementation of H47, AW5, and S118 mutants

expressed by transfection of human 293 cells. Shown is a fluorogram prepared from a Western immunoblot probed with a mixture of anti-N2, anti-C2, and anti-N1. Cells were transfected with the indicated plasmids, with or without An

or Ac, and harvested 3 days later. Lysates were subjected to SDS-PAGE in 10%

polyacrylamide gels and analyzed by Western immunoassay as described in Materials and Methods. BJ1 expresses wild-type assemblin and coexpression of An1Acresults in formation of an active two-chain assemblin (14). AW1

[image:5.612.60.298.70.212.2]

(en-codes the SCMV pAP) was present in all of the transfections. H47 is an abso-lutely conserved histidine found in CD2 (Fig. 1A) that is required for proteolytic activity (39); H47A and H47Q are two inactive mutants. S118 is the serine nucleophile of SCMV assemblin, and the S118A mutant is proteolytically inac-tive (39). AW5 encodes an assemblin mutant that lacks its carboxy-terminal 70 amino acids and is proteolytically inactive (40). The positions of the assembly protein precursor (pAP) and product (AP) and of assemblin (A) are indicated in the right margin and were determined by correspondence with marker proteins (“A” in lane 1; pAP and AP in a lysate of AW1-transfected and NP-40 nuclei of CMV-infected cells, as shown in Fig. 5, lanes 1, 2, 3, and 5). Trace assemblin band in lane 9 is due to spillover from adjacent lane.

FIG. 5. Intermolecular complementation of insertion mutation, LM3.S. Shown is a fluorogram of a Western immunoblot probed with a mixture of anti-N2, anti-C2, and anti-N1. Transfected cells were prepared and processed as described for Fig. 4; lysates were subjected to SDS-PAGE in a 10% polyacryl-amide gel and subjected to Western immunoassay. LM3.Sincludes a 15-amino-acid insertion in assemblin that results in loss of proteolytic activity and a shift in electrophoretic mobility (indicated byD3A). Proteins of interest are indicated to the right, abbreviated as in Fig. 1A; asterisks are explained in the legend to Fig. 2. BJ1 encodes SCMV wild-type assemblin; Mock is a lysate of cells transfected with a plasmid encoding the SV40 large antigen T alone. Preparations in lanes 4 to 8 contained SCMV pAP (encoded by AW1).

TABLE 1. Complementation of SCMV assemblin mutants

a

Assemblin mutant

Coexpressed protein

Substrate cleavageb

G116V

c

_A

n

2

G149V

c

_A

c

2

G216V

c

_A

c

2

G216V

c

_S118A

₂

G216V

c

_H47A

₂

S118A

H47A

2

S118A

AW5

₁

H47A

AW5

1

a_{Summary of results from at least three intermolecular complementation}

experiments per coexpressed protein pair. Plasmids encoding mutant proteins were cotransfected into HEK cells along with a plasmid encoding SCMV pAP. Cells were harvested, processed, and analyzed by immunoassay with anti-N1 to detect pAP3AP cleavage.

b_{“1”, pAP3AP cleavage detected;2, no pAP3AP cleavage detected.} c_{Assemblin mutants obtained from Carlos Lopez and Elcira Villarreal, Eli}

Lilly Research Laboratories.

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[image:5.612.317.555.559.661.2]

neither complementation of the LM3.

S

insertion mutant by A

n

(Fig. 5, compare lane 6 with lane 4) nor complementation of

the AW5 deletion mutant by A

c

yielded detectable

wild-type-size assemblin (Fig. 4, compare lane 1 with lane 13), even after

much longer exposures, also argues against homologous

re-combination between plasmids as the mechanism of the

inter-molecular complementation observed here.

I-site cleavage is not required for intermolecular

comple-mentation.

Two additional experiments were done to examine

the requirement for I-site cleavage in intermolecular

comple-mentation of assemblin. First, the S118A mutant was

coex-pressed with its complementing subunit, A

n

, to determine

whether intermolecular complementation resulted in I-site

cleavage of the mutated enzyme. I-site cleavage was clearly

evidenced in this experiment by the presence of A

c

(Fig. 6, lane

7), which could have come only from assemblin cleaved at its I

site. The presence of A

n

in this lane is due in part to its

synthesis from plasmid MH28 and in part to assemblin

cleav-age; other bands are either degradation products of the

inac-tive assemblin (also present in Fig. 6, lane 5) or

immunologi-cally cross-reactive host proteins (e.g., two bands above A

c

are

also present in (Fig. 6, lane 3). Controls showed that there was

no I-site cleavage by the inactive S118A mutant alone (i.e., no

A

n

or A

c

) (Fig. 6, lane 5) or when it was coexpressed with the

noncomplementing A

c

subunit (i.e., no A

n

seen in Fig. 6, lane

6; A

c

detected is expressed from noncomplementing MH29

plasmid). Thus, complementation of the S118A assemblin

mu-tant by the A

n

subunit does result in I-site cleavage, yielding

mutant A

n

and wild-type A

c

. I-site cleavage also occurred

dur-ing complementation of LM3.

S

by the A

_n

subunit and explains

the lower amount of

D

3A in lane 7 of Fig. 5 than in lanes 6

(enzyme alone) and 8 (noncomplementing A

c

subunit) (data

not shown from Western immunoassays for A

n

and A

c

done

with 14% polyacrylamide gels).

The occurrence of I-site cleavage during intermolecular

complementation of assemblin mutants raised the question of

whether this cleavage might be required for the restoration of

proteolytic activity observed. We tested this possibility by

con-structing assemblin mutants that are both proteolytically

inac-tive and noncleavable at their I sites. The H47A and H47Q

assemblin mutants were combined with one of two I-site

mu-tations. The first I-site mutation was a deletion of five I-site

residues. We have shown before that deleting residues Ile125

to Asp129 in the full-length proteinase (I125-D129

2

.

L

)

elimi-nates I-site cleavage without loss of proteolytic activity (39).

When this deletion was subcloned into assemblin (BJ12), I-site

cleavage was eliminated (data not shown); however, a new

cleavage product was observed (Fig. 7, lane 15, open circle),

which has a counterpart that is detected when the I site is

mutated in HCMV assemblin (16). In HCMV assemblin, this

[image:6.612.136.220.69.247.2]

'

24-kDa fragment arises from cleavage at a cryptic (C)

cleav-age site in the sequence VDA209

2

SG (16). A potentially

FIG. 6. Intermolecular complementation of S118A leads to I-site cleavage.

Shown is an autoradiogram of a Western immunoblot probed with a mixture of anti-N2 and anti-C2. Transfected cells were prepared and processed as described for Fig. 4; lysates were subjected to SDS-PAGE in a 14% polyacrylamide gel and analyzed by Western immunoassay as described in Materials and Methods. BJ1 encodes SCMV wild-type assemblin. Mock, Col. Cyto., and Col. Nuc. are defined in the legends to Fig. 4 and 5. Proteins of interest are indicated on the right by abbreviations used in Fig. 1A.

FIG. 7. Intermolecular complementation in the absence of I-site cleavage. Shown is an autoradiogram of a Western immunoblot probed with a mixture of anti-N2, anti-C2, and anti-N1. Transfected cells were prepared and processed as described for Fig. 4; lysates were subjected to SDS-PAGE in a 14% polyacrylamide gel, and analyzed by Western immunoassay as described in Materials and Methods. BJ1, Mock, Col. Cyto., and Col. Nuc. are defined in the legends to Fig. 4 and 5. The presence (1) or absence (2) of plasmids encoding SCMV pAP (AW1), An, and Acis indicated below each lane number. I125-D1292indicates the deletion of those residues

that constitute the I site in SCMV assemblin (39). A127 is the P1 (29) residue of the SCMV I site (39). H47 is an absolutely conserved histidine that is essential for proteolytic activity (39). An1Acis a cotransfection of Anand Acthat yields active two-chain assemblin (14). Proteins of interest are indicated to the right, abbreviated

as in Fig. 1A. An open circle is used to mark the position of the new cleavage product resulting from self-cleavage of an I-site mutant at its cryptic site; asterisks are explained in the legend to Fig. 2.

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[image:6.612.138.481.493.658.2]

homologous sequence in SCMV assemblin, ESSA200

2

AA,

could be the corresponding cryptic site. The cryptic site

cleav-age product is not easily detected when wild-type assemblin is

expressed alone (Fig. 7, lane 2, and reference 16) or in

SCMV-infected human foreskin fibroblasts (Fig. 2, lanes 1 and 2).

The second I-site mutant was a point mutation at the P1 (29)

residue of the I site (i.e., A127Q in SCMV) and had a

pheno-type similar to that of the deletion mutant, that is, no detected

I-site cleavage (data not shown) and the appearance of a new

self-cleavage product (Fig. 7, lane 16, open circle), presumably

resulting from cleavage at the same cryptic site revealed by the

I125-D129

2

deletion mutant. The smaller size of the fragment

from the deletion mutant is consistent with the loss of five

amino acids. Both of these I-site cleavage mutants still

pro-cessed pAP

3

AP (Fig. 7, lanes 32 and 33).

The two I-site mutations were combined with the H47A and

H47Q mutations described above to create three double

mu-tations: one in which the I-site residues were deleted from

H47A (i.e., H47A, I125-D129

2

), and two in which the I-site

point mutation, A127Q, was combined with either the H47A or

H47Q mutation (H47A, A127Q and H47Q, A127Q,

respec-tively). When each of the doubly mutated assemblins was

ex-pressed alone, neither I-site cleavage (i.e., no A

n

or A

c

pro-duced) (data not shown) nor C-site cleavage (Fig. 7; compare

lanes 10 to 12 with lanes 15 and 16) occurred, as expected

because of the inactivating H47 mutations. For the same

rea-son, no processing to AP was observed when each mutant was

coexpressed in transfections with pAP (Fig. 7, lanes 17 to 19).

Controls demonstrated pAP

3

AP cleavage by wild-type

assem-blin (Fig. 7, lane 8) and recombinant two-chain assemassem-blin (Fig.

7, lane 9). The presence of a band at the position of assemblin

in the two-chain proteinase sample must be due to spillover

from the adjacent lane, as no corresponding band has been

observed in other experiments (Fig. 2, lane 7, and Fig. 4, lane

2) as well as previously published experiments (see Fig. 3, 4, 6,

7, and 9 in reference 14). Controls also showed that the H47A

and H47Q single mutants were unable to cleave pAP

3

AP

(Fig. 7, lanes 20 and 21) unless they were complemented with

by the A

n

subunit (Fig. 7, lanes 30 and 31).

When the new doubly mutated assemblins were coexpressed

with the A

n

subunit, pAP

3

AP cleavage occurred (Fig. 7, lanes

27 to 29), indicating that intermolecular complementation of

inactive assemblin mutants by the wild-type A

n

subunit does

not require I-site cleavage of the mutant. Interestingly, neither

of these mutants showed C-site cleavage during

complemen-tation, even though both parental I-site mutants did. No

pAP

3

AP cleavage was detected when these mutants were

coexpressed with the A

c

subunit (Fig. 7, lanes 22 to 24).

Al-though, in complementations with A

n

, the mutants having a

cleavable I site (I site positive) showed more pAP

3

AP than

the double mutants lacking a cleavable I site (I site negative),

they also showed correspondingly more inactive assemblin

(compare intensities of AP and A bands in lanes 30 and 31 of

Fig. 7 with those in lanes 27, 28, and 29). Thus, the enzymatic

efficiency of the complementation reaction, as estimated from

the amount of pAP

3

AP relative to mutant assemblin, was not

appreciably enhanced by the presence of a cleavable I site.

DISCUSSION

We have found that the proteolytic activity of an inactive

CMV assemblin mutant (e.g., S118A) can be rescued by

coex-pressing it with an appropriate fragment or subunit (e.g., A

n

)

of wild-type assemblin. Subunit-specific rescue of three point

mutations, an insertion mutation, and a deletion mutation was

demonstrated and shown to occur in both BV-infected insect

cells and plasmid-transfected human cells. These results

indi-cate that the independently cloned A

n

and A

c

subunits of

assemblin can interact with their partner domains in a mutant

form of the enzyme to restore its proteolytic activity by

inter-molecular complementation.

Intermolecular complementation is characteristic of

pro-teins that form multimers, such as

b

-galactosidase (37, 41) and

interleukin-converting enzyme (13), and its occurrence in the

experiments reported here suggests that assemblin is able to

form multimers. This conclusion, based on interactions of the

proteins in vivo, is consistent with recent evidence from in vitro

experiments that the active form of HCMV assemblin is a

dimer (6, 23). Intermolecular complementation was also

ob-served with an inactive mutant form of the full-length

assem-blin precursor, pNP1 (Fig. 1 and 3), demonstrating that

inter-action of the complementing subunit with assemblin does not

require that the nonproteolytic carboxyl end of the precursor

(e.g., NP1

c

) first be shortened or removed by M- or R-site

cleavage (Fig. 1A).

Although the stoichiometry of the wild-type subunit and

mutant assemblin in the complex formed during

intermolecu-lar complementation is unknown, the minimal structure would

be a heterodimer composed of one complementing subunit

and one mutant assemblin molecule, as suggested in the

hypo-thetical model presented in Fig. 8. If assemblin absolutely

requires dimerization to become active (6, 23), then the

prod-ucts of A

n

plus mutant assemblin (Fig. 8, line 1) could include

(i) the heterodimer shown, (ii) a homodimer composed of two

mutant assemblin molecules, and (iii) a possible heterotrimer

composed of two mutant assemblins and a complementing

wild-type subunit. If still higher-order complexes can form, the

potential products would be more numerous and complicated.

We demonstrated that formation of the complementation

complex (e.g., mutant assemblin/A

n

subunit shown in Fig. 8,

line 1) results in I-site cleavage of the assemblin mutant (Fig.

6, lane 7) but that this cleavage is not required for proteolytic

activity (Fig. 7, lanes 27 to 29).

When I-site cleavage can occur during complementation, it

could lead to formation of wild-type two-chain enzyme in at

least two ways (Fig. 8). One would be through I-site cleavage of

the active complementation complex, either by a similar

com-plex (Fig. 8, arrow a) or by a wild-type two-chain assemblin that

resulted from complementation (Fig. 8, line 3). A second

would be through (i) sequential cleavage of inactive mutant

assemblin (Fig. 8, line 1), either by a complementation

com-plex (Fig. 8, arrow b) or by a previously formed wild-type

two-chain enzyme, to yield two-chain mutant assemblin (Fig. 8,

line 2), followed by (ii) direct or indirect displacement of the

mutant A

n

subunit by a wild-type recombinant subunit to form

wild-type two-chain assemblin (Fig. 8, arrow c). Although this

figure depicts the hypothetical interactions between monomers

for simplicity of illustration, analogous interactions could take

place between complexes. Our finding that I-site cleavage did

not noticeably enhance the efficiency of pAP

3

AP (Fig. 7)

indicates either that little wild-type two-chain enzyme is

formed in these complementation reactions or that the

enzy-matic activity of the complementation complex is comparable

to that of the wild-type two-chain enzyme. Because rescue of

CMV assemblin by intermolecular complementation did not

require I-site cleavage, similar results would be expected with

the assemblin homologs of non-CMV herpesviruses that have

no I site.

If the complementation complex evidenced here is reflective

of an assemblin dimer structure, then our results make several

predictions. First, because a heterodimer composed of S118A

and an A

n

subunit would have just one S118 nucleophile (i.e.,

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in the complementing subunit) and therefore only one

func-tional active site, our results indicate that the wild-type enzyme

does not require two functional active sites for proteolytic

activity. Second, the inability of two probable active-site

mu-tants, S118A and H47A, to complement one another by

form-ing an active dimer indicates that these putative catalytic

res-idues are not shared in a single active site formed between two

assemblin monomers.

We also showed in this report that intermolecular

comple-mentation can occur between HCMV and SCMV assemblins,

as HCMV A

n

rescued the activity of an SCMV assemblin

mutant (Fig. 3). This finding is consistent with our previous

report that HCMV and SCMV assemblin subunits can

com-bine to form active intertypic two-chain proteinase (e.g.,

HCMV A

n

/SCMV A

c

) (14). However, the extent of SCMV

pAP processing was reduced when HCMV A

n

was used in

place of SCMV A

n

to complement mutant SCMV assemblin

(Fig. 3). This contrasted with our finding that pAP cleavage by

an intertypic two-chain assemblin (e.g., HCMV A

n

/SCMV A

c

)

was comparable to that of the homotypic two-chain enzyme

(i.e., SCMV A

n

/A

c

) (Fig. 10 in reference 14). We interpret

these results as indicating that intertypic complementation

in-volves competition (by displacement or simple occupancy) of

the complementing subunit (e.g., HCMV A

n

) for access to its

partner domain within the assemblin mutant (e.g., SCMV A

c

)

and that the decreased pAP processing in these intertypic

complementation experiments may result from the HCMV A

n

subunit being less able than the SCMV A

n

subunit to compete

for SCMV A

c

interactions. This finding implies that there are

differences in the interactions between HCMV and SCMV

subunits. These differences were not apparent in our earlier

studies (14) on the formation of cross-species two-chain

re-combinant assemblins, presumably because no competing

same-species domains were present.

The in vivo intermolecular complementation of CMV

as-semblin mutants by wild-type asas-semblin subunits reported

here, and the recent reports that purified HCMV assemblin

functions as a dimer in vitro (6, 23), indicates that the

protein-ase can form active multimers. Although the biological

sig-nificance of these higher-order structures remains to be

determined, the finding that the pAP substrate of assemblin

interacts with itself to form multimers (42) raises the possibility

that assemblin dimerization both activates it and promotes

more efficient recognition and cleavage of its multimeric

sub-strate. A better understanding of the structure of these

assem-blin complexes and how they form is likely to contribute to our

understanding of how this novel proteinase works.

ACKNOWLEDGMENTS

We thank Jenny Borchelt, Kendra Clopper, and Becky Magno for

excellent technical assistance, and we thank Carlos Lopez and Elcira

Villarreal, Eli Lilly Research Laboratories, for help in obtaining the

antipeptide antisera anti-N2 and anti-C2 as well as for providing the

three glycine mutants of SCMV assemblin (G116V, G149V, and

G216V).

M.R.T.H. was a fellow in the Biochemistry, Molecular, and Cell

Biology Training Program. This work was supported by Public Health

Service research grants AI13718 and AI32957.

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