Table Of ContentADVANCESINVIRUSRESEARCH,VOL64
BACTERIOPHAGE HK97: ASSEMBLY OF THE CAPSID
AND EVOLUTIONARY CONNECTIONS
Roger W. Hendrix
PittsburghBacteriophageInstituteandDepartmentofBiologicalSciences
UniversityofPittsburgh,Pittsburgh,Pennsylvania15260
I. TailedPhages:Overview
II. InitialStagesofCapsidAssembly
III. RoleofProteaseandPortal
IV. DNAPackagingandExpansion
V. Cross-Linking
VI. EvolutionaryRelationships
VII. Conclusions
References
I. TAILED PHAGES: OVERVIEW
Tailedbacteriophagesarepossiblythemostabundantorganismson
theplanet(WommackandColwell,2000).Ifmaterialfromanenviron-
mental source such as soil or water is plated on a given laboratory
strain of bacteria the number of plaques obtained is generally quite
small, but direct examination of such material for morphologically
identifiabletailedphagesbyelectronmicroscopytellsquiteadifferent
story.Coastalseawater from a variety oflocations aroundthe globe is
seentohavearound107particlespermilliliter,particlecountsinsome
freshwater sources can be orders of magnitude higher than that, and
tailed phages in surface soil have been measured at around 108 parti-
cles per gram. In all these cases, the number of phage particles is
fivefold to tenfold higher than the number of prokaryotic cells. The
total number of tailed phages on Earth is estimated to be about 1031
individual particles, an almost unimaginably large number that
implies that if all the phages were laid end to end the line of phages
would stretch about 200 million light years.
Phages have been studied in the laboratory since their discovery
nearlyacenturyago,butthenumberofdistinctphagetypessubmitted
todetailedexaminationbygeneticandbiochemicaltechniquesisquite
small,perhapsasfewas20,andthenumberforwhichdetailedstruc-
ture and mechanism of assembly of the virion has been scrutinized is
even smaller. It has generally been assumed that what is learned
1 Copyright2005,ElsevierInc.
Allrightsreserved.
0065-3527/05$35.00
DOI:10.1016/S0065-3527(05)64001-8
2 ROGER W. HENDRIX
about one tailed phage will likely apply to other phages as well. This
hasbeenausefulapproach,butithasonlybeeninmorerecentyears,
with the advent of both environmental and genome sequencing
approachesinphagebiology,thatwehaveontheonehandappreciated
theextremesparsenesswithwhichwehavesampledtheglobalphage
populationandontheotherhandbeguntohavethemeanstoestimate
how well our work to date has illuminated more general questions
about how virions are constructed and what assembly strategies are
used to achieve those structures. Bacteriophage HK97, the subject of
this article, illustrates a number of assembly mechanisms, among
them mechanisms that appear to be shared widely with other tailed
phagesunderstudyandothersthat,ifnotuniquetoHK97,areatleast
unusual. Comparisons of capsid protein sequences argue, moreover,
that HK97 is a member of an evolutionarily related family of phages
that may encompass the entirety ofthe tailed phages.
II. INITIAL STAGES OF CAPSIDASSEMBLY
HK97isatailed,double-strandedDNA(dsDNA)phageofEscherichia
coli, with an isometric head 60nm in diameter and a 180-nm-long
noncontractile tail (Fig. 1). The head (capsid) has a triangulation
number of 7 (Conway et al., 1995) and a corresponding 420 copies of
its single major capsid subunit (415 copies when the portal structure
and attached tail take the place of one pentamer of the capsid sub-
unit).Aswithmostsuchviralcapsids,thestructurecanbeparsedinto
12 rotationally symmetric pentamers, occupying the corners of the
FIG 1. Electron micrograph of HK97 and HK022, negatively stained with uranyl
formate. These two phages have identical capsid subunits but different tail proteins.
HK97isthephagewiththelongertail.Scale:theHK97tailis(cid:1)180nmlong.
BACTERIOPHAGE HK97 3
icosahedralshell,and60hexamers,regularlyarrayedbetween,with3
hexamersoneachofthe20 triangularfaces oftheicosahedron. HK97
has the additional unusual feature that covalent bonds form between
adjacent subunits in the shell, linking all the subunits into a unitary
nondissociable structure. This article reviews what we know about
how the HK97 capsid subunits become assembled into a shell and
transformed into a mature head. In addition, it considers to what
extent we can expect the lessons learned from HK97 head assembly
to be applicable more generally to other viruses.
The capsid subunit of HK97 is a 384-amino acid (42-kDa) protein
(Dudaetal.,1995b).TheproteinfoldswiththehelpoftheGroEL–GroES
chaperone system, and although the interaction with GroEL–
GroES involves only one HK97 subunit per GroEL–GroES complex,
the first detectable form of the folded protein is oligomeric, a mixture
of soluble pentamers and hexamers (Ding et al., 1995; Xie and
Hendrix, 1995). The two types of oligomers, collectively known as
capsomers, correspond to the pentameric and hexameric groupings in
theassembledcapsid.Thefreecapsomersappeartobeproducedinthe
cell at a ratio of pentamers to hexamers corresponding to this ratio
in the assembled T ¼ 7 capsids. Pentamers and hexamers can be
interconverted in vitro in either direction by adjusting the properties
of the solvent (Xie and Hendrix, 1995). It may be that pentamers and
hexamers are in equilibriumin vivo.
PentamersandhexamerswillassembleaccuratelyintoT¼7icosa-
hedral shells in vitro if they are mixed and concentrated (Xie and
Hendrix,1995).Anexperimentinwhichinvitroassemblywascarried
outwithdifferentpentamer-to-hexamerratiosbutwithconstanttotal
protein concentration showed that both pentamers and hexamers are
required for shell assembly (Xie and Hendrix, 1995). This experiment
also ruled out assembly mechanisms in which the capsomers disas-
semble into smaller oligomers or monomers before they form the
capsid. These observations led to assembly models, still not tested
experimentally, in which the interactions between pentamers and
hexamers form intermediate structures in which the proteins of the
complexaremarkedbyaconformationalchange(Conwayetal.,1995).
This conformational change (according to this speculation) constrains
thepossibilitiesforsubsequentassemblystepsandtherebydirectsthe
proteins toward forming “correct” T ¼ 7 shells and away from other
possible assemblies.
Assembly from pentamers and hexamers is in striking contrast to
theextensivelycharacterizedinvitroassemblyofthecapsidsofphage
P22, which also makes a T ¼ 7 shell. The P22 capsid protein is found
4 ROGER W. HENDRIX
as a soluble monomer, and it seems clear that it assembles in vitro
directly into shells without forming pentamer or hexamer inter-
mediates (Prevelige and King, 1993). Another apparent difference
between HK97 and P22 with regard to capsid assembly is that P22
assembly requires a second abundant protein, the scaffolding pro-
tein, which coassembles with the capsid subunit (King and Casjens,
1974). However, this difference may be more apparent than real in
that the scaffolding protein function in HK97 may be carried out by
the amino-terminal 102 amino acids of the capsid subunit. This
amino-terminalpart,knownasthedregion,isremovedbyproteolysis
andexitsthecapsidduring capsidmaturation,asdoesthescaffolding
protein ofP22 and many other phages.
III. ROLEOF PROTEASE AND PORTAL
The HK97 capsid protein will assemble efficiently and accurately
into shells in the absence of any other phage-encoded proteins, as for
example when the capsid protein is expressed at a high level from a
plasmid clone of its gene, or when shells are assembled in vitro from
purified pentamers and hexamers (Duda et al., 1995b; Xie and
Hendrix, 1995). Thus the capsid protein by itself carries all the infor-
mationneededtoaccuratelyspecifyT¼7capsids.However,innormal
in vivo assembly there are two other phage-encoded proteins that
also become part of the structure. These are the protease, encoded by
the next gene upstream from the capsid gene, and the portal protein,
encoded by the next gene upstream from the protease gene (Fig. 2).
FIG2. (A)SchematicHK97headassemblypathway.(B)HeadgenesofHK97.
BACTERIOPHAGE HK97 5
Theportalproteinisfoundintheheadasa12-subunitgrommet-like
structure sitting on one of the corners (fivefold symmetry axes) of the
icosahedral shell and occupying the position that is occupied by a
capsid protein pentamer at each of the other 11 corners. Although
the function of the portal has not been studied extensively in HK97,
it is clear in other systems that it has a central role in packaging
the DNA into the preformed capsid, possibly rotating in its socket
as DNA is pumped into the capsid under the motive force of ATP
hydrolysis. The portal also serves as the site of tail joining, typically
with one or more adaptor proteins located between the portal and the
tail. This picture likely also applies to HK97, although the only rele-
vant evidence in the case of HK97 is that the portal can be visualized
by electron microscopy, attached to the top of the tail in virions that
havelosttheirDNA.Along-standingmysteryforallthetailedphages
is how they end up with exactly 1 portal in the capsid, despite the
presenceof12fivefoldcornersonthecapsid,eachofwhichinprinciple
could accommodate a portal. The simplest model that has been pro-
posed is that the portal serves as a nucleator of capsid assembly,
and that once nucleation is accomplished the rest of the shell assem-
bles from capsid protein. Although plausible, this model has not been
clearly confirmed for any phage. For HK97, if the portal acts as a
nucleator, we need to explain how efficient assembly can occur both
invivoandinvitrointheabsenceofportalprotein.Acurrentspecula-
tionisthattheneedforaportalnucleatorcanbebypassedifthecapsid
protein concentration issufficiently high.
Thesecondproteinrequiredfortheassemblyofcompleteprocapsids,
beyond the capsid subunit itself, is the phage-coded protease. The
protease is a 25-kDa protein that cleaves off the first 102 amino acids
of each of the capsid proteins, reducing their mass from 42 to 31kDa.
The protease also degrades itself, and the resulting fragments of the
proteaseaswellasthefragmentsoftheamino-terminalportionofthe
capsid protein are lost from the capsid, presumably by diffusion
through small holes in the shell that are seen in cryoelectron micros-
copy (cryo-EM) images (Conway et al., 1995). If proheads are assem-
bled with a mutant form of the protease that is catalytically inactive
butcompetentforassembly,thereareroughly50moleculesofprotease
per prohead, and this is presumed to be the same as the number of
wild-typeproteasesperprohead.Thisnumbercouldplausiblyindicate
that there is 1 protease for each of the 72 capsomers in the prohead,
but the actual locations of the proteases are not known. By a mecha-
nismthatisnotyetclear,theproteaseisinactiveuntilitisassembled
into the prohead.
6 ROGER W. HENDRIX
Theproteolysisthatremoves102aminoacids(thedregion)fromthe
amino terminus of the capsid protein and leads as well to autoproteo-
lysisoftheproteaseisthefirststepinanelaborateseriesofrearrange-
ments of the capsid subunits. This conformational and covalent
transitiontakesthestructurefromaloose,easilydissociablegrouping
of hexamers and pentamers through a series of steps to a tough,
unitary shell that is held firmly and irreversibly together by a combi-
nation of intricate intertwining of protein subunits and covalent
cross-linksthatbindtheentireshellintoasinglestructurethatcannot
be dissociated without breaking covalent bonds (Duda et al., 1995a).
Before proteolysis, the capsid structure is known as Prohead I.
Prohead I is ordinarily a transient structure, but it can be produced
inareasonablystableformifproteaseactivityisabsent.Thissituation
canbeachievedeitherbyexpressingthecapsidproteinintheabsence
oftheproteasegeneorbyusingamutantproteasethatlackscatalytic
activity. Prohead I can be dissociated into a mixture of capsid protein
pentamers and hexamers and those capsomers can subsequently be
reassembledintoProheadIthroughrathermoderatemanipulationsof
solvent conditions and protein concentration. In contrast, the struc-
ture found after proteolysis of the d region, known as Prohead II, is
considerably more stable than Prohead I and in fact has been seen to
disassemble only under strongly denaturing conditions. Despite the
considerable difference in biochemical properties between Prohead I
and Prohead II, the two structures have similar morphologies as
viewed in moderate-resolution cryo-EM reconstructions (Conway
etal.,1995).However,ProheadIIispoisedtocarry outthenextsteps
in the capsid rearrangements.
IV. DNA PACKAGING AND EXPANSION
Prohead II is almost certainly the structure that packages phage
DNA. DNA packaging has not been studied directly in HK97, but the
strong conservation of the DNA-packaging mechanism across all
the tailed phages studied, together with the conserved complement of
head genes, leads to the strong expectation that DNA packaging in
HK97 will be similar to that in phage l, which is well studied in this
regard. Assuming that to be the case, we would predict the following
sequenceofeventsinHK97DNApackaging:theterminase,composed
ofproteins gp1and gp2,binds to the replicated,concatemeric DNA at
thecospackaginginitiationsiteandcutstoformthe10-base30 exten-
sions of the mature DNA ends; the terminase remains bound to the
BACTERIOPHAGE HK97 7
newly created endcorresponding tothe“left”end ofthemature DNA,
and that complex docks on the prohead, at the vertex containing the
portal (gp3). During some of this part of the process, protein gp6 is
associated with the terminase–DNA complex and probably serves as
an assembly factor to assist in the docking of the terminase–DNA
complex on the portal. Once the complex is docked, the terminase
andportaltogetherformtheDNA-packagingmotor,whichhydrolyzes
ATPandpumpstheDNAintothehead.Whenthepackagingmachine
has finished pumping the full-length chromosome into the head and
hasreachedthenextcossite,theterminasecutstheDNAandleaves,
remainingcomplexedtotheendoftheconcatemerattheoutboardside
of the new cut. The final step in head assembly is thought, again by
analogy with phages such as l, to be the addition of a hexamer of
proteingp7 totheportal,whereit servesasa specificattachmentsite
for the separately assembled tail.
IncontrasttoDNApackaging,amaturationprocesstermed“expan-
sion” that accompanies DNA packaging has been extensively studied
inHK97(Ganetal.,2004;HendrixandDuda,1998).Expansion,asthe
name suggests, entails a change in the dimensionsof the shell. Inthe
case of HK97 this results in an approximately twofold increase in
the internal volume of the shell, without any change in the number
ofproteinsubunits.Inadditiontothis,thereisamarkedchangeinthe
overall shape and surface texture ofthe shell, from a nearly spherical
structure with a lumpy surface to an angular icosahedron with a
smooth surface. Other tailed phages that have been studied in this
regard also undergo an analogous expansion reaction. In these cases
the change in internal volume of the shell can be quite different from
the twofold increase seen in HK97, with the internal volume of phage
f29, for example, changing essentially not at all;what does appear to
be common to all the expansion reactions is that the expanded struc-
tureissubstantiallymoreresistanttodisruptionthantheunexpanded
prohead. Because of these facts, it is assumed that the function of the
expansion reaction is to increase the stability of the shell rather than
to increase its internal volume. The detailed motions of the capsid
subunits inferred for HK97 (see later) lendsupport to this view.
With HK97 it has been possible to carry out the prohead expansion
reactionwithpurifiedproheads.Expansionistriggeredbyavarietyof
changes in the solvent, the most easily controlled being the lowering
ofpHtoaround4(Lataetal.,2000).Therateoftriggeringisstrongly
dependentonpHinthevicinityofpH4,suggestingthatanimportant
componentofthetriggeringmaybetitrationofacarboxylategroupin
the protein. Under conditions of acid expansion the capsids pause at
8 ROGER W. HENDRIX
four distinguishable intermediate states, and it has been possible to
obtain structures for each of these by cryo-EM. This has made it
possible in turn to infer a dynamic view of the expansion reaction at
moderate cryo-EM resolution, in the form of a movie produced by
interpolating between these structures (Lata et al., 2000). A high-
resolution X-ray structure is known for the mature Head II form of
the capsid (Helgstrand et al., 2003; Wikoff et al., 2000), and this has
been fit into the lower resolution movie to infer a “pseudo-atomic
resolution” version of the movie (Conway et al., 2001). The main
feature of the expansion reaction, so visualized, is that the subunits
move largely en bloc, with little or no refolding of the central domain
that makes up the bulk of the subunit. This is remarkable in that
individual subunits move as much as 58A˚ and turn by as much as
39(cid:4)relativetotheirstartingpositions,implyingsubstantialchangesin
the intersubunit interactions. Two regions of the subunit, in contrast,
engage in significant refolding during expansion. The amino-terminal
(cid:1)30 amino acids are not visible and are presumably unfolded in the
proheadbutfoldedagainstandintertwinedwithneighboringsubunits
in the mature head. The second mobile region is a long b hairpin,
consisting of 34 amino acids and named the “E loop,” which moves by
ahingingmotionatitsbase.TheEloopislocatedontheoutersurface
of the capsid and in the prohead is free of contact with the rest of the
subunit and pointing away from the surface. During expansion it
descends and lands on the surface of the capsid with the tip of the E
loop extending across the subunit boundary to lie on a part of the
neighboring subunit.
V. CROSS-LINKING
The capsid subunits in the prohead are joined exclusively by non-
covalent interactions, but by the time the capsid is transformed into
thematureheadeachofthe420subunitsisjoinedbycovalentbondsto
two of its neighbors. The bond is an amide bond—sometimes referred
to in this context as an isopeptide bond—between the side chains of
Lys-169 on one subunit and Asn-356 on the neighboring subunit
(Duda et al., 1995a). It was shown first in biochemical experiments
and subsequently with the X-ray structure of the head that the indi-
vidualsubunits arejoinedcovalentlyintoclosedringsofeither five or
sixsubunitsandthattheseringsarelinkedtopologicallytoeachother
into a unitary fabric called “viral chain mail” (Duda, 1998; Wikoff
et al., 2000). A surprising and elegant feature of the geometry of the
BACTERIOPHAGE HK97 9
chain-mail structure is that it is not the six (or five) subunits of an
individual capsomer that join each other covalently to make a six (or
five)-membered covalent circle. Rather, it is the six (or five) subunits
immediately surrounding the capsomer, each derived from a different
one of the neighboring capsomers, that form the covalent circle.
A necessary consequence of this scheme of cross-linking is that the
covalent protein circles are topologically linked at the 140 sites of
threefoldlocal and global icosahedral symmetry on the capsid.
Formation of the covalent cross-links is autocatalytic (Duda et al.,
1995a). That is, there is no separate enzyme to provide the necessary
catalytic and positioning functions, which are instead supplied by the
capsidproteinsthemselves.Itisalsoclearthatcross-linkformationis
triggered by the subunit rearrangements of the expansion reaction
(Duda et al., 1995a). The structural information available for the
expansion reaction makes it possible to understand at least in broad
termshowthisworks.Thecross-linkinglysineresidue islocatednear
thetipoftheEloopandisthereforewellawayfromthecapsidsurface
intheprohead.DuringthecourseoftheexpansiontheEloopdescends
ontothe surface ofthecapsidwiththecross-linking lysine juxtaposed
with the asparagine on the neighboring subunit with which it will
react. In addition, during the course of expansion a third subunit
moves in such a way as to position the side chain of Glu-363 within
about 2A˚ of the cross-link bond (Wikoff et al., 2000). This positioning
suggests that the glutamate may have a catalytic role in the cross-
linking, a supposition that gains support from a mutant in which the
glutamate is changed to alanine. The mutation does not prevent
the expansion reaction, but it is completely defective in cross-link
formation (Wikoff et al., 2000). Taken together, these observations
argue that a function of expansion is to assemble the catalytic site for
cross-linking, bringing together the three crucial residues (K169,
N356, and E363), which are widely separated in the prohead, into a
proximity that allows reaction.
Thisviewoftheroleofexpansioninallowingcross-linkingsuggests
that cross-linking can commence once a catalytic site is assembled.
Furthermore,itappearsfromthemovieofexpansionthatnotallseven
ofthequasi-equivalentclassesofcross-linkingsitesachievetheirfinal
conformation at the same time. In particular, it appears that the E
loops on the subunits of the pentamer capsomers are the last to de-
scend and might therefore be expected to be the last to cross-link.
Biochemical and structural experiments confirm this expectation
(Gan et al., 2004), providing corroboration for important features of
the dynamic model ofexpansion embodied in the movie.
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