assembly and maturation of the bacteriophage lambda procapsid: gpc is the viral protease

doi:10.1016/j.jmb.2010.06.060 J. Mol. Biol. (2010) 401, 813–830

Available online at www.sciencedirect.com

Assembly and Maturation of the Bacteriophage LambdaProcapsid: gpC Is the Viral Protease

Elizabeth Medina1, Doug Wieczorek2, Eva Margarita Medina1,Qin Yang3, Michael Feiss2 and Carlos Enrique Catalano1⁎
1Department of MedicinalChemistry, University ofWashington School ofPharmacy, H172 HealthSciences Building,Campus Box 357610, Seattle,WA 98195-7610, USA2Department of Microbiology,University of Iowa, 51 NewtonRoad, Iowa City, IA 52242,USA3Department of PharmaceuticalSciences, University ofColorado Health SciencesCenter, C238-L15, Aurora,CO 80045, USA
Received 18 April 2010;received in revised form22 June 2010;accepted 25 June 2010Available online8 July 2010

0022-2836/$ - see front matter © 2010 E

*Corresponding author. E-mail [email protected] address: D. Wieczorek, Pr

2800 Woods Hollow Road, MadisonAbbreviations used: cos, cohesive

pulsed-field gel electrophoresis; dsDDNA; EDTA, ethylenediaminetetraatrifluoroacetic acid; pfu, plaque-formNational Center for Biotechnology I

Viral capsids are robust structures designed to protect the genome fromenvironmental insults and deliver it to the host cell. The developmentalpathway for complex double-stranded DNA viruses is generally conservedin the prokaryotic and eukaryotic groups and includes a genome packagingstep where viral DNA is inserted into a pre-formed procapsid shell. Theprocapsids self-assemble from monomeric precursors to afford a matureicosahedron that contains a single “portal” structure at a unique vertex; theportal serves as the hole through which DNA enters the procapsid duringparticle assembly and exits during infection. Bacteriophage λ has served asan ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the λ procapsid assemblypathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify andcharacterize the protease responsible for λ procapsid maturation andpresent a structural model for a procapsid-bound protease dimer. Theprocapsid protease possesses autoproteolytic activity, it is required fordegradation of the internal “scaffold” protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Ourdata demonstrate that these proteolytic maturation events are not requiredfor procapsid assembly or for DNA packaging into the structure, but thatproteolysis is essential to late steps in particle assembly and/or insubsequent infection of a host cell. The data suggest that the λ-likeproteases and the herpesvirus-like proteases define two distinct viralprotease folds that exhibit little sequence or structural homology but thatprovide identical functions in virus development. The data further indicatethat procapsid assembly and maturation are strongly conserved in theprokaryotic and eukaryotic virus groups.

© 2010 Elsevier Ltd. All rights reserved.

Keywords: virus assembly; viral capsids; procapsid assembly; bacteriophagelambda; herpesvirus
Edited by J. Karn
lsevier Ltd. All rights reserve

ess:

omega Corporation,, WI 53711, USA.end site; PFGE,NA, double-strandedcetic acid; TFA,ing unit; NCBI,nformation.

Introduction

The assembly of an infectious virus from macro-molecular precursors represents a temporally orches-trated biochemical pathway that is conserved in botheukaryotic and prokaryotic viruses.1–3 For example,the assembly pathway of complex double-strandedDNA (dsDNA) viruses such as the herpesvirusesand many bacteriophages is highly homologous andin particular the DNA packaging step is stronglyconserved.2–6 In these examples, the viral genome isphysically translocated into the confines of a pre-assembled procapsid structure by a terminase

d.

http://dx.doi.org/10.1016/j.jmb.2010.06.060

mailto:[email protected]

Fig. 1. Bacteriophage λ procapsid assembly. Currently accepted model for the assembly and maturation of the λprocapsid. Details are provided in the text.

814 Assembly of the Bacteriophage λ Procapsid

enzyme.2 Bacteriophage λ is representative of thesecomplex dsDNA viruses, and the wealth of genetic,biochemical, and biophysical information availablefor λ makes it an ideal model system in which tostudy the essential features of virus assembly.6–8

The mature λ genome (found in the infectiousvirus) consists of a 48.5-kb linear duplex withcomplementary 12-base single-stranded “sticky”extensions at each end.9,10 Upon injection intoEscherichia coli, the linear duplex circularizes viathese cohesive ends forming the intact cohesive endsite, or cos.11 During the latter stages of infection,circular DNA is replicated by a rolling circlemechanism that gives rise to linear concatemers ofthe individual genomes linked in a head-to-tailfashion.9,12 Expression of viral “late” genes yieldsthe capsid proteins that self-assemble into icosahe-dral procapsid shells.8,13 Genome packaging, whichrepresents the intersection of the DNA replicationand procapsid assembly pathways, involves theexcision of a single λ genome from the concatemericprecursor and simultaneous translocation of viralDNA into the pre-formed procapsid structure; thisprocess is catalyzed by the terminase enzyme and isfueled by ATP hydrolysis.2,5,14

Upon packaging 15–20% of the viral genome, theprocapsid undergoes an expansion process, whichincreases the capsid volume ∼2-fold and affords amore angularized icosahedral structure†.15–17 Theviral gpD protein adds to the surface of theexpanded capsid lattice and terminase continues totranslocate DNA until a unit-length genome hasbeen packaged.6,18 Terminase then cleaves theduplex at cos, releasing the DNA-filled capsid towhich “finishing proteins” and a tail are added toyield the infectious virus.The procapsid assembly pathways are strongly

conserved in the complex dsDNA viruses, bothprokaryotic and eukaryotic.14,19–23 In general,portal proteins assemble into a dodecameric ring

†We use the term “procapsid” to describe thecontracted, empty shell and the term “capsid” to describethe expanded, angularized shell that results from packa-ging viral DNA.

structure, which then nucleates polymerization ofthe major capsid protein into an icosahedral shell.Shell assembly is controlled by co-polymerizationwith an internal scaffold protein; in the absence ofscaffold, the capsid proteins polymerize intoaberrant and non-functional structures.24 Thescaffold proteins exit from the procapsid interiorprior to or at the time of DNA packaging, often byproteolytic degradation by a procapsid-boundprotease. The packaging-competent procapsidstructure contains a single portal ring situated ata unique vertex in the icosahedron (the portalvertex), which provides a hole through which viralDNA can enter the capsid during packaging andexit during infection.The accepted model for λ procapsid assembly,

while generally similar to most complex dsDNAviruses, is significantly more elaborate as follows(Fig. 1).7,13,25 The model proposes that the portalring (a.k.a. pre-connector) assembles from 12copies of the viral protein gpB. Next, 10–12 copiesof the gpC protein add to the portal ring to affordthe initiator structure; appropriate assembly of theinitiator is presumably mediated by the viralgpNu3 protein and host GroELS chaperonins.13

The initiator structure nucleates co-polymerizationof the gpNu3 scaffold protein and the gpE majorcapsid protein into an immature procapsid shellwith the scaffold at the interior. At this point, theprocapsid is “matured” in several unusual stepsunique to the λ virus (Fig. 1); (i) the gpC proteinsbecome covalently cross-linked to an equal num-ber (10–12) of gpE major capsid proteins, presum-ably at the portal vertex; (ii) the resulting “pY”proteins are proteolyzed to afford the pX1 andpX2 proteins (5–6 copies each), and the portalprotein is proteolyzed, removing 20 residues fromthe N-terminus to yield pB⁎. Finally, the gpNu3scaffold protein is proteolyzed and the degrada-tion products exit from the procapsid interioryielding a mature, packaging-competent procapsidstructure. This model for λ procapsid assemblyand maturation was developed over two decadesago and is based on genetic experiments andcomplementation studies using bacterial extractsin vitro. Many aspects of the model remain

Fig. 2. Sequence alignment of λ gpC and the S49 serine protease family. Multiple sequence alignments were constructed as described in Materials and Methods and selectedproteins are displayed in the figure. In addition, the N-terminal and C-terminal domains of E. coli SppAEC, as defined in Materials and Methods, were included in the alignment.The darker shade of blue represents amino acids that are conserved among all the sequences and the lighter shade of blue represents those that are less conserved. The arrowsindicate the putative gpC active-site serine (S166, black), the orienting serine (S188, blue), and the lysine catalytic base (K218, red). Note that these residues are strictly conserved inthe top 114 hits obtained in the sequence alignments (data not shown). The position of gpC Lys249 is indicated with an asterisk.

815Assem

blyof

theBacteriophage

λProcapsid

image of Fig. 2

Table 1. Mutation of gpC Ser166 is lethal to λdevelopment in vivo

ProphageaComplementing

plasmidVirus yield(SEM)b

λ cI857 None 129 (13)λ cI857 Cam20 None 1.29×10−4 (1.03×10−5)λ cI857 Cam20 pDW1Δ2A (gpC-WT)c 1.4 (0.03)λ cI857 Cam20 pDW1Δ2B

(gpC-S166A)c3.02×10−5 (2.30×10−5)

a Lysogenic derivatives of R594. All prophages contained thered3 mutation as indicated in Table 2.

b The results represent the average virus yield from at leastthree independent experiments with SEM indicated inparentheses.

c The pDW1Δ2A and pDW1Δ2B plasmids containedmutations creating the silent Q169Q change, as described inMaterials and Methods. Control experiments showed that theQ169Q changes were innocuous (data not shown).Complementing gpC was expressed from the indicated plasmidcontaining the relevant C gene under expression from the λ latepromotor PR′.


obscure and/or speculative; the protease respon-sible for degrading the gpNu3 scaffold and forprocessing of gpB and pY proteins remainsuncertain and the chemistry of the putativecross-link between gpC and gpE proteins remainsa complete mystery. Further, the role of pB⁎, pX1,and pX2 proteins in the assembly and function ofthe procapsid remains unknown. Thus, the pres-ent study was initiated to identify the viralprotease required for procapsid maturation andto define the roles of the matured procapsidproteins in the structure and function of the λprocapsid. The results of these studies demon-strate that gpC is the protease responsible for λprocapsid maturation and suggest that proteolyticmaturation is essential for late steps in virionassembly and/or for subsequent infection of theE. coli cell. Finally, our data suggest that assemblyand maturation of the λ procapsid is remarkablysimilar to the eukaryotic herpesvirus groups.

Results

The mutation changing gpC residue 166 fromserine to alanine is lethal

The λ gpC protein is incorporated into the nascentprocapsid, but the function of the protein has notbeen firmly established. Primary sequence analysishas revealed that gpC possesses homology to theS49 family of serine proteases and indicates thatserine 166 is strongly conserved (Fig. 2).26 Thisobservation has been interpreted to indicate thatgpC represents the viral protease responsible formaturation of the λ procapsid,7 and consistentwith this hypothesis, the λ Cam20 amber mutationis lethal to virus development in vivo.27 Based onsequence homology, we hypothesized that S166represents the catalytic serine residue. Here, weuse an in vivo complementation assay to define therole of S166 in the function of gpC in λdevelopment.First, we confirm that induction of λ cI857

Cam20, a lysogen that harbors an amber mutationin the C gene, does not afford significant virus yield(Table 1); however, when wild-type gpC wassupplied in trans with a complementing plasmid,the yield of λ cI857 Cam20 virus was increased by 4orders of magnitude (Table 1)‡. In contrast,supplying the gpC-S166A mutant protein in transdoes not complement λ cI857 Cam20 and in factdecreases virus yield 10-fold (Table 1). We interpretthese data to indicate that (i) gpC is essential forvirus development in vivo and (ii) Ser166 is criticalto gpC function.

‡While impressive, we note that the complementationwas incomplete, giving a virus yield of about 1% that ofwild-type λ cI857 virus (Table 1). The reason for this is notclear but is likely due to inappropriate expression of wild-type gpC from the plasmid.

Wild-type gpC possesses autoproteolytic activity

We next examined expression of the isolatedgpC protein in E. coli using plasmids that encodegpC-WT and gpC-S166A mutant protein asdescribed in Materials and Methods. Inductionof gpC-WT expression does not yield any signif-icant protein product identifiable in crude extractsof cells analyzed by SDS-PAGE (Fig. 3a). Incontrast, a protein of appropriate molecularweight is clearly evident when the gpC-S166Amutant protein is expressed in E. coli. Westernblot analysis (Fig. 3b) and mass spectrometry (notshown) confirm that the expressed protein isindeed full-length gpC. Importantly, immunoreac-tive bands are clearly evident in the induced gpC-WT sample, but they are significantly smaller thanthe full-length protein. We interpret these data toindicate that gpC-WT is expressed in E. coli butthat full-length protein is not observed due toautoproteolysis. Further, the data indicate thatSer166 is essential to autoproteolytic activity.

gpC is the viral protease responsible for scaffolddegradation

To further define the role of gpC in procapsidmaturation, we constructed a vector that expressesgpC in the context of the λ portal (gpB) andscaffold (gpNu3) proteins. The accepted modelpredicts that initiator structures will be assembledupon expression of these three proteins (Fig. 1) andwe refer to this vector as the initiator construct(pT7Init; Table 2). Induction of initiator-WT ex-pression does not yield any significant proteinproducts identifiable in the crude cell extractsanalyzed by SDS-PAGE (Fig. 3c). Notwithstanding,Western blot analysis using an anti-gpC antibodyreveals immunoreactive bands that are significant-ly smaller than full-length gpC and that are of a

§The Nu3 gene is embedded within the C gene in the λgenome. The Shine–Dalgarno sequence in pETC5 andpETC9 (Table 2) was modified to prevent expression ofgpNu3 from these constructs (Fig. 3a and b). In contrast,the initiator constructs retained a wild-type Nu3 Shine–Dalgarno sequence and scaffold expression is robust, asexpected (Fig. 3c and d).

Fig. 3. Expression of isolated gpC proteins and the initiator constructs. (a) Cells expressing wild-type gpC or gpC-S166A mutant protein (indicated at top of gel) were induced as described in Materials and Methods. Relevant genes inthe expression vector are depicted above the gel. Note that the Shine–Dalgarno sequence for the Nu3 gene has beendisrupted by mutation as described in Materials and Methods, which abrogates gpNu3 expression from theseconstructs (the Nu3 gene is shaded gray to depict this feature). SDS-PAGE of whole-cell lysates stained withCoomassie blue is shown. M, protein molecular standards; Pre, pre-induction sample; Post, 2 h post-induction sample.The position of full-length gpC protein (45.9 kDa) is indicated. The identity of gpC was confirmed by massspectrometry (data not shown). (b) Western blot of the gel presented in (a). Note that a small amount of proteolysis ofthe gpC-S166A mutant protein is observed in the Western analysis. We attribute this to nonspecific degradation bycellular proteases. (c) Cells expressing wild-type gpC or gpC-S166A initiator proteins (indicated at top of gel) wereinduced as described in Materials and Methods. Relevant genes in the expression vector are depicted above the figure.Note that both vectors contain a wild-type Nu3 Shine–Dalgarno sequence and strong gpNu3 expression is anticipated(the Nu3 gene is colored purple to depict efficient expression of the protein in this construct). SDS-PAGE of whole-celllysates stained with Coomassie blue is shown. M, protein molecular standards; Pre, pre-induction; Post, 2 h post-induction. The positions of full-length gpC protein (45.9 kDa) and gpNu3 protein (13.4 kDa) are indicated. Theidentities of gpC and gpNu3 were confirmed by mass spectrometry (data not shown). (d) Western blot of the datapresented in (c).

817Assembly of the Bacteriophage λ Procapsid

size comparable to those observed when gpC-WTis expressed in isolation (compare Fig. 3b and d).We next introduced the gpC-S166A mutation intothe initiator construct (pT7Init-gpC-S166A); instark contrast to the expressed wild-type initiators,full-length gpC is clearly evident by SDS-PAGE(Fig. 3c), by Western blot analysis (Fig. 3d), and bymass spectrometry (not shown). Furthermore, thegpNu3 scaffold protein is clearly visible in theinduced gpC-S166A initiator extracts, but it iscompletely absent in the induced gpC-WT initiator

sample (Fig. 3c)§. We interpret these data toindicate that (i) gpC possesses autoproteolyticactivity, (ii) gpC is also responsible for proteolysis

image of Fig. 3

Table 2. Bacteria, phages, and plasmids used in this study

Relevant properties

Bacterial strainsC600 Permissive host for λ amber mutants, due to glnV44 suppressor mutation.27

R594 Non-permissive host for λ amber mutants.27

BL21 (DE3) Expresses T7 RNA polymerase upon induction with IPTG, for gene expression from pET vectors.28

Phage strainsλ cI857 red3 Thermo-inducible strain of wild-type λ virus used for the in vivo complementation studies.29,30

λ cI857 red3 Cam20 λ cI857 red3 virus with a lethal amber mutation in the C gene.27

NS428 [N100 (λ cIts857Aam11 b2red3 Sam7)]

λ with lethal mutation in terminase gene A. Produces wild wild-type procapsidsbut fails to package DNA.31

PlasmidspDW1Δ2A gpC-WT complementation plasmid. Derivative of pDW1Δ2 with gene C codon 169 mutations generating

Q169Q silent mutation and an MfeI site. This work.pDW1Δ2B gpC-S166A complementation plasmid. Derivative of pDW1Δ2A with gene C codon 166 changes that produce

the gpC S166A mutant protein. This work.pETC5 Expression plasmid for gpC-WT. Expresses hexaHis-gpC-WT but not gpNu3. This work.pETC8 Expression plasmid for gpC-WT. Expresses hexaHis-gpC-WT that contains a Q169Q silent mutation; does not

express gpNu3. This work.pETC9 Expression plasmid for gpC-S166A mutant protein. Also contains a Q169Q silent mutation; does not express

gpNu3. This work.pET15b-H6gpE Hexahistidine-tagged gpE overexpression vector. A. Davidson, personal communication.pT7Init Wild-type initiator expression vector; expresses gpB, gpC-WT, and gpNu3. This work.pT7Init-gpC-S166A gpC-S166A mutant initiator expression vector; expresses gpB, gpC-S166A, and gpNu3. This work.pT7Cap Wild-type procapsid expression vector; expresses gpB, gpC-WT, gpNu3, gpE, gpD.32

pT7Cap-gpC-S166A gpC-S166A mutant procapsid expression vector; expresses gpB, gpC-S166A, gpNu3, gpE, gpD. This work.


of the gpNu3 scaffold protein, and (iii) Ser166 isrequired for proteolytic activity. We note thatportal structures are assembled upon induction ofboth wild-type and gpC-S166A mutant initiatorconstructs as evidenced by electron microscopy(data not shown).

Lambda gpC is the viral protease required forprocapsid maturation

We previously constructed pT7Cap, a vector thatexpresses the λ portal (gpB), gpC, scaffold (gpNu3),and major capsid (gpE) proteins in E. coli anddemonstrated that biologically active procapsids canbe isolated from cells expressing these proteins.32

Here, we show protein expression from this vector incrude cell lysates analyzed by SDS-PAGE. TheCoomassie blue-stained gel shows significant expres-sion of gpE, but no discernable expression of gpNu3,gpB, or full-length gpC (Fig. 4a)∥; notwithstanding,Western blot analysis shows the presence of gpCproteolysis products, but not full-length gpC in thecrude cell extract (not shown).Wenext introduced thegpC-S166A mutation into pT7Cap (pT7Cap-gpC-S166A) and examined the crude cell lysate of inducedcells. The Coomassie blue-stained gel shows that gpEexpression is unchanged in the gpC mutant vector(Fig. 4a); in contrast to wild-type pT7Cap, however, agpNu3 band is clearly visible and close inspection ofthe gel suggests that full-length gpC is also present;

∥The scaffold protein and minor capsid proteins gpCand gpB must in fact be expressed because functionalprocapsids can be isolated from these cells.28 They areexpressed at low levels, however, and are not visualizedin the crude extracts.

this was confirmed by Western blot analysis (notshown).Procapsids were purified from the induced cells as

previously described,32 which routinely yieldedequivalent amounts of wild-type and gpC-S166Amutant procapsid preparations (not shown). Electronmicroscopy confirms that the yield and gross appear-ance of the two procapsid preparations is essentiallyidentical (data not shown) and Fig. 4b shows that themutant procapsids are morphologically indistinguish-able fromwild type. Analysis of the protein content inthe purified procapsids demonstrates that while littlefull-length gpC is found in the wild-type procapsids, asignificant band of appropriate molecular weight isclearly present in the gpC-S166A mutant procapsids(Fig. 4c and d). Further, while 50–70% of the gpB isproteolyzed to pB⁎ in the wild-type procapsids, closeinspection of the gel indicates that gpB is notproteolyzed in the mutant procapsids (Fig. 4c andd). The identities of gpB and pB⁎ in the gel wereconfirmed by mass spectrometry, which furtherconfirmed the absence of pB⁎ in the mutant procapsidpreparations (data not shown).It is presumed that proteolysis of the gpNu3 scaffold

protein is required for its exit from the procapsidinterior.7,13,25 A functional (wild-type) gpC protease isrequired to degrade gpNu3 (see Fig. 3c) and weanticipated that the gpC-S166A mutant procapsidswould retain significant quantities of the gpNu3scaffold protein. The amount of gpNu3 associatedwith freshly purified procapsids, both wild type andmutant, varies between preparations (not shown).While freshly prepared gpC-S166A mutant procap-sids generally retain more gpNu3 than do wild type,the content in both decreases during purification andupon storage at 4 °C, ultimately yielding procapsids

Fig. 4. Expression of wild-type and gpC-S166A mutant procapsids. (a) Cells expressing all of the procapsid proteinsand either gpC-WT or gpC-S166A (indicated at top of gel) were induced as described in Materials and Methods. Relevantgenes in the expression vector are depicted above the figure; both vectors contain wild-type Nu3 Shine–Dalgarnosequences. SDS-PAGE of whole-cell lysates stained with Coomassie blue is shown. The positions of full-length gpCprotein (45.9 kDa), gpE (38.2 kDa), and gpNu3 protein (13.4 kDa) are indicated. The identities of gpC, gpE, and gpNu3were confirmed by mass spectrometry (data not shown). The gpD protein (11.6 kDa) also expresses strongly32 but is notshown in this figure. (b) Wild-type and mutant procapsids were purified as described in Materials and Methods.Representative electron microscopy images for wild-type and gpC-S166A mutant procapids (in duplicate) are shown. (c)SDS-PAGE of the purified preparations stained with Coomassie blue is shown and the positions of gpB, pB⁎, gpC, andgpE are indicated at right in black; their identities were confirmed by mass spectrometry (data not shown). Theanticipated positions of the pY (84.1 kDa) and pX1/pX2 (29 kDa/31 kDa) fusion proteins are also indicated in gray. (d)Expansion of the 50-kDa region of the SDS-PAGE gel shown in (c); the gel was stained with SYPRO® Ruby Protein GelStain (Lonza) for enhanced sensitivity. The anticipated positions of gpB (59.5 kDa), pB⁎ (57.3 kDa), and gpC are indicated.(e) Western blot analysis of the gel presented in (c) using an anti-gpE antibody. (f) Western blot analysis of the gelpresented in (c) using an anti-gpC antibody. Note that gpNu3 does not react to the anti-gpC antibody.


essentially devoid of scaffold as shown in Fig. 4c. Thisleads to the conclusion that scaffold proteolysis is notrequired for its exit from the λ procapsid interior. Thisis discussed further below.

The pX1 and pX2 proteins

The currently accepted model for λ procapsidmaturation proposes that each of the 10–12 copies ofgpC incorporated into the immature procapsidforms a covalent cross-link with a gpE major capsidprotein, presumably at the portal vertex (Fig. 1).7,13,25The resulting pY protein (84.1 kDa) is then sequen-tially proteolyzed to pX1 (31 kDa) and pX2 (29 kDa)proteins that remain associated with the maturedprocapsid structure. The role of these proteins in λprocapsid assembly and function remains unknownand we sought to characterize these mysteriousproteins. Unfortunately and despite considerableeffort, we could not conclusively identify the

putative pY, pX1, or pX2 fusion proteins in ourpurified procapsid preparations, by Coomassie bluestaining (Fig. 4c), by SYPRO® Ruby Protein Gel Stain(not shown), or byWestern blot analysis (Fig. 4e and f).Wenote that boiling of the purified procapsids in SDS-PAGE load buffer, even briefly, generates multiplegpE degradation bands in the molecular weight rangeof the pX proteins (see Fig. 4e). Further, gpCproteolysis products are observed in this molecularweight range (Fig. 4f), which complicates the analysis.We attempted a number of alternative protocols fordissociation and denaturation of the procapsid pro-teins prior to loading onto the gel, including incuba-tion with urea and/or acetonitrile under a variety ofconditions. Although these gentler approaches werenot as effective in dissociation of the procapsids, theycompletely abrogated the generation of gpE degrada-tion bands detectable by Western blot analysis; gpCbands in the ∼30-kDa region were still visible,however, as a result of autoproteolysis (data not

image of Fig. 4


show). In sum, we were unsuccessful in demonstrat-ing co-migrating bands that were immunoreactivewith both the anti-gpE and anti-gpC antibodies, asrequired of a bona fide cross-linked species, in any ofour purified procapsid preparations—wild-type ormutant. We also searched for these elusive proteins inprocapsids isolated from an induced packaging-deficient lysogen (λ Aam11 mutant, Table 2) andfrom intact, infectious virus; these attempts wereuniformly unsuccessful (data not shown).Finally, we utilized mass spectrometry to identify

and characterize the putative pY/pX proteins in the λprocapsids.33,34 Gentle cross-linking of the purifiedprocapsids with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (zero length cross-linker)or bis(sulfosuccinimidyl)glutarate (11.4 Å cross-linker)affords specific inter- and intramolecular cross-links inthe gpE major capsid protein that can be readilyidentified by mass spectrometry (P. Singh, E. Naka-tani, D. Goodlett, and C.E.C., unpublished results).Mass spectrometry data for both the wild-type andgpC-S166Amutant procapsids, control and chemicallycross-linked were scrutinized to identify gpC–gpEcross-links to provide evidence for the pY and pXproteins. We specifically probed for disulfide (Cys-Cys, loss of H2) and isopeptide (Lys-Asn/Gln, loss ofNH4

+; Lys-Arg/Glu, loss of H2O) cross-linked species,but all attempts were unsuccessful. In sum, analysis ofa number of λ procapsid preparations and infectiousvirus by SDS-PAGE, Western blot, and mass spec-trometry failed to provide evidence for a cross-linkedgpC–gpE species.

Biological activity of the gpC-S166A mutantprocapsids

The data presented above demonstrate that thegpC-S166A mutation abrogates procapsid matura-tion (i.e., proteolysis of gpB, autoproteolysis of gpC,

Fig. 5. Activity of purified wild-type and S166A mutant procapsids.(a) DNA packaging activity of puri-fied wild-type and gpC-S166A mu-tant procapsids. (b) Genomepackaging activity of purified wild-type and mutant procapsids. Insetshows PFGE analysis of the reactionmixtures stained with ethidium bro-mide. Note that the predominantpackaging product with both pro-capsid preparations is full-length λDNA, which indicates processivepackaging. (c) Virus assembly activ-ity of the wild-type and mutantprocapsids. Infectious viruses as-sembled in vitro were quantified byplaque assay; a value of 100%

represents a virus yield of 1.8×106 pfu/ml. We note that the procapsid preparations used in all of these studies wereevoid of scaffold protein (see Fig. 4c). The virus yield in the absence of procapsids or terminase was essentially zero5 pfu/ml; not shown). All of the reactions in this figure were performed as described in Materials and Methods and the

esults represent the average of at least four independent experiments with standard deviation indicated.

d(br

and proteolysis of gpNu3) but does not affectprocapsid assembly. We next examined the biolog-ical activity of the purified procapsids using a DNApackaging assay.32 In this assay, the terminaseenzyme translocates DNA into the procapsid inte-rior, rendering it resistant to degradation by DNase.We first examined packaging using pCT-λ, a cos-containing substrate in which an 8.7-kb duplexDNA is translocated into the capsid.35,36 Packagingof this fragment does not trigger procapsid expan-sion, which occurs only when ∼15 kb DNA istranslocated into the capsid.17,37 Thus, this assayexamines (i) the capacity of the portal (gpB/pB⁎) torecognize the terminase·DNA complex and initiateDNA translocation and (ii) the capacity of theprocapsid to accept the DNA; we refer to this as aDNA packaging activity. Figure 5a shows that DNApackaging by the gpC-S166A mutant procapsidsdoes not significantly differ from that of the wild-type procapsids.We next examined the reaction using a full-length

λ genome as a packaging substrate. This genomepackaging assay measures the capacity of theprocapsid (i) to package DNA, (ii) to undergo theexpansion step, and (iii) to stably accept the entiregenome length.32 The data presented in Fig. 5bdemonstrate that the mutant procapsids show amodest defect in genome packaging compared towild-type procapsids. Inspection of the reactionmixtures by pulsed-field gel electrophoresis (PFGE)(Fig. 5b, inset) shows that the predominant packag-ing product in both reactions is full-length λ DNA.This indicates that the packaging reaction is highlyprocessive with both wild-type and gpC-S166Amutant procapsids.35,37

Finally, we examined the activity of the procap-sids in the in vitro virus assembly assay in which aninfectious virus is assembled from purified proteinsand commercially available λ DNA.18 This assay

image of Fig. 5


examines the capacity of the procapsid (i) to acceptthe entire genome, (ii) to add the finishing proteinsand a tail, and (iii) to subsequently infect an E. colicell. The data presented in Fig. 5c demonstrate thatthe mutant procapsids exhibit a severe (2 orders ofmagnitude) defect in their capacity to assembly aninfectious virus in vitro.

Discussion

The assembly of complex dsDNA virusesincludes a DNA packaging step where a terminaseenzyme translocates the viral genome into theinterior of a pre-assembled procapsid structure.2

Terminase enzymes are among the most powerfulbiological motors characterized to date; theypackage viral DNA to near liquid crystallinedensities, which generates internal capsid pres-sures of more than 20 atm.17,38–40 Thus, viralprocapsids are robust macromolecular structuresthat must withstand both external environmentaland internal packaging stresses.Packaging-competent procapsids self-assemble

from monomeric protein precursors in a definedsequential pathway that is remarkably conserved inboth prokaryotic and eukaryotic viruses.14,19,21–23

Within this context, the presumed assembly path-way for the λ procapsid is remarkable in itscomplexity, as outlined in Fig. 1. Characterizationof this unusual pathway is the goal of this work.

The procapsid maturational protease

The assembly of a functional procapsid shellrequires co-polymerization of the major capsidprotein with an internal scaffold protein. In somecases, such as in bacteriophages ϕ29 and P22, thescaffold proteins exit the immature procapsidstructure and are reused. In others, including theherpesviruses and bacteriophage λ, the scaffoldingproteins are degraded by a procapsid-bound matu-rational protease.14,19–23 The MEROPS databaseclassifies protease enzymes according to sequenceand structural features.41,42 Protease family mem-bers share primary sequence homology, while clanmembers are presumed to share a common ancestry(based on structural homology). Accordingly, thephage proteases have been classified into fourfamilies: U9 (T4-like), U35 (HK97-like), S14 (D3-like), and S49 (λ-like)¶; the eukaryotic herpesvirusproteases have been grouped into the S21

¶MEROPS family names denote the catalytic type:serine (S), aspartate (A), cysteine (C), and so forth;families in the U-series have unknown catalytic mechan-isms. Some family members within a clan use differentactive-site residues. For instance, the crystal structures ofClpP and SppAEC are superimposable and both belong tothe SK clan; however, they utilize Ser/His/Asp and Ser/Lys catalytic centers and thus belong to the S14 and S49families, respectively.39,40

family.26,41,43 These families have been furthergrouped into two clans: the SH clan includes theU9, U35, and S21 families while the SK clan includesthe S14 and S49 families. Although the homologydata suggested that gpC was the λ procapsidprotease, this had not been directly demonstrated.Our data clearly show that gpC is indeed a proteaseand that Ser166 is required for catalytic activity. Theenzyme possesses autoproteolytic activity, it isresponsible for degradation of the gpNu3 scaffoldprotein, and it is required for the proteolyticmaturation of gpB assembled into the portalcomplex.

Structural model for the gpC protease

A structural homology search for gpC using thePhyre fold recognition engine44 identified E. colipeptide peptidase SppAEC as a strong structuralhomologue (Materials and Methods). SppAEChomologues exist in eubacteria, archaea, and thethylakoid membrane of chloroplasts, and theycollectively belong to the SK clan, S49 family ofsignal peptidases.45 These enzymes utilize a cata-lytic dyad composed of an active-site Ser aided by aLys catalytic base to degrade the N-terminal signalpeptide in secreted proteins. Using SppAEC as atemplate, we constructed a structural model for thegpC polypeptide as described in Materials andMethods. Of the five lysines in gpC, only Lys249 isclose to the catalytic Ser166 in the structural modeland this could represent the active-site base; thisresidue is not conserved in the sequence alignments,however (see Fig. 2).We note that SppAEC is composed of N-terminal

and C-terminal domains that appear to be tandemrepeats, each of which shows structural homologyto gpC.45,46 The catalytic dyad in SppAEC isformed at the domain interface with Lys209 andSer409 contributed by the N-terminal and C-terminal domains, respectively (Fig. 6a). It istempting to speculate that the catalytically compe-tent gpC protease is a dimer (or higher-orderoligomer) and that the Ser-Lys catalytic dyad isassembled at the subunit interface. We thereforeconstructed a structural model for a gpC dimerbased on the crystal structure of SppAEC, asoutlined in Materials and Methods. The resultingmodel (built without any a priori assumptions)positions Ser166 of one subunit at the dimerinterface, juxtaposed to Lys218 in the secondsubunit, appropriately positioned to function as acatalytic base (Fig. 6b). Importantly, both of theseresidues are strongly conserved in the sequencealignments (Fig. 2).Many of the Ser/Lys proteases utilize a third

catalytic residue (typically Ser or Thr) that helpsorient the catalytic base for catalysis.47 In the case ofSppAEC, the “orienting” serine corresponds toSer431.45 Thus, the “catalytic triad” is assembledfrom Lys209 (catalytic base) in the N-terminaldomain and Ser409/Ser431 (catalytic serine/orient-ing serine) in the C-terminal domain of the SppAEC

Fig. 6. Structural and functionalmodel for the λ gpC protease–scaffold protein. (a) Crystal structureof SppAEC protease from E. coli(accession #P08395). Note thatSppAEC is a single subunit com-posed of two domains repeated intandem; the N-terminal and C-ter-minal domains are depicted in blueand cyan, respectively.45,46 The ac-tive-site residues are indicated inspheres and colored as follows:catalytic serine (Ser409, red), generalbase (Lys209, light blue), and coor-dinating Ser (Ser431, purple). (b)Structural model for a dimeric gpCprotease constructed as described inMaterials and Methods. The twosubunits, which are homologous tothe N-terminal and C-terminaldomains in SppAEC, are indicatedin blue and cyan, respectively. Theactive-site residues are colored asfollows: catalytic serine (Ser166, red),general base (Lys218, light blue), andcoordinating lysine (Lys188, purple).(c) Analysis of the λ portal protein(gpB) sequence for signal peptidecleavage sites was performed asdescribed inMaterials andMethods.Every second residue is displayedalong the x-axis. Red bars indicatelikely proteolysis sites; blue curveindicates homology with signal pep-tide sequences. The gpC proteasespecifically cleaves the gpB portalprotein at residue Glu20 (arrow);proteolysis of the portal at Asp61 isnot observed in vivo and may indi-cate that this region of the protein isprotected within the confines of theprocapsid.


polypeptide (see Fig. 6a). Close inspection of thesequence homology data reveals that gpC Ser188 isstrongly conserved in the alignments (Fig. 2).Interestingly, this residue is juxtaposed to theputative Ser166/K218 catalytic dyad in the gpCdimer model (Fig. 6b). In direct analogy to SppAEC,we propose that the catalytic triad in gpC isassembled at a dimer interface with the catalyticbase (Lys218) contributed by one subunit and thecatalytic serine and orienting serine (Ser166/Ser188)

contributed by the second. Indeed, the superposi-tion of the catalytic triads in SppAEC and the gpCdimer model is striking (compare Fig. 6a and b).It is noteworthy that many of the signal

peptidases function as oligomers with the catalyticsite assembled at a subunit interface.45,46 Theoligomeric nature of the catalytically competentgpC enzyme remains unknown, but the structuralmodel presented in Fig. 6b provides an attractivemechanism to control the protease activity of gpC

image of Fig. 6

bThe expression of free scaffold protein is in vast excessof the protease–scaffold fusion protein in both λ and theherpesviruses. In the case of λ, this results from strongertranslation of the scaffold relative to protease, while in the


such that activation does not occur until incorpo-ration and presumably oligomerization within theconfines of the assembled procapsid. Whileadmittedly speculative, the model suggests thatgpC mimics both the structure and function of theS49 family of signal peptidases and provides amodel for mutagenesis studies that are currentlyunderway in our laboratory.

Maturation of the gpB portal protein

The λ gpC protein is required for the proteolyticmaturation of gpB assembled into the portalcomplex. This is a specific reaction in which the20 N-terminal residues of gpB are degraded toafford pB⁎ (Fig. 1). The basis for this specificity isunclear and could represent sequence and/orstructural features within the N-terminus of gpB.Given the similarity of gpC to the signal peptideproteases, we analyzed the primary sequence ofgpB for signal peptide homology using the SignalP servera. This analysis predicts that a signalpeptidase would cleave gpB remarkably close tothe cleavage site actually observed in the portalprotein (Fig. 6c). Thus, the gpC protease is relatedto the S49 signal peptidase enzymes and specif-ically degrades an N-terminal protein sequencethat bears similarity to a consensus signal peptide.Whether this specificity extends to autoproteolyticand gpNu3 proteolytic sites is currently underinvestigation.

The minor λ procapsid proteins

A major goal of this work was to characterizethe structure and function of the mysterious pXproteins. Unfortunately, we were unable to iden-tify these proteins in our purified procapsidpreparations. The accepted model for λ procapsidassembly predicts that the pY protein, whichrepresents a covalent fusion of gpC with the gpEmajor capsid protein, would accumulate in thegpC-S166A mutant procapsid preparations (seeFig. 1); however, we could not obtain evidence forthis putative intermediate. The reason for this isunclear, but we note that the evidence for theseelusive proteins was originally obtained usingradiolabeled cell extracts. This approach differsfrom those described here and it is feasible that ahigher level of sensitivity is required. We note,however, that we could not identify these proteinsusing Western blotting or mass spectrometry.How can we reconcile our results with previouslypublished data? The most parsimonious explana-tion is that while the pY/pX proteins may beformed, they are present in the procapsids inextremely limiting amounts. Thus, we concludethat they are not critical components in either thestructure or function of the λ procapsid.

a http://www.cbs.dtu.dk/services/SignalP

Model for procapsid assembly

In the absence of the pX proteins, the λ procapsidassembly pathway is remarkably similar to thatobserved in the eukaryotic herpesvirus groups andmay be described as follows (Scheme 1). Assemblyof the portal ring initiates co-polymerization of thescaffold and major capsid proteins into an icosahe-dral shell. A limited number of protease–scaffoldfusion proteins are incorporated into the procapsidby virtue of the C-terminal scaffold domain and on astochastic basis reflecting their relative concentra-tion in the cellb. Finally, the procapsid proteasedegrades itself and the scaffold and the proteolysisproducts exit the procapsid interior to afford apackaging-competent procapsid structure. In thecase of λ, the procapsid protease also processes theportal structure, removing 20 residues from the N-terminus of the portal proteins.It has been suggested that the herpesvirus-like

proteases (S21 family) and the λ-like proteases(S49 family) define two distinct maturation prote-ase folds that exhibit little sequence homology butthat provide identical functions in virusdevelopment.26 Consistently and despite a lack ofstructural homology, the domain organization ofthe two enzymes is virtually identical; in bothcases, an N-terminal protease domain is fused to aC-terminal scaffold domain that is identical withthe free scaffold protein (see Scheme 1). Thefunctional herpesvirus proteases are homodimers48

and we suggest that the λ protease is similarly adimer (Fig. 6b). Both enzymes possess autoproteo-lytic activity and both are required for degradationof the internal scaffold protein during procapsidmaturation. Thus, the two enzyme systems exhibitremarkable functional similarity despite theirevolutionary independence.Bacteriophage P2 also encodes a procapsid prote-

ase–scaffold fusion protein that shows functionalsimilarities to the λ and herpesvirus enzymes.49 TheP2 gpO protein is similarly composed of N-terminalprotease and C-terminal scaffold domains that arerequired for procapsid maturation and assembly,respectively.50,51 Unlike λ and the herpesviruses,however, P2 does not express a separate scaffoldprotein and pO exclusively serves this function. Theprotein possesses autoproteolytic activity that spe-cifically cleaves itself, releasing the N-terminalprotease domain (pO⁎), which then degrades thescaffold domain, the 31 N-terminal residues of themajor capsid protein, and the 26 residues of theportal protein.51,52 Specific to P2, the protease

herpesviruses, this is the result of strong transcription ofscaffold mRNA relative to that of protease. The amount ofprotease incorporated into the nascent procapsid issignificantly less than free scaffold in both virus systems 7,21.

http://www.cbs.dtu.dk/services/SignalP

Scheme 1. Revisedmodel for the assembly andmaturation of the λ procapsid. Details are described in the text. Shownbelow the proposed pathway is the conserved domain organization of the protease–scaffold fusion proteins and majorscaffold proteins from λ, the herpesviruses and phage P2. In both cases, the scaffold is expressed in significant excess ofthe protease–scaffold fusion protein. In contrast, the phage P2 gpO protein is expressed exclusively as a protease–scaffoldfusion protein without a separate scaffold protein (see the text for details).


domain is not degraded and remains in the viralparticles. The P2 protease belongs to the U35 familyof serine proteases43 and utilizes a “classical” serineprotease catalytic triad (Ser-His-Asp); it likelyfunctions as an oligomer.51 The protease domain isdispensible for procapsid assembly, but essential tovirus viability, indicating that proteolysis is anessential step in the assembly of an infectiousvirus.51 Thus, while sequence, structural details,and specific catalytic mechanisms may differ, thefundamental features of the protease–scaffold pro-teins appear to be recapitulated in all of complexdsDNA viruses.

Biological role for proteolytic maturation of the λprocapsid

The gpC protease is responsible for proteolyticdegradation of the internal scaffolding protein, yetour data indicate that proteolysis is not strictlyrequired for its exit from the procapsid. We note,however, that protease defects are significantly morelethal in vivo than in vitro. Early studies on lysates ofλCnull mutants found that the vast majority of particleswere unexpanded procapsid structures thatcontained gpNu3 and unproteolyzed gpB;53–55 essen-tially, no expanded capsidswere observed, indicatingthat although procapsid assembly occurred, theywere not active for DNA packaging. In contrast, ourstudies with gpC-S166A mutant procapsids indicatethat genome packaging is quite efficient.How can these contrasting observations be recon-

ciled? We suggest that timely removal of the gpNu3

scaffold protein from the procapsid interior is anessential step prior to DNA packaging. We note thatwhile unproteolyzed gpNu3 can exit the purifiedprocapsids, this is a relatively slow process thatoccurs over a span of days to weeks. This requiressignificantly more time than allowed in the 40-mintime frame required for virus development in vivo.Thus, a functional gpC protease is essential toaccommodate the time frame for virus developmentin the infected cell.A second role for gpC is proteolytic maturation of

the portal protein. λ is one of the few viruses inwhich portal proteolysis is observed during procap-sid maturation (phage P2 is another).50,52 Our dataindicate that proteolysis of gpB is not required forprocapsid assembly or for DNA translocation intothe procapsid. The gpC-S166A mutant procapsidsexhibit a significant defect in the in vitro virusassembly reaction, however, consistent with thelethal effect of C deletion mutations in vivo. Whilewe cannot rigorously exclude the possibility that asmall (and undetectable) amount of scaffold remainswithin the mutant procapsids and that this affectsvirion completion, we disfavor this explanation. It isfeasible that full-length gpC trapped within thegpC-S166A mutant procapsids affects packaging ofthe entire viral genome in subtle ways and that thisdisrupts viral particle completion; however, prelim-inary studies with purified procapsids containing agpC deletion mutation indicate that they behavesimilar to the gpC-S166A mutant procapsids (E.M.and C.E.C., unpublished results). We suggest thatproteolysis of gpB is required for downstream

image of Scheme 1


assembly steps, which could include (i) addition offinishing proteins (gpW/gpFII), (ii) addition of theviral tail, and/or (iii) efficient DNA ejection toinitiate the next round of infection. These possibil-ities are not mutually exclusive and all may play arole. Work currently underway in our laboratoryseeks to further define the nature of the functionaldefect in the mutant procapsids to provide amechanistic framework for viral capsid assembly.

Materials and Methods

Tryptone, yeast extract, and agar were purchased fromDIFCO. Molecular biology enzymes and mature λ DNAwere purchased from Invitrogen and New EnglandBiolabs. All of the synthetic oligonucleotides used inthese studies were purchased from Invitrogen and wereused without further purification. All chromatographymedia were purchased from GE Healthcare. Custom gpCand gpE polyclonal antibodies were obtained fromAnaspec on a fee for service basis using 5 mg/ml purifiedprotein in 6 M guanidinium hydrochloride as the antigen.Goat anti-rabbit fluorescein conjugate antibody waspurchased from Upstate. All other materials were of thehighest quality available.Bacterial cultures were grown in shaker flasks utilizing an

Innova 4430 incubator shaker. All protein purificationsutilized the Amersham Biosciences ÄKTA purifier core 10system from GE Healthcare. All of the purified proteinpreparations were homogenous as determined by SDS-PAGE (not shown). UV–VIS absorbance spectra wererecorded on a Hewlett-Packard HP8452A spectrophotome-ter. PFGE utilized a Gene Navigator® system (GE Health-care). Video images were captured using an EpiChemi3

darkroom system with a Hammamatsu camera (UVPBioimaging Systems); the video images were quantifiedusing either the LabWorks 4.6 (UVP Bioimaging Systems) orthe ImageQuant (Molecular Dynamics) software packages.Sucrose density gradients were poured using a BiocompModel 107 ip Gradient Master® and gradient centrifugationutilized a Beckman L-90K ultracentrifuge.

SDS-PAGE and Western blot analysis

Unless otherwise indicated, protein samples were boiledfor 30 s in SDS-PAGE load buffer (3.12 mM Tris, pH 6.8,50% glycerol, and 0.05% bromophenol blue) and thenfractionated by 15% SDS-PAGE. The proteins werevisualized by staining with Coomassie blue using standardmethods and by Western blot analysis, as follows. Thefractionated proteins were transferred to an Immobilon-FL® transfer membrane (Millipore) using a Fisher Biotechsemi-dry blotting apparatus (80 mAmp for 90 min). Themembrane was incubated in TBST buffer (10 mM Tris,pH 8, 150 mM NaCl, and 0.2% Tween) containing 5% low-fat milk for 1 h at room temperature with gentle agitationand then washed thrice for 5 min with TBST buffer. Theblocked membrane was incubated as above in TBST buffercontaining a 1:1000 dilution of either anti-gpC or anti-gpEpolyclonal antibody as indicated in each individualexperiment. The membrane was again washed thrice for5 min with TBST buffer and finally incubated in TBSTbuffer containing goat anti-rabbit fluorescein conjugateantibody (1:2000 dilution). The membranes were analyzedfor immunoreactive bands using a Storm 840 scanner in thefluorescence mode.

Expression and purification of gpE

E. coli BL21(DE3)[pLys S] cells were transfected withpET15b-H6gpE (Table 2, a generous gift of Dr. AlanDavidson, University of Toronto) and grown overnighton LB plates containing 50 μg/ml of ampicillin at 37 °C. Asingle bacterial colony was used to inoculate 10 ml 2X-YTmedia containing 50 μg/ml of ampicillin and the culturewas maintained at 37 °C overnight. The overnight culturewas used to inoculate 1 l of 2X-YT media, pH 7.2,containing 25 mM sodium phosphate, 5 mM glucose, and50 μg/ml ampicillin, and the culture was maintained at37 °C until an OD600 (optical density at 600 nm) of 0.5 wasobtained. Protein expression was then induced with 1 mMIPTG, the culture was maintained at 37 °C for 2 h, and thecells were then harvested by centrifugation. The cell pelletwas resuspended in 25 ml of ice-cold TSB buffer [20 mMTris buffer, pH 8, containing 20 mM NaCl, and 7 mM β-mercaptoethanol (β-ME)], and unless otherwise indicated,all subsequent steps were performed with ice-cold buffersat 0 to 4 °C. The cells were partially disrupted by sonication(10×10 s bursts), lysozyme was added to a concentration of80 μg/ml, and an ethylenediaminetetraacetic acid (EDTA)-free Complete® Protease Inhibitor Cocktail tablet (Roche)was added to themixture, whichwasmaintained at 4 °C for1 h. The cells were then lysed by sonication and insolublematerial was removed by centrifugation (6000g, 20 min).SDS-PAGE analysis revealed that H6-gpE partitioned intoboth the supernatant and the pellet fractions (data notshown); therefore, the supernatant was dialyzed in buffer A(10 mM Tris, pH 7.7, containing 6 M guanidine hydrochlo-ride, 100mM sodium phosphate, and 5mM imidazole) andthe pellet was resuspended in 25 ml of the same buffer. Thesamples were pooled and then loaded onto a Ni-NTAcolumn (4 ml) that had been equilibrated with buffer A, thecolumn was washed with buffer A containing 20 mMimidazole, and bound material eluted with a step gradientof 50, 100, 200, and 500 mM imidazole in buffer A; gpEeluted with 50 mM imidazole. The appropriate fractionswere pooled and then concentrated using an Amicon Ultra-15 filtration device® (Millipore). An extinction coefficient of36,330 M−1 cm−1 was determined using the Edelhochmethod.56

Construction of gpC expression plasmids

We constructed pETC5 to (i) express gpC bearing a C-terminal His6 tag that could be removed by thrombindigestion and (ii) express the full-length gpC-gpNu3fusion protein, but not free gpNu3; the thrombin cleavagesite was not used in the present study. pETC5 wasconstructed by sequentially inserting a proximal (5′) NdeI-to-EagI C gene segment followed by a distal (3′) EagI-to-XhoI segment into the polycloning site of pET24b(Novagen, Inc.). Briefly, the proximal (5′) segment wasproduced by PCR amplification of the λ DNA segmentextending from λ base pairs 4416 to 5340, where the leftprimer was 5′-GAC ATA TGA CAG CAG AGC TGC-3′,which introduced anNdeI site (in bold and italics), and theright primer was 5′-TTC GGC CGC CGG AGA GACGGG ATT TAC GTG-3′, which introduced changes in λbase pairs 5332, 5335, and 5338 (underlined) that (i)created an EagI site (in bold and italics) and (ii) changedthe Nu3 rbs sequence from 5′-AGGA-3′ (wild type) to 5′-CGGC-3′. The resulting PCR product was double-digested with NdeI and EagI.Next, the distal (3′) segment of C was amplified using

the left primer 5′-GGCGGC CGA ATG ACC AAA GAG


ACT CA-3′, which introduced an EagI site (in bold anditalics) and the right primer, 5′-TG CTC GAG GGA ACCGCG TGGCAC CAGCAC TGG TGT GTT CAG-3′, whichintroduced an XhoI site (in bold and italics) and thecodons for a thrombin cleavage site (underlined;LVPRGS). The PCR product was double-digested withEagI and XhoI. Finally, the two PCR products were tripleligated into NdeI/XhoI-digested pET24b to afford pETC5.pETC8 and pETC9 are derivatives of pETC5 in which an

MfeI site was introduced along with the same-senseQ169Q change by altering codon 169 from CAA to CAG.In pETC9, an additional S166A change was created byaltering codon 166 from AGT to GCA; this also created anFspI site. These changes were introduced into pETC5using the Ex-Site kit (Novagen, Inc.). Briefly, pETC5 wasused as template for PCR reactions using phosphorylatedprimers whose 5′-ends originated from adjacent base pairsin the C gene. Following the PCR reaction, the templateDNA was digested with DpnI and the linear, plasmid-length product molecules were ligated and used totransform E. coli DH5α cells. For pETC8, the forwardprimer was the 5-phosphorylated sequence 5′-TGC AGTGCA GGT CAA TTG CTT GCC AGT GCC GCC T-3′(MfeI sequence in bold and italics) and the reverse primerwas 5′-GTT CAT GTC GTT GGC AAG CGC CCA TA-3′.For pETC9, the forward primer, the 5-phosphorylatedsequence 5′-TGC GCA GCA GGT CAA TTG CTT GCCAGT GCCGCC T-3′ (FspI andMfeI sequences in bold anditalics) was used with the same reverse primer. Candidateplasmids were sequenced to verify the presence of thedesired mutations and to confirm that no extraneousmutations were introduced.

Expression and purification of gpC-S166A

E. coli BL21(DE3) cells were transfected with eitherpETC8 (gpC-WT) or pETC9 (gpC-S166A, Table 2) andgrown overnight on LB plates containing 50 μg/ml ofkanamycin at 37 °C. A single bacterial colony was usedto inoculate 25 ml 2X-YT media containing 50 μg/ml ofkanamycin and the culture was maintained at 37 °Covernight. The overnight culture was used to inoculate2 l of 2X-YT media, pH 7.2, containing 25 mM sodiumphosphate, 5 mM glucose, and 50 μg/ml kanamycin andthe culture was maintained at 37 °C until an OD600 of0.5 was obtained. Protein expression was then inducedwith 1 mM IPTG, the culture was maintained at 37 °Cfor 2 h, and the cells were harvested by centrifugation.SDS-PAGE analysis revealed that while expression of thegpC-S166A mutant protein was robust, expression ofwild-type gpC was not detectable. Thus, only themutant protein was purified, as follows.The cell pellet was resuspended in 100 ml of ice-cold

TSEB buffer (20 mM Tris buffer, pH 8, containing 100 mMNaCl, 2 mM EDTA, 7 mM β-ME, and 10% glycerol), andunless otherwise indicated, all subsequent steps wereperformed with ice-cold buffers at 0 to 4 °C. An EDTA-freeComplete® Protease Inhibitor Cocktail tablet (Roche) andlysozyme (80 μg/ml) were added to the mixture and thecells were partially disrupted by sonication (10×10 sbursts). The sample was maintained at 4 °C for 2 h andinsoluble material was then removed by centrifugation(6000g, 30 min); SDS-PAGE analysis revealed that all ofthe gpC-S166A protein partitioned into the pellet. Thelysis pellet was resuspended in 20 ml of buffer B (100 mMsodium phosphate, pH 7.6, containing 6 M guanidinehydrochloride) and incubated at 4 °C for 1 h. Insolublematerial was removed by centrifugation (10,000g, 30 min),imidazole (15 mM) was added to the supernatant, and

proteins were batch adsorbed to 3 ml Ni-NTA resin (3 h onice). The mixture was then loaded into a 15-ml column andadsorbed protein eluted in a step gradient of 20, 50, 100,200, 300, 400, and 500mM imidazole; gpC-S166A eluted inthe 50- and 100-mM imidazole fractions. The appropriatefractions were pooled, dialyzed against buffer B, and twicemore purified by Ni-NTA to obtain a protein preparationthat was greater than 95% pure as determined by SDS-PAGE (not shown). The appropriate fractions were pooledand concentrated using an Amicon Ultra-15 filtrationdevice® (Millipore). An extinction coefficient of140,930 M−1 cm−1 was determined using the Edelhochmethod.56

Construction of gpC complementation plasmids

Plasmids for in vivo complementation studies werederivatives of pSF1, which is pBR322 containing the λDNA insert extending from the HindIII site at λ base pair44,141 through cos to the BamHI at λ base pair 5505.57 Thisinsert contains the λ late gene promoter PR′; the lysis genescos, Nu1, A, and W; and the 5′ end of the B gene. To pSF1was inserted a PCR-amplified λ DNA segment extendingfrom the BamHI site at λ base pair 5505 to λ base pair6105; this segment contains the 3′ end of B, C, Nu3, and Dgenes. An artificially introduced SalI site was included inthe downstream primer for insertion into pSF1, whichyielded the plasmid pDW1. The plasmid pDWΔ1 wasobtained by deleting the λ DNA segment extending fromthe BstXI site at 46,441 in Rz to the BstXI site at 2862 inW ofpDW1. pDWΔ1 retains the late gene promoter and capsidgenes from B through D.pDW1Δ2was produced by digesting pDWΔ1with PshAI

and PpuMI, blunt-end filling with Klenow enzyme, andreligation. The PshAI to PpuMI deletion removes thepBR322 segment containing the NruI site, which facilitatedfurther DNA manipulations. pDW1Δ2A is a derivative ofpDW1Δ2 in which mutations generating the Q169Q (MfeI)changes replace the wild-type sequence. The appropriatesegment of pETC8 was PCR-amplified, digested with NruIand BsgRI, and used to replace the corresponding wild-type segment of pDW1Δ2. pDW1Δ2B is analogous topDW1Δ2A but has in addition the codon 166 changes thatproduce the S166A change in gpC. pDW1Δ2B wasconstructed identically as pDW1Δ2A except that pETC9was used in the PCR amplification.

Complementation tests

Lysogens alone or with the indicated complementationplasmids were grown with aeration in tryptone broth at31 °C to a density of 1×108 cells/ml. Aliquots wereremoved, diluted, and spread on tryptone broth agarplates that were incubated at 30 °C to determine cell titers.The remainder of the culture was shifted to 42 °C for15 min to induce prophages and then shaken at 37 °C for70 min to allow phage development and cell lysis.Chloroform was then added to complete cell lysis andaliquots were diluted and titered for plaque-forming units(pfu) by plating in tryptone broth soft agar with 200 μl ofan overnight culture of permissive C600 cells. Virus yieldswere calculated as the virus titer/viable cell ratio.

Construction of initiator expression vectors

Thewild-type initiator expressing operonwas constructedusing PCR methods previously described.32,58 Briefly,


mature λ DNA was used as a PCR template with thefollowing primers: forward primer, 5′-TTC TCC TCT AGAGAC CGG CAT GAC ACA GCG-3′; reverse primer, 5′-GGGG AAG CTT TTA CAC TGG TGT GTT CAG-3′.Complementary sequences in the forward primer (λsequence 2791–2808) and the reverse primer (λ sequence5720–5737) are underlined. The stop codon in the reverseprimer is indicated in bold type. The XbaI and HindIIIrestriction sequences are italicized in the forward and reverseprimers, respectively. PCR amplification yielded theexpected 2.9-kb product containing the phage lambda B, C,and Nu3 genes, along with the appropriate Shine–Dalgarnosequences. Amplified DNA was double-digested with XbaIand HindIII and cloned into similarly digested pKKT7; thisplasmid is a derivative of pKK223-3 originally developed byMeyer et al.59 The resulting plasmid (pT7Init) was used totransform E. coli DH5α cells, and transformants wereselected by ampicillin resistance. Candidate plasmids weresequenced to verify that no extraneous mutations werepresent and then used to transform BL21(DE3)[pLysS] cellsfor protein expression.The plasmid pT7Init-gpC-S166A is a derivative of

pT7Init in which codon 166 was mutated from TCA toGCA (Ser→Ala). The mutation was introduced usingthe Stratagene QuikChange® Site-Directed MutagenesisKit and the following primers: forward primer, 5′-GCCAAC GAC ATG AAC TGC GCA GCA GGT CAA TTGCTT GCC-3′; reverse primer, 5′-GGC AAG CAA TTGACC TGC TGC GCA GTT CAT GTC GTT GGC-3′; theposition of the introduced mutation is underlined. Theresulting plasmid was used to transform E. coli DH5αcells and transformants were selected by ampicillinresistance. Candidate plasmids were sequenced to verifythat no extraneous mutations were present and thenused to transform BL21(DE3)[pLysS] cells for proteinexpression.

Expression and analysis of wild-type and gpC-S166Amutant initiators

E. coli BL21(DE3) cells were transfected with eitherpT7Init or pT7Init-gpC-S166A (Table 2) and grownovernight on LB plates containing 50 μg/ml of ampicillinat 37 °C. A single bacterial colony was used to inoculate10 ml 2X-YT media containing 50 μg/ml of ampicillin andthe culture was maintained at 37 °C overnight. Theovernight culture was used to inoculate 1 l of 2X-YTmedia, pH 7.2, containing 25 mM sodium phosphate,5 mM glucose, and 50 μg/ml ampicillin and the culturewas maintained at 37 °C until an OD600 of 0.5 wasobtained. Protein expression was then induced with 1 mMIPTG, the culture was maintained at 37 °C for 2 h, and thecells were harvested by centrifugation. The pellets weretaken into gel load buffer and protein expression wasanalyzed by SDS-PAGE and Western blot.

Construction of pT7Cap-gpC-S166A

The plasmid pETC9 (Table 2) was double-digested withNruI and AatII, and the 575-bp fragment was isolatedfrom a 1% agarose gel using the Qiagen QIAquick® GelExtraction Kit according to the manufacturer's protocol.This fragment contains the mutant S166A codon of the Cgene. The fragment was cloned into similarly digestedpT7Cap using standard procedures affording pT7Cap-gpC-S166A, a vector that expresses gpC-S166A in thecontext of all of the remaining capsid proteins. Expression

and purification of wild-type and gpC-S166A procapsidswere performed as previously described.32

Sequencing of procapsid proteins by massspectrometry

The proteins in the purified procapsid preparationswere fractionated by 15% SDS-PAGE gel and visualizedby staining with a non-fixing Coomassie stain (PierceImperial Protein Stain). The gels were washed overnightwith water and the desired protein bands were excisedwith a clean razor blade. The gel slices were soaked withgentle agitation in 500 μl of 100 mM ammoniumbicarbonate at room temperature, the liquid was dis-carded, and 500 μl of Optima® grade acetonitrile (Fisher)was added. The samples were incubated as before, theacetonitrile was discarded, and the 100-mM ammoniumbicarbonate/acetonitrile wash steps were repeated twicemore. The gel slices were then dried at room temperaturein vacuo (Barnstead/Genevac miVac Duo concentrator®)and the proteins were digested in situwith trypsin (20 μg/ml in 50 mM ammonium bicarbonate) overnight at roomtemperature with gentle shaking. The solution wasremoved, placed in a clean tube, and the gel slice wastwice extracted with 50 μl of 5% acetonitrile/0.1%trifluoroacetic acid (TFA) for 15 min at room temperatureand then once with 50 μl of 50% acetonitrile/0.1% TFA. Allof the acetonitrile/TFA extracts were combined with theinitial ammonium bicarbonate digestion solution and thepooled extracts were concentrated in vacuo to a finalvolume of 10 μl (miVac Duo). The samples were thendiluted with an equal volume of 5% acetonitrile/0.1%formic acid for mass spectrometry analysis.Mass spectrometry sequence analysis was performed

on a fee-for-service basis by the University of WashingtonMass Spectrometry Center. Peptide digests were ana-lyzed by electrospray ionization in the positive ion modeon a hybrid linear ion trap/Orbitrap mass spectrometer(Thermo Scientific, San Jose, CA) interfaced to a WatersnanoACQUITY UPLC (Milford, MA). Peptides wereapplied to a pre-column consisting of a 100-μm-internal-diameter fused silica capillary packed with ∼2 cm of200Å C18 particles (Michrom Bioresources, Auburn, CA)and held in place by a sintered glass frit. Peptides were thenseparated on an analytical column consisting of a 75-μm-internal-diameter fused silica capillary with a gravity-pulled tapered tip packed with ∼12 cm of 100Å C18particles (Michrom) and eluted at 250 nl/min using anacetonitrile gradient of 5–35% B in 55 min. The mobilephase consisted of A, water (0.1% formic acid), and B,acetonitrile (0.1% formic acid). The electrospray voltagewas applied via a liquid junction located in between thepre-column and analytical column. The heated capillaryinlet was maintained at 200 °C. For the MS “survey” scan,Orbitrap resolution was set to 60,000 (m/z 400) and ionpopulation was 500,000 (2.0 s maximum fill time). ForMS/MS, precursor ions were isolated and fragmented inthe linear ion trap using an ion population of 10,000(200 msmax ion fill time), an isolation width of 2.0 Da, anda normalized collision energy of 35%. All data wereacquired in the profile mode using an MS “survey” scanover the m/z 400–2000 range followed by MS/MS data-dependent selection of the five most abundant precursorions contained in the spectrum. Singularly charged ionswere excluded and data redundancy was minimizedfurther by excluding previously selected precursor ions(−0.1 Da/+1.1 Da) for 45 s (140 list maximum) followingtheir selection for MS/MS.


Tandemmass spectrometry data were converted into theappropriate file format (.mzXML) using the instrumentvendor's software (extract_msn.exe; Thermo). Peptides forgpB, pB⁎, gpC, gpE, and gpNu3 were identified bysearching the all-virus-proteins database using the searchengine SEQUEST (University of Washington). The para-meters utilized were tryptic specificity and a parent ionerror tolerance set at 2.0 Da. Methionine oxidation wasconsidered as a variable modification. Peptides andcorresponding protein identifications were analyzed fur-ther using Peptide/ProteinProphet (Institute for SystemsBiology) and filtered using a protein probability of 0.95.

Electron microscopy

A 2-μl drop of the purified procapsid preparation(0.5 nM) was applied to a 400-mesh carbon grid (GliderGrid®, Ted Pella) for 20 s. Excess sample was removed bygentle blotting followed by aspiration, and the grid waswashed thrice with distilled water and then stained with2% uranyl acetate for 20 s. Excess stain was removed bygentle blotting followed by aspiration and the sample wasair-dried. Electron micrographs were obtained on an FEIMorgagni 268 100-kV electron microscope.

DNA packaging assay

This assay was performed based on a previouslypublished procedure.32,35 Briefly, the reaction mixtures(20 μl) contained 50 mM Tris–HCl buffer, pH 7.4, 9 mMNaCl, 10 mM MgCl2, 2 mM spermidine, 1.3 mM β-ME,1 mM ATP, 150 nM integration host factor, 30 nMprocapsids, and 2 nM pCT-lambda DNA. The reactionwas initiated with the addition of terminase holoenzymeto a final concentration of 100 nM and the mixture wasincubated at room temperature for 30 min. DNase wasthen added to a final concentration of 10 μg/ml and thereaction was left at room temperature for 5 min. To stopthe reaction, we added 21 μl of phenol:chloroform andloaded 20 μl of the aqueous solution onto a 0.8% agarosegel. The 8.7-kb packaged product (DNase resistant) wasvisualized with 0.5 μg/ml of ethidium bromide that wasadded directly into the gel during electrophoresis andpackaged DNA quantified by video densitometry aspreviously described.32

c http://blast.ncbi.nlm.nih.gov/d http://www.uniprot.org/help/uniprotkbe http://www.sbg.bio.ic.ac.uk/phyref http://www.pymol.org/

Genome packaging assay

This assay was conducted as described above except thatthe concentration of procapsids was increased to 40 nM andfull-length λ DNA (48.5 kb) was used as the packagingsubstrate.37 Packaged DNA was analyzed by agarose gelassay as above and by PFGE assay where indicated.37 Forthe PFGE assay, the samples were loaded onto a 1.2%agarose gel in 0.15× TBE buffer (13.5 mM Tris, pH 8.0,containing 13.5 mM boric acid and 0.3 mM EDTA)maintained at 8 °C. The gels were run for 3 h at 365 Vusing a hexagonal electrode array with a 0.8-s north/southand east/west pulse. DNA was visualized by staining with0.5 μg/ml of ethidium bromide for 30 min and DNA wasquantified by video densitometry as described previously.37

The in vitro virus assembly assay

This assay was performed based on a previouslypublished procedure.18 Briefly, the reaction mixtures

(12 μl) contained 20 mM Tris buffer, pH 7.4, 25 mMpotassium glutamate, 16 mMNaCl, 5.16 mMMgCl2, 2 mMspermidine, 200 μMATP, 3 nMmature λ DNA, 4 μM gpD,10 nM gpFI, 50 nM integration host factor, and 30 nMpurified procapsids. The packaging reaction was initiatedwith the addition of terminase holoenzyme to a finalconcentration of 100 nM and the mixture was incubated atroom temperature for 20min. To complete virion assembly,we added 10 μM gpW, 10 μM gpFII, and 10 nM purified λtails and incubated the reaction mixture at 37 °C for 1 h.Assembled infectious viruswas quantified by infection of E.coli LE392 using a standard plaque assay.18

Protein sequence alignment and gpC structural modelconstruction

Primary sequence homology analysis was performedusing the National Center for Biotechnology Information(NCBI) BlastP programc. The full-length gpC sequence(accession #P03711) was used as the query sequence andall non-redundant protein sequences in the NCBI databasewere analyzed; default parameters were used to identifythe top 250 candidate proteins. Essentially identical resultswere obtained using the UniProt Knowledgebased.Multiple protein sequence alignment utilized the NCBICOBALT multiple alignment tool using default para-meters. Representative alignments are displayed in Fig. 2.A structural model for gpC was constructed using the

Protein Homology/Analogy Recognition Engine (Phyree)44

using full-length gpC as the query sequence. This analysisreturned E. coli SPPAEC (a.k.a., protease IV, accession#P08395) as the top structural homologue (13% sequenceidentity, E-value=2.2×10−26, 100% estimated precision). Asecond analysis using gpC residues 81–306,which deletes theC-terminal gpNu3 scaffold domain and predicted N-terminal disordered residues, yielded a virtually identicalstructures for this region (21% sequence identity, E-value=1.8×10−22, 100% estimated precision); the latter gpCmonomer structure was used without further modification.A gpC dimer structural model was constructed by

superposition of the gpC monomer model (residues 81–306) onto the N-terminal domain (residues 56–316) and theC-terminal domain (residues 317–549) of the SPPAECcrystal structure (based on Refs. 45 and 46) using theAlign command in the MacPymol Molecular Viewerpackagef.60

Acknowledgements

The authors wish to express their gratitude toDrs. David Goodlett and Scott Shaffer, and PragyaSingh for their help in the mass spectrometrystudies. We are also indebted to Dr. Tamir Gonenfor his help in electron microscopy. We furtherwish to thank Dr. Alan Davidson for the generousgift of the H6-gpE overexpression plasmidpET15b-H6gpE. This work was supported by theNational Science Foundation grant MCB-0111066

http://blast.ncbi.nlm.nih.gov/

http://www.uniprot.org/help/uniprotkb

http://www.sbg.bio.ic.ac.uk/phyre

http://www.pymol.org/


(C.E.C.) and 0717620 (M.F.) and National Institutesof Health grant GM-51611 (M.F.).

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